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... Recipes From An Italian American Kitchen By Jordan Nussear Honors Project Thesis Submitted in Partial Fulfillment Of the Requirements for a Baccalaureate Degree With Distinction In the Ron and Laura Strain Honors College of THE UNIVERSITY OF INDIANAPOLIS May 2020 Faculty Advisor: Randi Frye, MFA Illustration Executive Director of Honors: James B. Williams, Ph.D. Jordan Nussear 2020. Copyright in this work rests solely with the author. Please ensure that any reproduction or re-use is done in accordance with relevant national copyright protection. i. Abstract: While language and other indicators have waned, food has remained a central symbol in Italian-American culture. From film depictions to memoirs and other forms of literature, ItalianAmericans are consistently connected to food. Food has provided a collective identity for ItalianAmericans that extends generations, and it has allowed the experiences and stories of immigrants to be shared. The connection food offers, whether its to family history or community ties, ignites pride in many people even though the culture has shifted over time. Although the evolution of ItalianAmerican culture has been examined, it has never been produced in a way that appeals to the mass of people that embrace this culture or have some connection to it. To engage larger audiences and inform them about the evolution of Italian-American culture in an interactive and entertaining way, a cookbook that shares traditional Italian-American recipes and their historical and cultural context has been developed. Accompanying the cookbook is a marketing plan with social media calendars, print, and digital collateral. Keywords: cookbook, Italian-American culture, marketing plan ii. Table of Contents Abstracti Table of Contentsii Introduction1 Project Process....2 Final Product...8 Reflection8 References..11 Appendices.....13 Appendix A: Recipes From An Italian American Kitchen Appendix B: Communication Plan Appendix C: Digital Ads Appendix D: Social Media Calendar Appendix E: Print Ads 1 Introduction: Emma DAmico, my great-grandmother, was raised by Italian immigrant parents in Brooklyn, New York throughout the 1930s and into the 1940s. An avid cook, Emma hoarded recipes to share with her friends, children, grandchildren, and eventually great-grandchildren. These recipes, scrawled in pencil on the backs of envelopes, bank statements, and old newspapers, tell the story of a young woman navigating the union of her parents Sicilian culture and her own American surroundings. Emmas story is one shared by many Italian-American immigrants. However, as time passes younger generations know less about family history and the triumphs and struggles of ItalianAmerican immigrants. This is exactly why Emmas story is a story that needs to be told, or better yet, tasted. The 1930s and 40s were an instrumental time in the development of Italian-American culture (Italians, 2013). New York in 1930 was home to over one million Italian-Americans that constituted approximately 17 percent of the citys entire population (Italians, 2013). New York was the cultural epicenter for Italian-Americans, with enclaves developing across all the citys boroughs and each section developing its own personality (Italians, 2013). The cultural activity that arose in Brooklyn in particular during this time helped shape modern perceptions of Italian-Americans, making it a valuable moment in history to be examined. Recipes From An Italian-American Kitchen is a creative project sharing the culture and history of Italian-Americans through food. Food has provided a collective identity for Italian-Americans that extends generations, and this book builds on that cultural history by outlining the origins and evolution of recipes in a format that can be easily shared. The overall goal of this project was to not only create a book that shares both recipes and cultural history, but to create an entire brand surrounding the book as well. Included in this project are a few different elements that work together to create the final product. The main component is the actual Recipes From An Italian- 2 American Kitchen cookbook that contains 20 recipes and some cultural and historical background that summarizes how they evolved upon their arrival in America. The second portion is the communication plan, outlining the goal, target audiences, and objectives of a strategic public relations campaign. The third element is the deliverables associated with the communication plan. Included in the plan is a list of deliverables that should be completed to successfully achieve the plan objectives. These deliverables include three digital ads, a flyer, a rack card, and a pitch letter. They work together to the target audiences awareness of the cookbook. This project contributes to the overall field by raising awareness and interest in the cultural evolution of a cultural segment that has extensive roots in America. Project Process The project development differed slightly from the proposed timeline based on the resources that were available to me throughout the semesters; however, the phases that the project had been broken up into remained the same. The cookbook development was divided into five phases, each focusing on a different element of the project. Phase 1 Phase I of the cookbook development process was a review of the literature in the field that would aid in the development of the books cultural history, graphic design, and marketing material. While cookbooks may seem like nothing more than a collection of recipes, they are, in fact, incredibly detailed and well-organized pieces of storytelling and art. Like a poem or painting, a cookbook tells its story through food and is a means of sharing culture. To do so effectively in this cookbook, knowledge of the history of recipes and an understanding of the time period was crucial. But that was not everything. An understanding of the fundamental principles of graphic design and their modern usage was necessary to ensure that the pages were both easy to understand and aesthetically appealing. In the development of a cookbook, knowledge of branding, public relations, 3 and marketing elements also come into play especially as an audience is carved out. These three elements guided this project and the following literature was utilized to gain a better understanding of the field. Culture is the backbone of any cookbook. As the inspiration for many kinds of cuisine, cultural elements should be interwoven into the narrative so that the recipes shared are authentic, no matter what region they are from. In his book The Italian-American Table: Food, Family, and Community, (2013) Simone Cinotto analyzes the Italian culture, traditions, and food present in New York in the 1920s until the 1940s and how those elements have shaped Italian-Americans, the inspiration for this cookbook. Cinotto connects historic events in American history with trends in the Italian-American community in New York and trends in the role of food consumption. His insights into the business of food and the popularization of ethnic cooking proved useful in connecting the recipes in the proposed cookbook with their origins in New York in the 1940s. Tracing the origin recipes farther back than New York was aided by the book Italian cuisine: a cultural history. In this book, culinary developments throughout Italian history are identified and can be compared to the developments in Italian-American cuisine (Capatti, 2003). These three sources, while emphasizing different aspects of Italian cuisine, provided an overview of how ItalianAmerican culinary trends evolved. Like culture, graphic design was a fundamental element of the development of this cookbook. While graphic design is constantly evolving, there are fundamentals that exist to guide the field. A book that outlines these principles and serves as a foundational text in many design classes is Paul Rands Thoughts on Design. Rand (2014) examines the purpose of design and designs ability to integrate beauty and function. Additionally, he offers suggestions on how to effectively use elements and principles of design such as typography, symbol, and color. While Rands book outlines the basic principles of design, Michael Bieruts book, How to use graphic design to sell things, explain things, 4 make things look better, make people laugh, make people cry, and (every once in a while) change the world, adopts a more modern take on the elements of graphic design found in creative materials today. With an extensive career in design, Bierut (2016) examines how modern design has evolved through his own sketches, revisions, and final products. Both of these texts provided reference materials in the actual design of the cookbook. Because each author has a distinct vision for the field, Rands being traditional while Bieruts is more modern, their contrasting perspectives had to be navigated throughout the design process. I opted to align my project to Bieruts modern perspective because I preferred the simplicity of the page design to contrast the colorful photos of food; however, Rands perspective was helpful in understanding some of the basic principles. Photography is another element that contributes greatly to the design process. Taking photographs, especially of food, required an understanding of lighting, angles, color and more to ensure an aesthetic and appetizing final image. The Ultimate Guide to Natural Light for Food Photography (2018) was utilized as a guide for staging food photography and composing photographs in the cookbook. Fran Flynn, the author of the lighting text, teaches in-person photography workshops for Le Cordon Bleu. Her experience and insight into food photography were very valuable as I used natural light for all the photographs included in the book, and this helped me determine what time of day to take photographs and what lighting conditions to work under. Branding is also very important when developing a campaign, or this case, product. Ensuring that all brand elements align, connect the finished book with the promotional materials. In Designing Brand Identity: An Essential Guide for the Whole Branding Team, Alina Wheeler explores the considerations that go into forming a brand strategy and reviews the steps needed to effectively execute such a strategy. The branding elements Wheeler outlines for companies extend to developing the brand identity a book might have as well. This project incorporates her ideas on a 5 cohesive brand identity by maintaining the same font and general aesthetic across all communication channels from print to digital. The marketing element of book publishing plays as critical a role in branding and design. Without marketing, the book may never be seen. In The Complete Guide to Book Marketing, David Cole touches on the importance of branding in relation to building a publishing identity. Cole (2003) outlines how the publishing identity distinguishes books and writers from other writers in the industry as they navigate traditional and non-traditional selling platforms. Cole (2003) also defines the tactics needed to create an effective public relations campaign and includes the importance of digital elements considering that social media has quickly become one of the largest tools for marketers. Understanding how to utilize online platforms is essential when developing effective public relations and marketing campaigns. In A Social Strategy: How We Profit from Social Media, Mikolaj Jan Piskorski outlines the different social media platforms and which of these platforms offer the greatest return for marketers. The book also includes case studies, analyzing other social strategies to provide a well-rounded view of how to maximize return through social media marketing (Pisjorski, 2014). This source was beneficial when developing a social media campaign promoting the cookbook because it offered insight into which platforms to utilize and how to do so effectively. For instance, I chose Instagram because it is the most visual platform and can showcase the books photography with more ease than Facebook or Twitter. In Writing Winning Proposals: Public Relations Cases, Gilliland and Hagley expand beyond social media strategies and describe how to develop strategies for every element of public relations. The book includes case studies depicting the importance of developing a public relations plan and the elements that need to be included to ensure that it is effective (Gilliland, Hagley, Cameron, & Veronda, 2018). The authors instruction on how to narrow the target audience and develop an objective for the overall campaign was the basis of the communication plan that accompanied the 6 book. While there are a number of Italian neighborhoods in cities across the United States, the Italian neighborhood of Fletcher Place was selected because it helped to narrow the target audience and keep the scope of the project manageable. Successful cookbooks were also examined for content organization, design, and brand identity to gain a better understanding of the industry as a whole. Ten published cookbooks were reviewed and analyzed for factors that make them successful. Initially, these were going to be cookbooks in the same niche market, but after looking at books in the area of Italian cooking, I found this was too limited. Therefore, as a part of the ten selected cookbooks, I decided to include a few books outside of the specific Italian cooking genre. Three of the ten cookbooks analyzed were written in relation to history, four were written by professional chefs, and three were written by modern celebrities. Dividing the cookbooks into separate categories ensured that a wide range of successful work was examined. These works were examined on the basis of content, layout and design, cultural elements and overall theme. Three of the ten cookbooks analyzed were written in relation to history. These books included How Italian Food Changed the World, A Taste of History, and A Square Meal. These books differed slightly in their formats but provided valuable insight into how to incorporate history with recipes. A Taste of History is what Recipes From An Italian-American Kitchen is closest to in terms of the format. The book features shorter blurbs of history before each recipe, which when researching reviews of the book, seemed to align with both reader preference and the time frame in which I had to complete this project. The four books written by professional chefs were Lidias Italy in America, Giadas Italy, Everyday Italian, and Cook Like A Pro. These books were selected because they were written by recognizable names in the culinary field and clearly had a high production value. These books were an aid to the cookbook development process because they were used as examples of how to properly write instructions for recipes, organization, layout, and overall tone. The three books written by modern celebrities, Magnolia Table, Cravings, and Super Tuscan were 7 viewed more on the basis of their design, layout, and photography. These resources were incredibly helpful in understanding how to stage the food for photos and deciding how to layout the pages. Overall, all of the aforementioned material contributed to the formation and execution of this project. Additional sources listed in the appendix section of the cookbook also provided content for the individual recipes. Phase II Phase II of the project was determining which recipes to include in the cookbook and researching their history. All of the recipes in this book were provided by my grandmother and great-grandmother. After extensive research was completed, some recipes proved to have a richer history than others. A total of twenty Italian-American recipes were included along with their cultural influences. Phase III Phase III of the development process contained the book outline. The research gathered and analyzed was paired with its corresponding recipe and organized into an outline. This outline, once approved by the project advisor, acted as a guide for Phase IV. Phase IV Phase IV of the development process was the photography and design portion. There were photographs taken of each recipe included in the cookbook. The photographs were sorted based on content and composition. Over 3000 photographs were taken throughout the entire process and the photographs that were not included in the book were evaluated for their ability to be used on social media in the books promotion. The design of the cookbook was done in conjunction with photography. The final product at the end of Phase IV was evaluated and edited as a whole by the project advisor. 8 Phase V Phase V of the development process was the development of a marketing plan, outlining target audiences, key messaging, and the current market environment. A three-month social media plan was developed, incorporating posts up until the books release. Included in the marketing plan was a series of print and digital materials that, when used effectively, could help persuade the target audiences to purchase a copy of the book. The final product was a clean, well-organized, and well-designed cookbook with the same quality of a professionally designed cookbook. The accompanying marketing materials were professional, aligned with the books theme, and effectively conveyed any key messages. Final Product: See the Appendix for the final project. Reflection: The overall goal of this project was to create a vessel for culture and history to be shared through food and that goal was met. The experience of Italian immigrants in New York City dramatically shaped modern perception of Italian-American food (Maranzi 2019), and Recipes From An Italian-American Kitchen helps to illuminate these experiences in a well-organized and beautifully designed cookbook with the same quality of a professionally designed cookbook. The history and recipes of 20 Italian-American dishes illustrate the social and cultural traditions during a monumental time in history. The strategic marketing and communications plan ensures that the final product could be successfully marketed to targeted audiences, while the social media calendar encourages engagement digitally. Print and digital advertisements aligning with the books theme convey the projects key messages to the target audiences. Most importantly, this cookbook connects people like me, the grandchildren and great-grandchildren of Italian immigrants, with a better understanding of their family history in an engaging and delicious way. 9 On a personal note, this project offered valuable insight into a multitude of elements surrounding the creation of both a cookbook and marketing campaign. A significant amount of research had to be conducted at the beginning of the project because my primary understanding of what goes into a cookbook came from watching the Food Network. I had very little knowledge of how to stage food for photography, how to design a book layout, and how to write a recipe people can follow. Throughout Phase I of the project when I gathered the research on proper lighting and design techniques and successful cookbooks, I was able to very clearly determine what I wanted my book to be. For example, Chrissy Teigens book Cravings was an example of what I did not want my book to look like. Flipping through the pages of that book helped me figure out what font sizes to use (hers were too large), what colors to use (hers were too bold), and how to organize the page (hers were too cluttered). On the contrary, the simplicity of Joanna Gaines Magnolia Table and Giada De Laurentiis' Giadas Italy I found to be striking. Subsequently, the page design for Recipes From An Italian-American Kitchen is clean and simple to contrast the bright and rich colors of the photographs. This project was also the first time I have ever had to complete a communication plan, so a multitude of the resources accumulated in Phase 1 contributed to that as well. Given the extensive nature of the project and the limited time frame, the communication was limited to the Indianapolis area only. The strategy behind the communication plan is that, if it were to be extended on a regional or national level, similar tactics still apply. For example, one of the elements of the communication plan is contacting food influencers to help promote the book. This was confined to Indianapolis influencers. If the book was to be promoted nationally, there are a number of food influencers and bloggers that could be used to promote the book. It would just be a matter of researching which blogger audiences align with the theme of Recipes From An Italian-American Kitchen. The same can be said about reaching out to Italian-American communities. Again, it is possible, but research would have to be conducted as to the geographic location, demographics, and interests of 10 these areas. With the advanced technology in place today, settings for Instagram advertisements can filter this information to promote the advertisement to the target markets. Overall, this project allowed for a connection between numerous disciplines including communication, marketing, graphic design, photography, history, and anthropology. This kind of crossover is not typically found in the classroom so the opportunity to actually implement a project including all these elements was educational and valuable. 11 References Bastianich, L., & Manuali, T. B. (2011). Lidias Italy in America. New York: Knopf. Bierut, M. (2016). How to use graphic design to sell things, explain things, make things look better, make people laugh, make people cry, and (every once in a while) change the world. New York: Harper Design. Capatti, A., & Montanari, M. (2003). Italian cuisine: a cultural history. Columbia University Press. Cinotto, S. (2013). The Italian-American Table: Food, Family, and Community in New York City. University of Illinois Press. Retrieved from http://www.jstor.org/stable/10.5406/j.ctt3fh46s Cole, D. (2003). The complete guide to book marketing (Rev. ed.) [Rev. ed.]. New York: Allworth Press. (2003). Retrieved December 22, 2018, from https://ezproxy.uindy.edu/login?url=http://search.ebscohost.com/login.aspx?direct=true& db=nlebk&AN=191812&site=ehost-live&ebv=EB&ppid=pp_C Corcos, G., Mazar, D., & Rodgers, R. (2017). Super Tuscan: heritage recipes and simple pleasures from our kitchen to your table. New York: Touchstone. Flynn, F. (2018). The ultimate guide to natural light for food photography. Australia: Fran Flynn Courses. Gaines, J. (2018). Magnolia table: a collection of recipes for gathering. New York, NY: William Morrow Cookbooks. Garten, I. (2018). Cook like a pro: recipes & tips for home cooks ; a Barefoot Contessa cookbook. New York: Clarkson Potter/Publishers. Gilliland, R. A., Hagley, T., Cameron, G. T., & Veronda, C. K. (2018). Writing winning proposals: Public relations cases. San Diego, CA: Cognella. Italians of New York City. (2013, October 01). Retrieved April 10, 2019, from https://www.walksofnewyork.com/blog/italians-new-york-city Laurentiis, G. D. (2006). Everyday Italian: 125 simple and delicious recipes. Prahran, Vic.: Hardie Grant. Laurentiis, G. D. (2018). Giadas Italy. New York: Clarkson Potter. 12 Mariani, J. (2011). How Italian Food Conquered The World (1st ed.). New York: Palgrave Macmillan. Piskorski, M. (2014). A Social Strategy: How We Profit from Social Media. Princeton University Press. Retrieved from http://www.jstor.org/stable/j.ctt6wpzxq Rand, P. (2014). Thoughts on design. San Francisco: Chronicle Books. Staib, W., & Murphy, M. W. (2019). A taste of history cookbook: the flavors, places, and people that shaped American cuisine. New York: Grand Central Life & Style. Teigen, C. (2017). Cravings. London: Michael Joseph. Wheeler, A. (2013). Designing Brand Identity: An Essential Guide for the Whole Branding Team, 4th Edition. Hoboken, NJ: John Wiley and Sons. Ziegelman, J., & Coe, A. (2017). A square meal: a culinary history of the Great Depression. New York: Harper. Appendix A Recipes From An Italian American Kitchen: Book http://online.fliphtml5.com/xlsfr/iwje/ Recipes From An I talian-American Kitchen Jordan Nussear Recipes From An I talian-American Kitchen Table of Contents 5 6 8 Authors Note Foreword Antipasti Antipasti Marinated Mushrooms Stuffed Artichokes Rice Balls 15 Pasta Cauliflower Sauce with Spaghetti Pasta with Meat Sauce Fettuccine Alfredo Creamy Fettuccine with Vegetables Spaghetti with Meatballs Tomato and Clam Linguine Sicilian Fettuccine Broccoli and Zucchini Pasta Fish Sauce 35 Main Dishes 39 Desserts Grandma Damicos Baked Chicken Simple Salad Eggplant with Marinara Eggplant Parmesan Chicken Parmesan Rice Pudding Italian Wedding Cookies Authors Note Its Christmas Eve. There is frost on the windows. The dining room table is set. My family is gathered around the island in the kitchen talking and laughing, excitedly awaiting our traditional Christmas Eve dinner and snacking on the array of antipasti my grandmother has spent the week preparing. This is what I remember when I think of my Italian roots. Food has a way of connecting people, even if we dont always realize it. My greatgrandmother Emma Damico, born to Sicilian parents, grew up in Brooklyn, New York during the 1930s and 40s. An avid cook, Emma collected recipes to share with her friends, children, grandchildren and eventually great-grandchildren. These recipes, scrawled on the backs of envelopes, bank statements and old newspapers, tell the story of a young woman navigating the union of her parents Sicilian culture and her own American surroundings. My grandmother, Maxine, was taught to cook by her grandmother, and when her sons married, she shared the family recipes with her new daughters. These recipes eventually made it into my hands and now yours. Enjoy. 7 Foreword While language and other cultural indicators have waned, food has remained central to Italian-American culture. From film depictions to memoirs and other forms of literature, Italian-Americans are consistently connected to food. Food has provided a collective identity for Italian-Americans and unites generations. It has also been an outlet for sharing their experiences and stories of immigration. True Italian-American culture began in the early 1900s with an influx of Italians immigrating to the United States. About 4 million Italians immigrated between 1880 and 1920, with roughly 85 percent coming from southern Italy. The political and economic turmoil in southern Italy left many in poverty, but it also allowed the cuisines of this region to thrive in the United States.1 By 1930, New York City was the cultural epicenter for Italian-Americans in the United States, with the group constituting approximately 17 percent of the entire citys population. Enclaves developed across all the citys boroughs and each section developed its own personality. The cultural activity that arose in Brooklyn during this particular time has dramatically shaped modern perceptions of Italian-Americans. 1 Food was a particularly important vessel that helped Italian culture to take root in America. Brooklyn and Queens led the charge with eight major macaroni plants operating by 1930. By 1938, the number of Italian-run grocery stores reached over ten thousand. Selling imported goods from Italy and macaroni made in the United States next to cans of Carnation Evaporated Milk truly conveys the convergence of cultures that was occurring during this time. Cheese makers, rather than using the milk of water buffaloes like they did in Italy, began using American cows milk. Bakers recreated the loaves native to their region and butchers made their own sausages rather than importing them from Italy. Because the ingredients were not identical to the food available in Italy, recipes took on their own unique traits. 2 The plethora of food in the United States was also a dramatic shift from what the immigrants had experienced in Italy. A majority of the Italian immigrants had left the impoverished southern regions like Abruzzo, Molise, Campania, Calabria and Sicily, where they were familiar with going hungry. In America, food was abundant and the prices were lower than anything in Italy. In his book, How Italian Food Changed the World, John Mariani described the new lifestyle Italians were met with when they 8 settled in AmericaThe immigrants access to everything that had once been available only to the rich or on a special holy daybig platters of pasta; tender beef, lamb, pork, and even veal; a chick that had not grown old and tough; and wonderful sweets and candiesnot only improved their diet but increased their new sense of being ItalianAmericans. Many proudly put on weight and the men patted their stomachs as a sign of the abbondanza of their new country. 2 This abundance caused the role of the Italian housewife to shift. No longer was she tasked with strictly feeding her family; the increased access to food put pressure on her to now be a good cook. Family life centralized around the kitchen table as food became a pivotal factor in the new ethnic identity Italians were forming. Selectively maintaining Old Country values, this new environment gave Italian-Americans the opportunity to create their own traditions, and many of these new rituals centered around food. 3 As time progressed the public perception of Italian food began to shift. Traditional recipes were altered to include ingredients found in America, the names of dishes changed and new dishes were developed. Each of these deviations, from subtle recipe changes to the introduction of entirely new meals, were part of the building blocks for an entirely new culture and one that lives on today. Food evokes powerful memories and feelings that span generations. We often carry on food traditions, sometimes never knowing why. If your parents or grandparents did it, you probably will too. Today, roughly 18 million Americans share Italian ancestry. While individual stories vary, Italian-Americans are united by a shared history. The intention of this cookbook is to connect individuals like myself, who have a distant connection to their heritage, with the recipes and stories of those that came before us. So much of the history of Italian-Americans, past and present, can be captured in food, and this book will share some of that rich and delicious history with you. 9 Antipasti Meaning before the meal, antipasti are a series of small appetizers served before a formal Italian meal. This colorful platter is typically used to signify to dinner guests that something special is about to begin. While Americans often view antipasti as a common household occurrence in Italy, it is actually often reserved for special occasions, holidays and family gatherings. Made up of meats, cheeses, marinated vegetables, and olives, antipasti are a beautiful and delicious way to begin a dinner party.4 Ingredients: Instructions: Mozzarella Provolone Sopressata Capocollo Prosciutto Ham Salami Black Olives Green Olives Roasted Red Peppers Crostini Collect all of the desired ingredients and arrange them on a platter. The beauty of antipasti is that it can be mixed to fit to your taste. Marinated Mushrooms Ingredients: Instructions: 1 pound mushrooms cup balsamic vinegar cup olive oil 1 teaspoon salt 1 clove garlic, minced 1 tablespoon parsley, dried Mix all the ingredients together and pour over mushrooms. Cover and marinate in refrigerator overnight. Serves 4 10 Stuffed Artichokes Stuffed Artichokes were brought to America by Sicilian immigrants. With so many Italian immigrants in New York recreating the food of their home country, artichokes were regularly being shipped to the city from California. Sicilian gangster and underboss for the Morello crime family, Circo Terranova, cornered the baby artichoke market by purchasing all of the shipments. Soon, baby artichokes became a symbol of the Morello crime family in New York. Terranova, The Artichoke King, sold these artichoke shipments for a 30 to 40 percent profit, terrorizing the distributors in the process. The Artichoke Wars began as Terranovas gang began frightening farmers into submission by attacking the California fields with machetes and hijacking trucks filled with the vegetable. New York gangs even released gas bombs on artichoke farms, attracting the attention of the Federal Bureau of Investigation and forcing New York City Mayor Fiorella LaGuardia to declare the sale, display, possession of artichokes illegal in New York on Dec. 21, 1935. The ban was lifted a week later after the racketeers surrendered. 5 Despite the rocky history in New York, Stuffed Artichokes continued to be a classic dish among Italian-American families. Ingredients: Instructions: 2 artichokes 1 cup Progresso Italian breadcrumbs 1 teaspoon salt teaspoon garlic powder teaspoon black pepper 2 tablespoons parsley, dried cup grated Parmesan 1 tablespoon garlic, minced Rinse the artichokes. Cut the stalk off at the bottom to get a flat base. Cut the top of the artichokes off and open the leaves. Flip the artichokes so that the tops are touching the cutting board and apply pressure to open the leaves further. Serves 2 Combine the breadcrumbs, salt, pepper, garlic powder, parsley and Parmesan. Sprinkle the breadcrumb mixture into the now open artichoke leaves. Start from the outside and work inward, spreading the petals as you go. Shake out any excess. Take a tablespoon of minced garlic and sprinkle it over the artichokes. Put the artichokes in a pot on a raised surface and fill the pot so it has about three inches of water. Cover and turn the stove on medium heat. Steam the artichokes for about an hour, adding more water every 15 minutes. Cook the artichokes until the flesh of the outer leaves can be scraped off easily with your teeth. 13 Rice Balls Rice Balls, or Arancini, are a Sicilian specialty with an extensive history dating back to the 10th century. Arancini, meaning little oranges, refers to the spherical shape and golden crust of these cheese-filled snacks. They can be stuffed with a variety of different ingredients including ham, mozzarella, peas or rag.6 Ingredients: Instructions: 2 cups uncooked Arborio rice 3 cups chicken broth 1 egg yolk 1/2 cup grated Pecorino Romano 1/3 pound ground beef 1 medium onion, chopped 1/2 cup tomato sauce 1/4 teaspoon salt 1/4 teaspoon pepper 1/2 lb mozzarella cheese, cut into inch chunks 1 1/2 cups seasoned bread crumbs 2 egg whites Oil, for deep-frying Cook the rice with chicken broth instead of water, according to the package instructions. Once the rice is cooked, allow to cool slightly, then stir in the Pecorino Romano and egg yolk. Cover and refrigerate until cooled, overnight if possible. Serves 12 To cook, place the oil in a large pot or deep fryer, making sure not to fill the pot more than halfway, to prevent spillage. Heat to about 370 degrees F. To make the rag filling for the rice balls, brown ground beef and onions in a skillet over medium heat until the meat is cooked. Drain excess fat from the mixture and add tomato sauce, salt and pepper. To shape the rice balls: divide the rice into 12 balls. Then take a ball of rice and split in half. With one half, flatten rice in the palm of your hand, forming rice into a cup. Add 1 tablespoon of rag and 1 square of cheese into the cup formed in the rice and use the other half of rice to cover the mixture, forming a ball. Continue until you have formed all 12 balls. Place the bread crumbs and egg whites in separate shallow bowls. Dip the rice balls in egg whites, then roll them in bread crumbs. Fry the rice balls, 2-3 at a time, until golden brown (5-7 minutes in order to melt the cheese in the middle). Remove from oil and drain on paper towels. Serve hot. 15 Cauliflower Sauce with Spaghetti New York City during the 1920s was bustling with growth as a wave of Italian-American immigrants began to settle into their new home. Unfortunately, this growth stalled when the Great Depression began. The abundance of food immigrants saw when they first arrived decreased as families struggled financially. Women were forced to be creative in the kitchen when pocketbooks could no longer cover the cost of meat. During this time, cauliflower was added to dishes as a substitute for meat and this recipe was created. Ingredients: Instructions: 1 large head of cauliflower, chopped 1 tablespoon olive oil 2 tablespoon garlic, minced 1 small onion, chopped 5 cans anchovies 2 (28oz) cans crushed tomatoes 1 (15oz) can tomato sauce cup water 1 tablespoon parsley flakes Red pepper flakes, to taste Salt and pepper, to taste Parmesan cheese, grated Heat a tablespoon of olive oil in a large saucepan. Saut the cauliflower, onions, garlic and undrained anchovies together for about 7 minutes or until the cauliflower is lightly browned. Add crushed tomatoes, tomato sauce, water, parsley flakes, salt, pepper and red pepper. Let simmer for about 1 hours until the cauliflower is tender. Serve over spaghetti and top with parmesan cheese. Serves 8 17 Pasta with Meat Sauce This dish is a fusion between the Rag alla Bolognese native to Bologna and Neapolitan rag. Incorporating both a ground meat mixture and Italian sausage, this dish represents the ease with which Italian immigrants in New York could access meat, a luxury many in the poor southern regions of Italy did not possess.2 Ingredients: Instructions: 2 (28 oz) cans crushed tomatoes 1 (15 oz) can tomato sauce 1 large white onion, chopped 4 cloves garlic, minced 3 tablespoons basil, lightly dried 3 tablespoons parsley, lightly dried Red pepper flakes, to taste Salt and pepper, to taste 2 pounds extra-lean ground beef 1 pound Italian sausage links cup of grated Parmesan cheese 2 eggs 4 slices of white bread 2 tablespoons garlic powder 1 teaspoon salt 2 teaspoons pepper 2 pounds of pasta (fusilli bucati pictured) In a large bowl, combine ground beef, salt, pepper, eggs, garlic powder and cheese. Wet the slices of bread thoroughly, squeeze and crumble the bread into the ground beef mixture. Mix well with your hands until the mixture is thoroughly combined. Set aside. Serves 8 In a large saucepan, combine the crushed tomatoes and tomato sauce. Bring to a boil, cover and reduce the heat on the sauce until it bubbles. Put a little oil in a frying pan and brown sausage links on both sides. Add the sausage links to the sauce, cover and let simmer slowly. In the same frying pan, brown the meat mixture together with the onions and garlic. Add the browned meat to the sauce. Add basil, parsley, salt, pepper and red pepper flakes to taste. Cover and let simmer for two hours, stirring occasionally. Cook pasta in salted water until al dente. Drain and top with sauce. Serve. 18 Fettuccine Alfredo This traditional Italian-American dish is credited to Italian chef Alfredo di Lelio. In 1914, when Lelios pregnant wife was experiencing severe nausea and morning sickness, he made her a plate of plain pasta and mixed it with butter and Parmesan cheese. This dish became a staple in the Lelio household and he added it to the menu of his restaurant. In 1920, American film actors Douglas Fairbanks and Mary Pickford visited Lelios restaurant, and after trying the fettuccine alfredo brought the recipe back to America where it was shared with Hollywoods elite as Alfredos Fettuccine. While the dish became popular in the United States, it did not receive the same acceptance in Italy where it was viewed as little more than buttered noodles. In the United States, the dish underwent a few modifications based on the resources available. The quality of the butter and Parmesan cheese available in the United States differed from its Italian counterpart, requiring the addition of cream to match the consistency of the original dish.7 Ingredients: Instructions: cup butter 1 cups parmesan cheese 1 cup heavy cream 1 pound fettuccine, cooked 2 tablespoons fresh parsley, chopped Strain cooked fettuccine and pour the noodles into a bowl. Immediately add butter and stir until melted. Mix in the cream, parmesan cheese and parsley. Stir until the pasta is thoroughly coated. Top with cracked pepper and grated parmesan cheese. Serves 4 21 Creamy Fettuccine with Vegetables After Fettuccine Alfredo became popular in the United States, deviations of the recipe were written. Soon, Italian-American restaurants across the east coast were featuring Fettuccine Alfredo with a twist, some including chicken or shrimp while others featured vegetables in this creamy sauce. Ingredients: Instructions: cup parmesan cheese 1 cup heavy cream 8 ounces fettuccine, cooked 2 tablespoons olive oil 2 cups broccoli florets 1 small onion, chopped 3 cloves garlic, minced Black pepper Saut the garlic and onion in olive oil over medium heat. Stir often until the onion becomes translucent. Add broccoli and cook for about 10 minutes or until the broccoli is soft. Cover and reserve. Cook the fettuccine. Drain and return the pasta to the pot. Add the cream and the reserved broccoli mixture, stirring over low heat. Add cheese and toss well. Remove from heat and top with black peper. Serves 4 23 Spaghetti with Meatballs In Italy, spaghetti and meatballs is not a dish offered at restaurants aside from those looking to draw in American tourists. This is because spaghetti and meatballs is an entirely Italian-American creation. While rooted in traditional Italian cuisine, the dishs components did not come together on Italian soil. Italy has its own version of the meatball, but it varies greatly from its American counterpart. The Italian version of meatballs are eaten alone or in soups, never on a plate of pasta. Typically, they are no larger than a golf ball. The reason meatballs have evolved to what we know them as today is due to the economic shift Italian families faced when they immigrated to America. After leaving Italy because of poor economic conditions and extreme poverty, immigrants went from spending 75 percent of their income on food in Italy to only 25 percent of their income on food in America. Now affordable, meat became a staple in the Italian-American diet. Meatballs, rather than being mostly bread like its Italian origin, consisted of mainly meat with a little bread, resulting in a denser final product. The red marinara sauce we are familiar with was popular in the United States because canned tomato sauce was easily accessible at local grocery stores.2 Ingredients: Instructions: 2 (28 oz) cans crushed tomatoes 1 (15 oz) can tomato sauce 1 large white onion, chopped 4 cloves garlic, minced 3 tablespoons basil, lightly dried 3 tablespoons parsley, lightly dried Red pepper flakes, to taste Salt and pepper, to taste 2 pounds extra-lean ground beef cup of grated Parmesan cheese 2 eggs 4 slices of white bread 2 tablespoons garlic powder 1 teaspoon salt 2 teaspoons pepper 2 pounds of spaghetti In a large bowl, combine ground beef, salt, pepper, eggs, garlic powder and cheese. Wet the slices of bread thoroughly, squeeze and crumble the bread into the ground beef mixture. Mix well with your hands until the mixture is thoroughly combined. Form into golf ball-sized balls. Serves 8 In a large saucepan, cook the onions and garlic in olive oil until tender. Add the crushed tomatoes and tomato sauce. Add basil, parsley, salt, pepper and red pepper flakes to taste. Bring to a boil, cover and reduce the heat on the sauce until it bubbles. In a frying pan, heat 2 tablespoons of olive oil and brown the meatballs. Only cook them about way through. Add the browned meatballs to the sauce. Cover and let simmer for two hours, stirring occasionally. Cook pasta in salted water until al dente. Drain and top with sauce. Serve. 24 Linguine with Clam Sauce Linguine with Clam Sauce is a signature pasta dish in Naples and Rome. In Italy, the clams are always cooked in their shells, but in the United States, the sauce is made with chopped clams. While over 75 percent of Italys border touches a body of water, areas within the United States have little access to fresh seafood and so canned, chopped clams were an acceptable alternative when recreating this dish.8 Ingredients: Instructions: 2 teaspoons chopped garlic 2 tablespoons olive oil 6 cans clams, canned with liquid Parsley Salt and pepper Red pepper 2 tablespoons of white wine 1 bottle of clam juice Saut the garlic in olive oil. Add the clams with the juices. Add of the bottle of additional clam juice if necessary. Add wine. Salt, pepper and red pepper flakes to taste. Add lots of freshly chopped parsley and let simmer until the linguine is al dente. Pour sauce over pasta and enjoy. Serves 4 27 Tomato and Clam Linguine Another version of Linguine with Clam Sauce incorporates tomatoes as well. In Italy, this red sauce utilizes the sweetness of plum tomatoes common throughout the country, but in the states, imported canned tomatoes purchased worked to mimic the traditional Italian flavors when the tomatoes grown in New York lacked the same depth and sweetness. Ingredients: Instructions: 1 (6.5 oz) can of minced clams 2 tablespoons of fresh parsley, minced 2 cloves of garlic, minced 1 (14.5 oz) can whole tomatoes drained and coarsely chopped 2 teaspoons tomato paste teaspoon basil, dried pound of linguine Drain the clams and reserve the juice. Set aside. Coat a large nonstick skillet with cooking spray. Heat over medium-high heat until hot. Add parsley and garlic. Saut until tender. Add clam liquid, tomatoes and tomato paste. Bring the sauce to a boil. Reduce heat. Add the clams and basil. Simmer uncovered for five minutes. Remove from the heat. Combine clam mixture and linguine in a bowl. Toss and serve. Serves 2 28 Sicilian Fettuccine Sicilian Fettuccine is a take on the traditional Sicilian dish Pasta alla Norma. Incorporating similar ingredients, the main difference is the absence of ricotta that makes Pasta alla Norma so distinct. New York Citys large Sicilian population made the evolution of recipes a natural occurrence based on taste and what was available, and this iteration of Pasta alla Norma is what was adopted by my family. Ingredients: Instructions: 1 pound fettuccine 1 pound peeled and diced eggplant 1 (28 oz) can peeled tomatoes 6 tablespoons olive oil 4 cloves of garlic 2 tablespoons fresh Italian herb mix (basil, oregano, and thyme) Salt and pepper, to taste Heat 3 tablespoons of olive oil in a frying pan. Saut the diced eggplant for 4 minutes. Remove the eggplant from the pan and place it on a paper towel to drain away any excess oil. Serves 4 Add 3 tablespoons of oil to the pan. Add garlic and saut. Add tomatoes and cook for 1 minute. After allowing tomatoes to cook, add fresh chopped herbs, eggplant and continue cooking for an additional one minute. Salt and pepper to taste. Drain pasta when al dente. Combine pasta with the contents of the frying pan and stir together over low heat. Serve. 31 Broccoli and Zucchini with Pasta This iteration of the Sicilian Spaghetti and Broccoli Soup incorporates zucchini and shelled noodles. Still maintaining the same texture and flavors of the soup, this dish is heartier and the zucchini adds an additional layer of flavor. Ingredients: Instructions: 2 heads broccoli, cut into small florets 5 zucchini, peeled and sliced 2 tablespoons garlic, minced 1 (16 oz) can chicken broth 1 pound medium shell pasta Olive oil Salt and pepper, to taste Red pepper flakes, to taste Parmesan cheese, grated In a large saucepan, brown the broccoli and garlic, stirring occasionally. Add sliced zucchini and continue to brown until the broccoli is tender. While the broccoli and zucchini are cooking, cook the pasta in salted water until chewy. Do not fully cook the pasta. Drain well and add the pasta to the broccoli mixture. Add chicken broth, salt, pepper, and red pepper flakes. Stir and mix well. Cover and let simmer for about 20 minutes until the pasta is tender and the broth is almost absorbed. Top with parmesan cheese and serve. Serves 4 33 Fish Sauce The Feast of the Seven Fishes is an Italian-American Christmas Eve tradition. Today, the meal typically consists of seven different seafood dishes, as many Roman Catholic households abstain from meat on Christmas Eve. The tradition comes from Southern Italy, where it is known simply as The Vigil (La Vigilia). This celebration commemorates the wait, the Vigilia di Natale, for the midnight birth of the baby Jesus. It was introduced in the United States by Southern Italian immigrants in New York Citys Little Italy in the late 1800s.10 Ingredients: Instructions: 4 (28 oz) cans crushed tomatoes 2 (6 oz) cans tomato paste 3 tablespoons olive oil 1 medium onion, finely chopped 6 cloves of garlic, minced 3 stalks of celery, finely chopped 2 pounds large shrimp 2 pounds large scallops 2 pounds scungilli 4 cans chopped clams drained (reserve juice) 1 pound calamari 2 pounds mussels 4 tablespoons basil, lightly dried 4 tablespoons parsley, lightly dried Red pepper flakes, to taste Salt and pepper, to taste In a large pot, heat 3 tablespoons olive oil. Saut celery, onions and garlic on a low heat until tender. Add tomatoes and paste to the mixture. Stir well. Cover and cook for 20 minutes. Add parsley, basil, salt, pepper and red pepper. Stir. Cover and cook for 2 hours. Clean all seafood and drain well. If the sauce is too thick, add some of the reserved clam juice. Avoid adding water or the sauce will be too thin. Add seafood and cook about 30 minutes or until the seafood is cooked. Do not overcook the seafood. Serve over linguine. Serves 12 34 Grandma Damicos Baked Chicken This is a Damico original that is perfect for a weeknight dinner or family gathering. Paired with a simple salad that incoporates Italian herbs and the typical oil and vinegar dressing, this combo is sure to please. Ingredients: Instructions: 1 pound chicken thighs 2 cups mushrooms, sliced 1 can beef or mushroom gravy Butter Garlic powder Oregano Salt and pepper Lay the chicken thighs in a baking dish. Sprinkle with salt, pepper, garlic powder and oregano. Bake at 375 degrees for 1 hour. Serves 6 Meanwhile, saut mushrooms in butter until soft. After chicken has baked for an hour, take it out of the oven and cover the chicken with the mushrooms. Pour the gravy over the mushrooms and chicken. Put the chicken back in the oven for a hour, turning the chicken after 15 minutes. Simple Salad Ingredients: Instructions: 1 head romaine lettuce, chopped 2 cups cherry tomatoes 1 can black olives 1 red onion, sliced thinly 1/4 cup olive oil 1/4 cup balsamic vinegar Italian seasoning Salt and pepper In a large bowl, mix the lettuce, olives, tomatoes and onion. Top generously with Italian seasoning and salt and pepper. Cover with olive oil and balsamic vinegar. Serves 6 37 Eggplant with Marinara This simplified version of Eggplant Parmesan is a great side with dinner or can be served over pasta as a full entre. Ingredients: Instructions: 1 (28 oz) can of crushed tomatoes 1 (15 oz) can of tomato sauce 1 medium white onion, chopped 3 cloves of garlic, minced 2 tablespoons fresh basil, chopped 1 large eggplant, sliced Salt and pepper, to taste Parmesan cheese In a large saucepan, saut onions and garlic until lightly browned and tender. Add tomatoes and sauce. Stir, add basil, salt, pepper and red pepper to taste. Cover and let simmer for about 1 hour. Slice eggplant. Fry eggplant in oil until lightly browned on each side. Put on paper towels to absorb the excess oil off the eggplant. Top the eggplant slices with sauce and sprinkle with Parmesan cheese. Serve. Serves 4 38 Eggplant Parmesan Eggplant Parmesan is a staple in southern Italian cuisine. Typically associated with Naples, Eggplant Parm is popular in the Campanian countryside and Calabria and Sicily as well. While its popularity is known throughout the region, the dishs origins are not. There are a few different theories about how this dish was created. The first was that the name is derived from Parmigiano cheese, although food writers have argued that Parmigiano is not native to southern Italy where Eggplant Parmesan is found. Some believe the dish originated in Parma in northern Italy, the birthplace of Parmesan cheese. Despite the dishs origins being unknown, its connection to Italian-American cuisine is simple. Like so many other recipes, southern Italian immigrants brought it with them to America, and the recipe has remained fairly true to its Italian roots. 11 Ingredients: Instructions: 1 large eggplant, peeled and sliced 1 (28 oz) can of tomato sauce 1 medium white onion, chopped 3 cloves of garlic, minced 2 eggs, beaten 2 tablespoons parsley, lightly dried 2 tablespoons basil, lightly dried Red pepper flakes, to taste Salt and pepper, to taste 1 (16 oz) package mozzarella cheese, shredded Parmesan cheese, grated In a saucepan, saut onions and garlic in olive oil until lightly browned. Add sauce, parsley, basil, salt, pepper and red pepper. Let simmer for 30 minutes. Serves 6 In a bowl, beat eggs and add a pinch of salt. Dip each slice of eggplant in the egg mixture. Heat olive oil in a frying pan and add the eggplant. Lightly brown on each side. Remove the eggplant from the pan and place it on a paper towel to absorb any excess oil. Take a 9x13 pan and spray it with non-stick cooking spray. Put a little sauce on the bottom of the pan. Put a layer of eggplant and cover it with mozzarella cheese. Top with sauce and parmesan cheese. Repeat layers ending with parmesan cheese on top. Cover with foil. Bake at 350 degrees for about 20 minutes or until the sauce is bubbling and the cheese is melted. 40 Chicken Parmesan Chicken Parmesan is a true Italian-American classic. Available at practically every Italian restaurant in the United States, this dish actually has no real connection to Italy aside from its inspiration. Inspired by Eggplant Parmigiana, Chicken Parmesan incorporates the abundance of meat available in New York with a traditional Italian dish. The lightly breaded and fried eggplant was replaced with chicken breast in this Italian-American adaptation. Italian immigrants created the dish due to the American preference for meat, the fact that Italian immigrant workers were now able to afford meat, and the lack of eggplants available in the United States. 12 Ingredients: Instructions: The Sauce 1 medium white onion, diced 1 tablespoon garlic, minced 1 (28 oz) can tomato sauce 1 tablespoon parsley flakes, lightly dried 1 tablespoon basil, lightly dried Salt and pepper, to taste Red pepper flakes, to taste In a saucepan, saut onion and garlic in olive oil until tender. Add tomato sauce and stir. Add parsley flakes, basil, salt, pepper and red pepper flakes. Mix well and let simmer for 30 minutes. The Chicken 8 skinless, boneless chicken breasts 2 eggs 1 cup Italian seasoned bread crumbs 3 tablespoons parsley flakes cup Parmesan cheese 2 teaspoons garlic powder 1 (16 oz) package mozzarella cheese, shredded Olive oil Serves 8 Beat two eggs in a bowl. Add one tablespoon of parsley flakes and salt. Put chicken breasts in the egg mixture and stir until they are well coated. Set aside. Mix bread crumbs with one to two tablespoons of parsley flakes, garlic and a cup of Parmesan cheese. Take the chicken breasts and pound them until they are about 1/2 inch thin. Coat the chicken breasts in the bread crumb mixture. Fry the chicken breasts in olive oil until they are lightly browned on each side. Lay the chicken breasts on paper towns to drain off any excess oil. Spray 9x13 pan with non-stick cooking spray. Put tomato sauce on the bottom of the pan. Next, put a layer of chicken breasts and top them with mozzarella cheese. Cover the cheese with sauce and top with parmesan cheese. Repeat the layers, ending with the parmesan cheese. Cover the pan with foil. Bake at 350 degrees for 30 minutes or until the sauce is bubbly and the cheese is melted. 43 Rice Pudding Rice pudding is an Italian dessert made from Arborio rice, milk, eggs, sugar and spices. This recipe incorporates the raisins that were traditionally found in Italian desserts but leaves out the orange peel, opting for cinnamon instead. Ingredients: Instructions: 1/2 cup Arborio rice 4 cups whole milk 1/2 cup sugar 1 egg 1/2 tsp cinnamon 1 tsp vanilla 2/3 cup raisins In a medium saucepan, combine rice and milk, and cook slowly for about 20 minutes uncovered, over low heat, stirring frequently. Serves 4 In a separate bowl, combine egg, sugar, cinnamon and vanilla. When rice is done, add egg mixture to rice very slowly and cook another 5 minutes. Add raisins, remove from heat. Can be served warm, room temperature or chilled. 44 I talian Wedding Cookies These cookies can be found all across Europe with names like Russian Tea Cookies or butterballs. They get their name because at some Italian weddings, these cookies are piled high and served as the cake. Ingredients: Instructions: 1 cup butter, softened cup granulated sugar 1 cup powdered sugar 2 teaspoons vanilla 2 cups of flour teaspoon salt 1-2 cups pecans, chopped In a medium bowl, cream the butter and sugar together. Serves 24 Mix in the vanilla, salt and flour. Add the pecans and mix until they are evenly distributed. Roll the dough into small balls and lay them on a nonstick cookie sheet. Place in the refrigerator for 1-2 hours. Bake cookies at 350 degrees for 10-12 minutes. Remove from the oven and let cool for about 5 minutes. Roll the cookies in powdered sugar until coated. 47 References Foner, N. (2000). From Ellis Island to JFK: New York`s Two Great Waves of Immigration. New Haven; London; New York: Yale University Press. Retrieved March 3, 2020, from www.jstor.org/stable/j.ctt1nptw2 1 Mariani, J. (2011). How Italian Food Conquered The World (1st ed.). New York: Palgrave Macmillan. 2 Cinotto, S. (2013). Introduction. In The Italian-American Table: Food, Family, and Community in New York City (pp. 1-16). University of Illinois Press. Retrieved March 3, 2020, from www.jstor.org/stable/10.5406/j.ctt3fh46s.6 3 Italian Tradition: Antipasti: Appetizer or Meal. (2014, August 23). Retrieved February 24, 2020, from https://www.delallo.com/blog/antipasti-meal-social-gathering-or-both/ 4 The Oddball Story of Ciro The Artichoke King Terranova. (2018, May 25). Retrieved March 1, 2020, from https://themobmuseum.org/blog/eighty-years-ago-ciroartichoke-king-terranova-died-broken-heart/ 5 Kilkelly, M. B. (2017, June 8). Behold the Baby Artichoke, or, Power to the Punies. Retrieved 2020, from https://www.archives.nyc/blog/2017/6/8/behold-the-babyartichoke-or-power-to-the-punies 6 Daily, K. (2012, October 18). Arancini: The Golden Fried Rice Of Italy. Retrieved February 28, 2020, from https://www.huffpost.com/entry/how-to-makearancini_n_1412240 7 Kohatsu , K. (2017, December 7). The Origins Of Fettuccine Alfredo. Retrieved January 12, 2020, from https://www.huffpost.com/entry/the-origins-offettuccine_b_4590831 8 Bastianich, L. (n.d.). Linguine with White Clam Sauce. Retrieved January 12, 2020, from https://lidiasitaly.com/recipes/linguine-white-clam-sauce/ 9 Trembath, B. K. (2015, December 18). Whats the Meaning of The Feast of Seven Fishes? Retrieved March 1, 2020, from https://history.denverlibrary.org/news/whatsmeaning-feast-seven-fishes 10 48 Wright, C. A. (n.d.). A History of Eggplant Parmesan. Retrieved February 28, 2020, from http://www.cliffordawright.com/caw/food/entries/display.php/topic_id/4/id/109/ 11 Gentile, D. (2017, December 7). The Surprising Origins Of 8 Italian-American Dishes. Retrieved January 12, 2020, from https://www.huffpost.com/entry/thesurprising-origins-of_b_7802486 12 49 Acknowledgements I want to take a moment to thank all the people that helped make this book possible. The biggest thanks goes to my grandmother, Maxine, who provided the recipes in this book and taught me about the joys of cooking for the family. Without you, this book would not have been possible. Thank you to my parents, David and Debbie and my brother, Camden, for letting me take over the kitchen for two weeks, helping me stage the food and eating all the leftovers. Many thanks to Randi Frye, Assistant Professor of Art & Design, for serving as my project advisor and providing valuable and constructive feedback throughout the entire process. Finally, thank you to my friends, Abby and Beka, and my Top Dog Team for editing the numerous drafts I sent you and putting up with my constant need for validation. 50 51 Recipes From An ItalianAmerican Kitchen is a cookbook detailing the recipes and cultural histories of 20 delicious dishes. Whether you are interested in learning more about your heritage or are just a fan of phenomenal food, this is the book for you and your family. Appendix B Recipes From An Italian American Kitchen: Communication Plan Strategic Communication Plan: Recipes From An Italian-American Kitchen Introduction: Launching in June 2021, Recipes From An Italian-American Kitchen is an exciting addition to the cookbook world. With a unique take on Italian-American culture and history, this book will provide readers with delicious and educational insight into their heritage. To ensure the cookbook is a success, it is vital that the proper audiences are made aware of the book, and to do so, a strategic communication plan is needed. Situation Analysis: According to articles in both T IME Magazine and U SA Today, genealogy is the second most popular hobby in the United States (Dalzell, 2017). Over 26 million people have taken an at-home ancestry test, highlighting the growing interest among Americans in understanding their heritage (Regalado, 2019). Many individuals are curious about connecting with their family history and deepening their sense of self. Comparatively, cookbook sales are on the rise in a declining industry. With cookbook sales in 2018 being 21% higher than sales in 2017, there is a growing desire for people to spend more time at home and in their kitchen (White, 2018). Cookbooks have also bridged the digital divide, with users being open to both hard copies and electronic versions. Pairing the growing interest in ancestry with the rise in cookbook purchases offers a great opportunity for this book. Consumers are interested in both cookbooks and genealogy so they need to be made aware of how R ecipes From An Italian-American Kitchen connects the two. Goal: For R ecipes From An Italian-American Kitchen to be purchased and enjoyed by readers across Indianapolis. Target Audience: Fletcher Place Residents: Fletcher Place is a neighborhood on the south side of Indianapolis. In the 1800s it was home to German and Irish settlers, but by the turn of the century, a wave of Italians, Jews, and other Central Europeans arrived. The neighborhood has held strong to their cultural upbringing. Fletcher Place is also home to the Holy Rosary Catholic Church, which especially serves the Italian-American community, even offering mass in Latin. The church also is well known for its Italian Street Festival held in June in the Fletcher Place. The festival, hosted since 1983, is the most well-attended church festival in Indianapolis and gives way to hundreds of visitors and dozens of vendors (About, n.d.). As the festival draws in hundreds of people across the city interested in consuming and engaging with Italian-American culture, this provides ample opportunity to connect with individuals that could have an interest in the contents of this cookbook. Indianapolis Foodies: Indianapolis is home to a diverse culinary scene with a variety of food bloggers and Instagrammers making Indy residents their primary demographic. These platforms typically have between 1,000 and 10,000 followers. While these numbers may seem small, they are the niche audience that the content of this cookbook is tailored to. The benefit of reaching out to these local influencers is that they have already established a loyal following who trust their opinion. If this book appeals to the blogger, then it will likely resonate with the audience, resulting in increased book orders. Objective: Fletcher Place Residents: General Objective: To pique the interest of Fletcher Place residents to purchase a cookbook. SMART Objective: Generate 25 cookbook orders within three months of the book launch. Indianapolis Foodies: General Objective: To pique the interest of Indianapolis food enthusiasts to order a cookbook. SMART Objective: Gather 10 media mentions on Indianapolis food blogs by the book launch. Strategy: Fletcher Place Residents: This objective can be accomplished by specifically targeting the Italian-American community in Fletcher Place through a variety of communication outlets with messaging encouraging individuals to connect with their roots and highlighting the content of the cookbook. Indianapolis Foodies: T his objective can be accomplished by strategically contacting Indianapolis food blogs and social media pages in an attempt to generate their interest and have them post about the book on my behalf. Collateral: Fletcher Place Residents: - Geotargeted Instagram ads specifically for the Fletcher Place neighborhood will help increase awareness of the cookbook and generate interest. - Flyers around and in the vestibule Holy Rosary Church will raise awareness of the book among the neighborhoods Italian-American population. - A rack card also located on the churchs bulletin will help gain awareness and interest. - A booth at the Italian Street Festival with books available for sale will attract the target audience and get book sales Indianapolis Foodies: - Thorough research about Indianapolis food blogs and a selection of which blog audiences would align with the theme of R ecipes From An Italian-American Kitchen. - Pitch letters to local bloggers requesting they read my book and endorse it to their followers. A copy of the book would be included. - Pre-selected photos and an information sheet so that if they decide to share their thoughts, they are properly informed about the book. Timeline: This campaign will take place between March 1, 2021 and July 1, 2021. Evaluation: The metrics for evaluating this campaign will be the number of book orders before and after the launch. References: About Holy Rosary. (n.d.). Retrieved February 3, 2020, from https://www.indyitalianfest.org/about_us Dalzell, R. (2018, June 15). Genealogy: the Second Most Popular Hobby in the US? Retrieved February 3, 2020, from https://blogs.ancestry.com/cm/genealogy-second-most-popular-hobby-us/ Regalado, A. (2020, January 24). More than 26 million people have taken an at-home ancestry test. Retrieved February 3, 2020, from https://www.technologyreview.com/s/612880/more-than-26-million-people-have-taken-an-a t-home-ancestry-test/ White, M. C. (2018, August 14). Recipe for success: Cookbook sales survive shift to digital media. Retrieved February 3, 2020, from https://www.nbcnews.com/business/consumer/recipe-success-cookbook-sales-survive-shift-d igital-media-n900621 Appendix C Recipes From An Italian American Kitchen: Digital Ads Recipes From An I talian-American Kitchen Recipes From An I talian-American Kitchen Recipes From An I talianAmerican Kitchen Appendix D Recipes From An Italian American Kitchen: Social Media Calendar RECIPES FROM AN ITALIAN-AMERICAN KITCHEN MARCH Date Content Get ready to eat! I am so excited to announce the launch of my new cookbook Recipes From An Italian-American Kitchen! Enjoy delicious food and learn about Italian-American culture at the same time! Stay tuned for sneak peaks, recipes, 3/1/21 mouth-watering photos and more! 3/2/21 3/3/21 3/4/21 3/5/21 3/6/21 Here is a photo of one of my favorite dishes from Recipes From An Italian-American Kitchen! This one is sure to please a crowd. To make it 3/7/21 yourself, pre-order the book now! Link in bio! 3/8/21 3/9/21 My grandmother and great-grandmother were my biggest inspirations for this cookbook. They keep family recipes scrawled on the backs of any paper they can find. Here is one on the back of a 3/10/21 bank envelope! 3/11/21 3/12/21 3/13/21 3/14/21 3/15/21 3/16/21 Photos Life isn't complete without dessert. These cookies are a staple at any family gathering whether its a birthday, Christmas or just a party, 3/17/21 they're always enjoyed by all! 3/18/21 3/19/21 3/20/21 Here's a little sneak peak of one of the recipes in the Recipes From An Italian-American Kitchen! Fun fact: one of the fonts I use in the book matches my great-grandmother's handwriting 3/21/21 almost exactly! 3/22/21 3/23/21 3/24/21 3/25/21 3/26/21 This might be my favorite photo ever. Learn how to make your own antipasti platter with Recipes From An Italian-American Kitchen! Link to 3/27/21 preorder in bio! 3/28/21 3/29/21 3/30/21 3/31/21 APRIL Date Content Photos Two more months until we can all enjoy Recipes From An Italian-American Kitchen! I can hardly wait to share my most delicious family recipes 4/1/21 with you all! 4/2/21 4/3/21 4/4/21 4/5/21 4/6/21 4/7/21 4/8/21 Rice pudding is so underrated! Sweet and cinnamony, this recipe is guaranteed to be one of 4/9/21 your new favorite desserts! 4/10/21 4/11/21 4/12/21 4/13/21 4/14/21 4/15/21 4/16/21 4/17/21 4/18/21 4/19/21 4/20/21 4/21/21 Meat. Carbs. What more could you ask for? 4/22/21 4/23/21 4/24/21 Fun Fact: Artichokes were once illegal in New York City. Check out Recipes From An ItalianAmerican Kitchen to find out why and to get a 4/25/21 stellar recipe for stuffed artichokes! 4/26/21 4/27/21 4/28/21 4/29/21 4/30/21 MAY Date Content One month to go! Recipes From An ItalianAmerican Kitchen releases June 1st! Link to 5/1/21 preorder in bio! 5/2/21 Photos 5/3/21 5/4/21 5/5/21 Who doesn't love Chicken Parm? This is the best Chicken Parm you'll ever have, hands down. 5/6/21 Don't believe me? Try it out for yourself! 5/7/21 5/8/21 5/9/21 5/10/21 5/11/21 Good 'ole Spaghetti and Meatballs. Such a classic and yet rarely done right. I've got a secret for perfect meatballs. Check out Recipes From An 5/12/21 Italian-American Kitchen to find out! 5/13/21 5/14/21 5/15/21 Tomato and Clam Linguine often flies under the radar, but make this for guests and they'll be 5/16/21 begging for more. Link in bio! 5/17/21 5/18/21 5/19/21 5/20/21 5/21/21 5/22/21 5/23/21 5/24/21 5/25/21 5/26/21 And the countdown begins! FIVE DAYS left to preorder Recipes From An Italian-American 5/27/21 Kitchen! FOUR DAYS LEFT! Make food for the whole family and enjoy a mini history lesson at the same 5/28/21 time! THREE MORE DAYS! It's almost here! Link in 5/29/21 bio! Can you believe there are only TWO DAYS LEFT until Recipes From An Italian-American Kitchen releases?! This has been such a long journey and I'm so excited for you all to get to 5/30/21 share in some amazing food and history! 5/31/21 TOMORROW! Today is the day! I am so excited to announce the launch of Recipes From An Italian-American Kitchen! I know you and your family will love these recipes as much as I do. Link to order in 6/1/21 bio! Appendix E Recipes From An Italian American Kitchen: Print Ads I talian Wedding Cookies Ingredients: Instructions: 1 cup butter, softened cup granulated sugar 1 cup powdered sugar 2 teaspoons vanilla 2 cups of flour teaspoon salt 1-2 cups pecans, chopped In a medium bowl, cream the butter and sugar together. Serves 24 Mix in the vanilla, salt, and flour. Add the pecans and mix until they are evenly distributed. Roll the dough into small balls and lay them on a nonstick cookie sheet. Place in the refrigerator for 1-2 hours. Bake cookies at 350 degrees for 1012 minutes. Remove from the oven and let cool for about 5 minutes. Roll the cookies in powdered sugar until coated. Enjoy. ITALIANAMERICANKITCHEN.COM Recipes From An I talian-American Kitchen Dear [Insert Name], As a fellow Hoosier and food-lover, Id love to share a copy of my new book Recipes From an Italian-American Kitchen with you! Inspired by my grandmothers love of food and my interest in family history, I decided to combine the two in an interesting and delicious way. This book is a collection of Italian recipes and their evolution in America. From funny stories about illegal artichokes to serious notes about how food was used during the Great Depression, my hope is that this book provides readers with a delicious dinner and a quick history lesson. Ive been following your blog/Instagram for a while now and I think that this book would be something that really resonates with your followers. It would be wonderful if you could help me spread the word about R ecipes From an Italian-American Kitchen so many more people can enjoy some of my favorite Italian-American recipes. Id be happy to send you a free copy for you to decide if youd like to share it on your blog/Instagram. And if youre interested in learning more about the book, feel free to check out my Instagram (@italianamericankitchen) or my website (www.italianamericankitchen.com)! I look forward to sharing these wonderful recipes with you! -Jordan Recipes From An I talian-American Kitchen FOOD THE WHOLE FAMILY CAN ENJOY. www.italianamericankitchen.com  ...

... Ultrastructural Study of Spermiogenesis in Masticophis flagellum, the Coachwhip Snake By Brenda Nunez Honors Project Thesis Submitted in Partial Fulfillment Of the Requirements for a Baccalaureate Degree With Distinction In the Ron and Laura Strain Honors College of THE UNIVERSITY OF INDIANAPOLIS May 2020 Faculty Advisor: Kevin Gribbins, Ph.D. Executive Director of Honors: James B. Williams, Ph.D. Brenda Nunez 2020. Copyright in this work rests solely with the author. Please ensure that any reproduction or re-use is done in accordance with relevant national copyright protection. i Abstract Currently, little is still known of spermatid development during spermiogenesis in snakes. There are only two complete ultrastructural studies on ophidians that detail spermatid ultrastructure within two venomous species: The Cottonmouth Snake and the Sea Snake. Therefore, this research was carried out with the goal of broadening our knowledge of the ontogenic steps of spermiogenesis within snakes, particularly in the Colubridae family since no other nonvenomous snake family has been studied. This was accomplished by studying spermatid maturation in the Colubridae snake Masticophis flagellum (Coachwhip snake) using transmission electron microscopy. M. flagellum specimens were gathered in the fall of 2013 in Arkansas, which is the time of year they are highly active spermiogenically. The testes were removed, placed in Trumps fixative, and tissues were processed for standard electron microscopy. Many of the ultrastructural spermatid details in the Sea Snake are similar to what has been described for squamates and ophidians. The hypothesis for this study was that spermiogenesis in the Coachwhip will have a number of notable differences in spermatid development compared to the two previously studied viperids. Most differences in spermatid development in the viperids studied to date occur in the proximal flagella, condensation pattern of the nuclear chromatin, and the acrosome complex. Other features of the spermatid architecture were similar between what has been observed in the Cottonmouth and Sea Snake. Although the spermatids within snakes appear to be well-conserved, there are differences in intracellular details between previously studied snakes, particularly in the development of the midpiece and principal piece of the flagellum, and in the elongation process of the ii spermatid. The significance of such key character differences during spermiogenesis in snakes is still unknown. Presently, there have not been enough species investigated to elucidate the phylogenetic implications of such fundamental character differences during spermiogenesis, which is the main purpose of such a study in M. flagellum. Keywords: spermiogenesis, ophidians, spermatid ultrastructure, ontogenic steps, transmission electron microscopy iii Table of Contents Cover Page. Abstract.i Table of Contentsiii List of Figures.iv Introduction..1 Statement of Purpose...1 Justification..1 Literature Review.2 Methods6 Research Design...6 Timeline...8 Faculty Advisor/Laboratory Used8 Results10 Analysis & Conclusions.17 Next Steps..19 Reflection...20 References..22 Appendices.25 Appendix A: Necessary Materials.25 Appendix B: CITI Training Certification..26 Appendix C: Material Safety Data Sheets.28 Appendix D: NCHC Project Poster...45 iv List of Figures Figure 1: Early stages of round spermatid development. Shows the development of the acrosome vesicle and basic structure of round spermatids in M. flagellum ....10 Figure 2: Elongation of acrosomal vesicle, defined development of subacrosomal space, beginning of elongation of the nucleus..12 Figure 3: Major elongation events represented. Condensation and elongation of the nucleus, development of flagellum, pronounced view of manchette and Sertoli cells 14 Figure 4: Final stages of condensation for a fully motile spermatid. The acrosomal complex forms protective apical head, the nucleus is uniform and condensed, the midpiece has pronounced mitochondrial spirals....16 1 Introduction Statement of Purpose The purpose of this project was to analyze the ultrastructural development of Masticophis flagellum spermatids during the process of spermiogenesis. This was accomplished by viewing M. flagellum testicular tissue samples using transmission electron microscopy and labeling the morphology of spermatids throughout the different developmental stages of spermiogenesis. These results will allow for a broader understanding of the changes that occur to the testis and developing sperm in M. flagellum. Now that the ultrastructural details of spermiogenesis within M. flagellum are well understood, they can be compared to those of other ophidians and to possibly serve as a histopathological model for the impacts of pesticides on snake reproductive cycles. Reptile spermiogenesis can serve as a model for sperm development in humans, particularly to predict the impact of pesticides in humans because the testes of snakes are more similar to humans than the amphibian models that have been used in the past. Justification and Honors Worthiness Partaking in this research will help expand the understanding of spermatid development in snakes and squamates, which as mentioned earlier, is lacking. This information can be used to compare normal snake spermatid development to spermatid development in snakes exposed to pesticides and other reproductive toxicants. Agriculturally speaking, the long-term impact of pesticides on animal reproduction can be studied using reptiles as a toxicological model (Gribbins, 2011). Additionally, the impact of pesticides on human spermatid development can be predicted since humans 2 rely greatly on water and pesticides can run off into water supplies. This can happen easily in water used for recreational activities, which would raise the risk of exposure. The results from this study can also provide data that may be used in future studies on phylogenetic relationships among snake species based on sperm/spermatid development/morphology once enough species have been studied. This project required many different skills that cannot be learned in a traditional classroom setting. Learning how to embed and section tissues are both valuable skills that can be used in other lab settings, and require a great deal of instruction and practice. Furthermore, I structured the project on my own and made sure that I was able to formulate results that give a comprehensive picture of how spermiogenesis occurs in Masticophis flagellum. There are many histological concepts that I mastered on my own that I have not learned in any of my other science classes. The skills I learned and the independence that I employed made this project truly honors worthy, and it will give me more opportunities than traditional undergraduate coursework in the future as I hope to go on to graduate school. Literature Review Pesticides Affecting Reproductive Cycles One of the most common threats to animal species is habitat loss, but pesticide use has also become a pertinent issue. Geographic areas that contain a large percentage of agricultural land with high pesticide use tend to have a greater loss of species diversity (Gibbs, et al., 2009). Reptiles and amphibians are two animal classes that are commonly affected by pesticides. This can be attributed to the susceptibility of these classes to 3 toxins. The normal nonlethal dosages of pesticides, such as carbaryl, can become more toxic to nonmammalian vertebrates that are undergoing stress (Relyea, 2003). With increasing levels of habitat loss and the constant threat of predation, these species become more vulnerable to pesticides because this effect is amplified by normal stress (Gibbs, et al., 2009). An organ system that can be greatly affected by pesticides is the male reproductive system. Some of the most common insecticides used in the agricultural industry are organophosphates, which can coincidentally be toxic to male reproductive organs (Joshi, et al., 2007). In fact, the testes were the only organ that had a visible size reduction in a study conducted that exposed lab rats to organophosphate pesticides (Joshi, et al., 2007). Additionally, the sperm counts were greatly decreased in rats exposed to these pesticides. These findings could hold further implications for human fertility as well, which is why understanding and studying the reproductive cycles of reptiles such as snakes is important. Since many of these species are semi-aquatic and are regularly exposed to these pesticides in natural water sources, they can be used as a model for repeated pesticide exposure for humans (Joshi, et al., 2007). Spermatogenesis in Snakes Spermatogenesis contains many stages, which include proliferation, meiosis, and spermiogenesis. In snakes, spermiogenesis contains four substages that include acrosome development, flagellum formation, elongation, and condensation of the DNA (Aldridge and Sever, Gribbins, 2011). However, before these can occur, it is important to know that genetic recombination has already occurred in order to produce unique haploid cells to 4 provide for greater genetic biodiversity observed within a species (Schulz, 2009). In acrosome development, the acrosome matures near the nucleus of the developing sperm. It does so to be able to cover the head of the mature sperm. The acrosome is in an apical position, while the developing flagellum takes on a basal developmental location. This helps establish polarity in the spermatozoa and these two locations of development have been utilized in phylogenetic studies among species that have similar acrosome position (Rheubert, et al., 2009; Gribbins, 2011). Additionally, there are manchette microtubules surrounding the nucleus that aid the spermatid in elongation, forming the manchette. Other microtubules are also working late in elongation to form a flagellum, which is vital for the mobility of the sperm cell (Aldridge and Sever, Gribbins, 2011). While elongation in the flagellum is occurring, the acrosome covers the nuclear head, which creates a thin rostrum that gives a more characteristic sperm shape. Since the nucleus is being forced into the acrosome, the chromatin inside the nucleus begins to condense, which is the final substage of spermiogenesis (Gribbins, 2011). While the steps above describe the general process of spermiogenesis in reptiles, there are differences in the spermiogenesis of snakes. In snakes, spermiogenesis occurs in the germinal epithelium. Additionally, the acrosomal vesicle that develops in early round spermatids forms from aggregating vesicles that come from the Golgi apparatus. The acrosomal granule is seen within the acrosomal vesicle before it even merges with the nucleus at the early to mid-round spermatid stage (Gribbins, Freeborn & Sever, 2016). The acrosomal complex continues to develop and is compartmentalized, with layers such 5 as: the subacrosomal space, perforatorium, and epinuclear lucent zone. During ophidian elongation, a manchette forms out of microtubules that aids in further nuclear elongation. Furthermore, it has been observed that snake spermatids only have parallel microtubules and not cylindrical fibers like other squamates have (Gribbins, Freeborn & Sever, 2016). These microtubules tend to run the full length of the flagellum, which is not common in other reptiles. Environmental Effects on Spermatogenesis While the steps of spermatogenesis mentioned above occur universally across snake species, different factors can impact the rate at which they appear. For example, the elevation at which the organism is found can greatly impact when it can reproduce, which directly impacts sperm reproduction (Cox, 2006). Another important environmental factor that influences sperm development is temperature. Cold environments in particular can affect reptiles in a positive way when concerning their reproductive abilities. This is mainly due to how reptiles born in colder weather are able to grow more efficiently since they learn to use resources more effectively (Cox, 2006). The enhanced growth also applies to their reproductive organs (Oufiero & Angilletta, 2006). However, Masticophis flagellum prefers to mate in warmer weather and the differences between spermatogenic development ultrastructurally within species occupying different environmental temperatures have not been explored. The reason for this lack of data again revolves around the lack of information on sperm development in snakes (Gribbins, 2011). But, if data were to be gathered, there can be more ties made between warmer weather triggering an endocrinal and spermatogenic reactions in snakes. This means that increasing 6 temperatures could cause male snakes to produce sex hormones, which would lead to greater sperm production. If this result is true in Masticophis flagellum, it could make them a great model species for the endocrine stress model in other species (Lutterschmidt & Mason 2009). Masticophis flagellum Masticophis flagellum is a species of snake, commonly known as the Coachwhip Snake. It varies greatly in size, making its smooth scales its defining feature. They are one of the most common snake species in any ideal warm temperature habitat, varying from deserts, grasslands, forests, creeks, valleys, and even swamplands (Stewart, 2018). Their preference for warmer weather extends to their reproductive habits. They breed in the warmer days of late summer and neonates hatch before the cold weather of late autumn starts (Stewart, 2018). Since Coachwhips are so common throughout the southern United States, they are a perfect model to use to study pesticide levels/effects throughout the country (Gibbs, et al., 2009). And even though their mating season is specific, their wide range of habitats can be used to study how spermatogenesis adapts in the snake species given different weather and elevation conditions (Oufiero & Angilletta, 2006). Methods Research Design Male Masticophis flagellum were gathered in Arkansas in the early fall of 2013. After being given a lethal overdose of anesthesia, the testes were removed, cut into 4mm pieces, and embedded in plastic. This was done using a standard Embed 812 kit. The embedded tissues were sectioned using a Leica Ultracut S Ultramicrotome located in the 7 histology lab in the basement of Lilly Science Hall. Glass knives were required for the microtome to be able to section the embedded tissue, and these knives were provided by Dr. Gribbins. The embedded testicular tissues from approximately five male M. flagellum were sectioned and placed on light microscope slides. Once the samples were placed on light microscope slides, they were then stained using toluidine blue (see Appendix C for safety data). This nontoxic stain was placed on the slides from 15 seconds-1 minute and then rinsed off using deionized water. Both the slides and the stain were provided by Dr. Gribbins and the Biology Department. Once the staining process was complete, the slides were analyzed under a light microscope that was connected to a desktop computer using Adobe Photoship CS7 to ensure that testes were in the spermiogenic stage of development. All of this equipment was located in Dr. Gribbins histology lab. Once testicular blocks were identified as containing spermiogenic stages by light microscopy analysis, they were thin sectioned (90 nm) and prepared for electron microscopy on a Zeiss 109 TEM. The samples were stained for TEM using uranyl acetate and lead citrate (see Appendix C for safety data). Two different kinds of grids were required to hold down different testicular thin sections during TEM, a 150 mesh gilder grid and two square mesh EMS grids (EMS, 2019). The spermatid cells were compared to those of the Cottonmouth and Sea Snake to examine how spermiogenesis differs between different species of snake. A comprehensive list of all the materials that were used in this research can be viewed in Appendix A. 8 Spermiogenic cell types were photographed from the thin sections using a Zeiss 109 electron microscope. The different developmental morphologies were identified from the thin sections and arranged in photograph plates using Adobe Photoshop CS7. Photoshop was also used to label the pictures and identify key features of Coachwhip spermiogenesis. Additional sectioning was required since not all of the stages of M. flagellum spermatid development were identified, and sectioning/staining was terminated once all spermiogenesis stages were found and photographed. Timeline My project began over a year ago in February, 2019, in order to obtain preliminary data. I presented at a research conference at Butler University in April, 2019, and I applied to present at the 2019 National Collegiate Honors Conference. Therefore, I had all of the embedding and sectioning for my project completed by March, and over half of my images labeled by early April, 2019. I also worked through last summer at home to prepare for my project before the fall semester began. This was done to ensure that I had ample time to comprehensively label the histological pictures obtained from the electron microscope for NCHC. All of my images were fully labeled and evaluated by Dr. Gribbins by the first week of March, 2020. My present manuscript was written between late November, 2019 through mid-March, 2020. Role of Advisor Dr. Kevin Gribbins, my advisor, was essential in guiding my research and helping me reach my goals. I scheduled meetings with him, and he periodically checked in with me to see if I needed anything. He edited and reviewed my work before any submissions 9 took place so that I presented accurate data. I worked predominantly in his lab, located in the basement floor of Lilly Science Hall. Even though he has previously trained me in using sectioning equipment, he still helped me construct the layout of my study, and provided me with adequate resources to label my histology pictures. We worked together during this past summer so that I could complete my research on time, and so that I was able to obtain images of all the stages of spermiogenesis in M. flagellum. 10 Results Go Figure 1 A-C. Early stages of round spermatid development. Shows the development of the acrosome vesicle and basic structure of early round spermatids in M. flagellum. NU- Nucleus, AV- Acrosomal Vesicle, AVS- Acrosomal Vesicle Shoulder, Go- Golgi Complex, Ve- Vesicles, Li- Lipid Droplet, Black Arrow- Prominent Acrosomal Granule, Mi- Mitochondria, LB- Lamellar Body The cells that enter the third and last step of spermatogenesis, spermiogenesis, have undergone incomplete cytokinesis at the end of meiosis (the second step of spermatogenesis), and have already undergone genetic recombination and are haploid. These early round spermatids have an especially pronounced nucleus (Fig. 1A & 1C, 11 NU), which begins to develop an adjacent acrosomal vesicle (Fig. 1A-C, AV). This vesicle formation marks the first step of spermiogenesis. The acrosomal complex will eventually form the apical head, or protective tip, of the mature spermatid. The Golgi body complex (Fig 1B & 1C, Go) produces the acrosomal vesicle and can be seen directly adjacent to the growing acrosomal vesicle. Mitochondria can be observed (Fig, 1A & 1C, Mi) near the Golgi complex and nucleus, providing needed energy to the production of the acrosomal vesicle. The acrosomal granule was developing as well (Figure 1A & 1C, black arrow). This granule was filled with proteolytic enzymes, which were responsible for the breakdown of proteins in a fully elongated spermatid. The Lamellar body (Fig. 1C, LB) function is unknown in other reptiles and forms during acrosome development. 12 Figure 2 A-C. Elongation of acrosomal vesicle, defined development of subacrosomal space, beginning of elongation of the nucleus. A- Annulus, NU- Nucleus, AVAcrosomal Vesicle, Black Star- Lipid Vesicles, PAG- Prominent Acrosomal Granule, White Arrow- Subacrosomal Granule, SAS- Subacrosomal Space, PC- Proximal Centriole, DC- Distal Centriole, NF- Caudal Nuclear Fossa. In the mid-stages of spermiogenesis, the nucleus became slightly more oval shaped (Fig. 2A-C, NU) as it prepares to elongate. The subacrosomal space develops between the nucleus and acrosomal vesicle (Fig. 2A & 2C, SAS), which will help with further partitioning of the acrosome complex. Within the subacrosomal space is a small cone-shaped structure known as the subacrosomal granule (Fig. 2A, White Arrow). This 13 granule will become cylindrical in shape and will further help the sperm cells nucleus obtain its characteristic oblong shape. Additionally, the acrosomal vesicle was spreading across the surface of the apical nucleus (Fig, 2C, AV), flattening out the nuclear head to form the apex of the spermatid. At the caudal end of the cell, the proximal centrioles (Fig. 2A & 2C, PC) the distal centriole (Fig. 2A, DC), and the annulus (Fig. 2A) can be seen. Figure 2A demonstrates how these structures begin to stick out from the basal nucleus, ultimately forming the flagellum that the sperm cell uses for motility. While the developing flagellum formed by the centrioles will protrude from the cell, the nuclear fossa (Fig. 2A, NF) will remain within the cell but still aid in the motility and anchoring of the flagellum. The prominent acrosomal granule (Fig. 2A & 2C, PAG) has become more prevalent in the acrosomal vesicle. 14 Figure 3 A-E. Major elongation events represented. Condensation and elongation of the nucleus, development of flagellum, pronounced view of manchette and Sertoli cells. NUNucleus, AV-Acrosomal Vesicle, PC-Proximal Centriole, DC-Distal Centriole, FS-Fibrous Sheath, PE-Perforatorium, SC-Sertoli Cell, SAS- Subacrosomal Space, SCM-Sertoli cell membranes, BP-Basal Plate, AVS- Acrosomal Vesicle Shoulder, ALR- Acrosomal Lucent Ridge, Li-Lipid droplet, AC- Acrosomal Cortex , MT- Microtubules, ELZ- Epinuclear Lucent Zone Elongation, the second stage of spermiogenesis, is especially prevalent in Figures 3A and 3E. The nucleus in particular appears (Fig. 3A & 3E, NU) elongated within the cytoplasm and shows condensation of its chromatin as noted by its intense dark stain. The flagellum has further developed and now protrudes fully from the mature spermatid. Even the early structures of the flagellum, such as the distal centriole and the proximal centriole (Fig. 3A & 3E, DC, PC), are still visible and serve as the base of the flagellum. The fibrous sheath (Fig. 3E, FS) that forms a protective and structural barrier around the flagellum can also be observed at this time. Additionally, Figure 3D shows a manchette 15 has properly formed around the nucleus and 3E has manchette microtubules, which aid in sperm elongation. A transverse section of the cell (Fig. 3C) displays how the acrosomal vesicle has developed into a more complex structure. The perforatorium (3B-3C, PE) is a rod-like structure at the center of the acrosomal complex. It can be seen that the apical head that is forming is composed of many layers surrounding the epinuclear lucent zone, such as the subacrosomal space from earlier stages of spermiogenesis (Fig. 3C, SAS), the acrosomal lucent ridge (Fig. 3B-3C, ALR), the acrosomal vesicle (Fig. 3B-3C, AV), and the Sertoli cell membrane (Fig. 3C, SCM). The subacrosomal space, the acrosomal lucent ridge, and the acrosomal vesicle all give the apical head of the spermatid its characteristic pointed shape and unique compartmentalization. 16 Figure 4 A-E. Final stages of condensation for a fully motile spermatid. The acrosomal complex forms protective apical head, the nucleus is uniform and condensed, the midpiece has pronounced mitochondrial spirals. NU-nucleus, Mi- Mitochondria, PCProximal Centriole, DC-Distal Centriole, FS-Fibrous Sheath, DB-Dense Body, PFPeripheral Fiber, PE-Perforatorium, White arrow- Nuclear Fossa, Red arrowMitochondrial Spiral, END- End Piece, Black arrow- Acrosome, Ma- Manchette, AVSAcrosomal Vesicle Shoulder, ALR- Acrosomal Lucent Ridge, ACM- Acrosomal Medulla The spermatid represented in Figure 4 is a fully matured spermatid right before spermiation. Figure 4A best displays the architecture of the body of the mature spermatid. The nucleus (NU) is fully elongated, and the fibrous sheath (FS) has fully formed around the principal piece and midpiece of flagellum. The mitochondrial spiral (red arrow) has finished forming, which is universally common in the midpiece of spermatid flagellum in snakes (Alberts, et al., 2002). Dense bodies, mitochondria, peripheral fibers, and the fibrous sheath can be seen in either the principal piece and midpiece of the flagellum cross sections of Figures 4B-4D. All of these structures aid in the motility of the mature 17 sperm cell. There are also over 20 tiers of mitochondria within the midpiece of the flagellum. The apical nuclear head has completely developed, as can be seen in Figure 4A (black arrow). The structures that make up the apical nuclear head, such as the acrosomal vesicle, perforatorium, subacrosomal space, Sertoli cell membrane, basal plate, acrosomal vesicle shoulder, acrosomal lucent ridge, and acrosomal cortex are now fully developed and visible (see Fig. 3B-C). The end piece of the flagellum in Figure 4D still has peripheral fibers, which is uncommon in reptiles. Figure 4E displays a sagittal section of a single mature spermatid. The manchette can be seen fully formed around the nucleus. While the flagellum cannot be seen, the distal centriole is still present and there is a clear view of the acrosomal vesicle shoulder and the acrosomal lucent ridge. Analysis/Conclusion Spermiogenesis in Masticophis Flagellum (Coachwhip Snake) is similar to other ophidians and reptiles, but the main differences typically occur during the elongation phase of spermatid maturation (Gribbins, 2011). While other squamates have their peripheral fibers terminate in the principal structure of the flagellum, the Coachwhip Snake, the Sea Snake, and Cottonmouth Snake have peripheral fibers that extend all the way through its end piece (see Fig. 4D). All three of these snakes also do not seem to have circum-cylindrical fibers, which means that they will have a more robust nucleus since only parallel fibers are aiding in elongation (Gribbins, Freeborn & Sever, 2016; Gribbins, et al., 2009). Another notable similarity in all three snakes is that the formation of the acrosomal cortex appears nearly identical. For example, there are clear granulated 18 protein layers, perforatorium, and a thinned apical nucleus (Gribbins, Freeborn & Sever, 2016; Gribbins, et al., 2009). All three snakes also have a prominent acrosomal granule while the acrosomal cortex is first forming, which is common in snake spermiogenesis. These similarities or possible synapomorphies within snakes during spermatid maturation could help determine phylogenetic relationships between snakes and other reptiles. However, there are also differences between Coachwhip, Sea Snake, and Cottonmouth Snake spermiogenesis. A difference between the Sea Snake and the Coachwhip Snake is that the dense collar piece is not visibly present between the distal and proximal centrioles in the Coachwhip Snake (Gribbins, Freeborn & Sever, 2016). The Cottonmouth Snake has areas of open nucleoplasm in its spermatid nucleus, indicating spiral chromosome condensation. There are also translucent areas on either side of the flagellum within the cell (located at the nuclear fossa) of the Cottonmouth snake (Gribbins, et al., 2009). However, neither the Coachwhip nor the Sea Snake appear to have this type of chromosome condensation due to the dark and dense staining of the nucleus in late stages of spermiogenesis. The Cottonmouth Snake also appears to have a much thicker acrosomal lucent ridge than either the Sea Snake or the Coachwhip (Gribbins, Freeborn & Sever, 2016; Gribbins, et al., 2009). Another distinction worth mentioning is the number of concentric mitochondria between the Sea Snake and Coachwhip. While the Coachwhip has 20 concentric rings of mitochondria around the midpiece of the flagellum, the Sea Snake has 40 rings of mitochondria, which are used to power the flagellum (Gribbins, et al., 2009). These differences again might be important differences 19 between species of snake that could aid in separating taxa of snakes from one another in phylogenetic analysis. However, it is too soon to draw any definitive conclusions since so much is unknown about spermiogenesis in snakes. Next Steps The image results obtained in this study could greatly benefit studies on the environmental impacts of pesticide use. Coachwhips are common throughout the entire United States, and are present in many different climates. Therefore, Coachwhips make an excellent model to see the broad environmental impacts of pesticides, as well as the impacts on specific climates and environmental characteristics on sperm development. Additionally, now that another model of spermiogenesis in snakes has been proposed, there can be a greater understanding of pesticide effects on spermiogenesis in human males. Specifically, whether or not certain pesticides are impacting the fertility and sperm development in human males may be explored by using snakes as their testicular architecture is similar to humans and, therefore, a possible histopathology model for how pesticide exposure affects human and other amniotes sperm production. The results obtained in this study are but one piece of a very large puzzle. Further research can certainly be done by analyzing spermiogenesis in snakes similar to the Coachwhip, and all snakes in general. This would lead to a more accurate understanding of phylogenetic relationships among snakes. In regards to this study, more images may be obtained to give a more comprehensive view of not just spermiogenesis, but the whole process of spermatogenesis in M. flagellum. One image that would aid in our analysis is a full sagittal image that would display all of the pieces of the flagellum and acrosome. 20 While some transverse sections were obtained (see Fig. 4B-4D), it would be extremely beneficial to obtain a sagittal image of the flagellum since the key differences between M. flagellum and other snakes were found in the flagellum. This research project has found a fairly comprehensive model for spermiogenesis in the Coachwhip Snake, but further research must be done in order for it to be applied in other research questions. Reflection This project was greatly beneficial to me because it allowed me to learn skills that I would not be able to learn in any other science classes. For example, I learned how to section tissues using a microtome, which could help me obtain future employment and research opportunities. I also learned how to prepare and stain tissues, which are valuable skills that are not part of the curriculum of other biology labs. In addition to these skills, I also learned how to employ time management while balancing a full-time class load and structuring my project around it. One of the greatest opportunities presented to me with this research was being able to present my research at two conferences. The NCHC in New Orleans was the opportunity of a lifetime for me, and I was able to participate in many growth and leadership activities. This conference was the culmination of my research, and quite honestly, most of what I have learned during my academic career. However, this project also came with its difficulties. I did not have much experience with histology coming into college, and many of my classes never focused on it. This required me to do a lot of learning on my own, which can be difficult on top of a traditional course load. Moreover, I began researching with Dr. Gribbins halfway through my first year at the University of Indianapolis. While I was thrust into the world of 21 research, I was also learning to navigate college classes. My project also shifted between three organisms before finally settling on M. flagellum. It was frustrating at the time, but I understand that some of the other organisms I had worked on were more on the lines of a graduate level project that would take much longer to complete than a traditional honors project. And while I tried to adhere to a strict timeline, life and other unforeseen circumstances kept appearing. One major obstacle that occurred near the end of my project was the Coronavirus pandemic. I was supposed to go in to the histology lab after spring break to find the images I am missing in my results. However, I was not able to do this. Further studies will have to complete some of the details that are missing for the spermatid flagellum and acrosome complex. Looking back on my project, if I could do it all again, I would have completed my manuscript last semester, but I have learned many lessons along the way. 22 References Alberts, B., Johnson, A., Lewis, J., et al. (2002). Sperm. Molecular Biology of the Cell. 4th edition. New York: Garland Science. Available from: https://www.ncbi.nlm.nih.gov/books/NBK26914/ Aldridge, R. D., & Sever, D. M. (2011). Reproductive Biology and Phylogeny of Snakes (Vol. 9, Reproductive Biology and Phylogeny). Enfield, NH: Science. Cox, R. M. (2006). A Test of the Reproductive Cost Hypothesis for Sexual Size Dimorphism in Yarrow's Spiny Lizard. Journal of Animal Ecology 75.6 (2006): 1361-1369. Print. Embedding Kit. (2019). Retrieved from https://www.emsdiasum.com/microscopy/products/chemicals/edta.aspx EMS and Gilder Grids. (2019). Retrieved from https://www.emsdiasum.com/microscopy/products/grids/gilder.aspx Gibbs, K., Mackey, R., & Currie, D. (2009). Human land use, agriculture, pesticides and losses of imperiled species. Diversity and Distributions, 15(2), 242-253. doi:10.1111/j.1472-4642.2008.00543.x Gribbins, K.M. (2011). Reptilian spermatogenesis: a histological and ultrastructural perspective. Spermatogenesis, 1(3):1-20. Gribbins, K. M., Freeborn, L. R., & Sever, D. M. (2016). Ultrastructure of spermatid development within the testis of the Yellow-Bellied Sea Snake, Pelamis platurus (Squamata: Elapidae). Spermatogenesis, 6(3). doi: 10.1080/21565562.2016.1261666 23 Gribbins, K. M., Rheubert, J., Anzalone, M.L., Siegel, D.S., Sever, D. M. (2009). Ultrastructure of Spermiogenesis in the Cottonmouth, Agkistrodon piscivorus (Squamata: Viperidae: Crotaline). Journal of Morphology, 000, 1-12. doi: 10.1002/jmor.10798 Joshi, S., Mathur, R., & Gulati, N. (2007). Testicular toxicity of chlorpyrifos (an organophosphate pesticide) in albino rat. Toxicology and Industrial Health, 23(7), 439-444. Lutterschmidt, D.I., and Mason, R.T. (2009) Endocrine mechanisms mediating temperature-induced reproductive behavior in red-sided garter snakes (Thamnophis sirtalis parietalis). Journal of Experimental Biology, 212: 31083118. Oufiero, C. E., and Angilletta, M.J. (2006). Convergent Evolution of Embryonic Growth and Development in the Eastern Fence Lizard (Sceloporus undulatus). Evolution, 60.5: 1066-1075. Relyea, R. (2003). Predator cues and pesticides: A double dose of danger for amphibians. Ecological Applications, 13(6), 1515-1521. Rheubert, J., Wilson, B., Wolf, K., & Gribbins, K. (2010). Ultrastructural study of Spermiogenesis in the Jamaican gray anole, Anolis lineatopus (reptilia: Polychrotidae). Acta Zoologica, 91(4), 484-494. doi:10.1111/j.14636395.2009.00446.x Safety Data Sheet: EMBED 812 RESIN. (2017). Retrieved from https://www.emsdiasum.com/microscopy/technical/msds/14900.pdf 24 Schulz, R. (2009). Spermatogenesis in fish. General and Comparative Endocrinology. Stewart, H. (2018). Masticophis flagellum. Animal Diversity Web. Accessed March 2, 2019 at https://animaldiversity.org/accounts/Masticophis_flagellum/ Ted Pella, INC. (2017). Safety Data Sheet: Product No. 19312, 19314 Lead Citrate, Trihydrate. Retrieved from https://www.tedpella.com/SDS_html/19312_19314_sds.pdf Ted Pella, INC. (2017). Safety Data Sheet: Product No. 19451 Toluidine Blue O, Certified. Retrieved from https://www.tedpella.com/SDS_html/19451_sds.pdf Ted Pella, INC. (2017). Safety Data Sheet: Product No. 19481 Uranyl Acetate, Dihydrate. Retrieved from https://www.tedpella.com/SDS_html/19481_sds.pdf Protopapas, N., Hamilton, L.E., Warkentin, R., Xu, W., Sutovsky, P., Oko, R., (2019). The perforatorium and postacrosomal sheath of rat spermatozoa share common developmental origins and protein constituents, Biology of Reproduction, Volume 100(6); 14611472, https://doi.org/10.1093/biolre/ioz052 25 Appendices Appendix A: Necessary Materials, Budget Materials Acquired Embed 812 Kit Price $96 Snake samples Microtome Light microscope (w/camera) Glass knives Microscope slides Slide covers Stains TEM Mesh gilder grids $25 EMS grids $46 26 Appendix B: CITI training Completion Date 22-Mar-2019 Expiration Date 21-Mar-2021 Record ID 29634279 This is to certify that: Brenda Nunez Has completed the following CITI Program course: Human Subjects Research (HSR) Group 3: Non-Health Related Research 1 - Basic Course (Curriculum Group) (Course Learner Group) (Stage) Under requirements set by: University of Indianapolis Verify at www.citiprogram.org/verify/?wd5cf10bc-283c-42c1-9ced-5fae9e50bd6e-29634279 27 Completion Date 06-Dec-2018 Expiration Date 05-Dec-2023 Record ID 29634280 This is to certify that: Brenda Nunez Has completed the following CITI Program course: Responsible Conduct of Research (RCR) Group 8: Responsible Conduct of Research 1 - Basic Course (Curriculum Group) (Course Learner Group) (Stage) Under requirements set by: University of Indianapolis Verify at www.citiprogram.org/verify/?w447ce709-3f12-406c-92be-e9ce28d469ed-29634280 28 Appendix C: Material Safety Data Sheets Embed 812 Resin 29 30 31 32 Toluidine Blue Safety Data Sheet Product No. 19451 Toluidine Blue O, Certified Issue Date (05-06-15) Review Date (08-31-17) Section 1: Product and Company Identification Product Name: Toluidine Blue O, Certified Synonym: Tolonium Chloride, Basic Blue 17 Company Name Ted Pella, Inc., P.O. Box 492477, Redding, CA 96049-2477 Inside USA and Canada 1-800-237-3526 (Mon-Thu. 6:00AM to 4:30PM PST; Fri 6:00AM to 4:00PM PST) Outside USA and Canada 1-530-243-2200 (Mon-Thu. 6:00AM to 4:30PM PST; Fri 6:00AM to 4:00PM PST) CHEMTREC USA and Canada Emergency Contact Number 1-800-424-9300 24 hours a day CHEMTREC Outside USA and Canada Emergency Contact Number +1-703-741-5970 24 hours a day Section 2: Hazard Identification 2.1 Classification of the substance or mixture GHS Pictograms GHS07 GHS Categories GHS07 - Irritant Acute Tox. 4 Skin Irrit. 2 H332: Harmful if inhaled. H315: Causes skin irritation. 2.2 Label elements Hazard Pictograms GHS07 Signal Word: WARNING Hazard statements H332 Harmful if inhaled. H315 Causes skin irritation. Precautionary statements P261 Avoid breathing dust/fume/gas/mist/vapors/spray. P280 Wear protective gloves/protective clothing/eye protection/face protection. P304+P340 IF INHALED: Remove victim to fresh air and keep at rest in a position comfortable for breathing. 33 34 35 36 37 Uranyl acetate 38 39 40 41 Lead citrate 42 43 44 45 Appendix D: NCHC Project Poster  ...

... Effects of Gender and ASD Diagnosis on Perceived Personality and Job Capability in Interviews By Darian J. Murray Honors Project Thesis Submitted in Partial Fulfillment Of the Requirements for a Baccalaureate Degree With Distinction In the Ron and Laura Strain Honors College of THE UNIVERSITY OF INDIANAPOLIS May 2020 Faculty Advisor: Melissa Loria, Ph.D. Executive Director of Honors: James B. Williams, Ph.D. Darian Murray 2020. Copyright in this work rests solely with the author. Please ensure that any reproduction or re-use is done in accordance with relevant national copyright protection. EFFECTS OF GENDER AND ASD DIAGNOSIS IN INTERVIEWS ii Abstract Gender and disabilities have long been identified as factors that contribute to discrimination in society today. The current study explores the interaction between these two factors in the specific context of a hiring situation. In this study, participants were asked to read a resume and job interview notes and then answer questions regarding perception of social skill, job capability, and personality. The resume and job interview notes were identical except two were male and two were female, and one of each gender had a stated diagnosis of Autism Spectrum Disorder (ASD). There were 116 total participants (mean age=36.32, SD=10.94), of which 65.5% were male. No significant interaction effects were found; however, the results revealed a number of interesting main effects within the constructs studied, including the factors hiring recommendation, overall performance, absenteeism, bad other characteristics, and job fit. Despite the lack of interaction effects, future research exploring the significant main effects observed in this study would likely contribute to the growing body of ASD literature by providing further avenues of research and insights into possible limitations that are found when studying this population in this regard. Keywords: Autism Spectrum Disorder (ASD), discrimination, job interview, perception, gender EFFECTS OF GENDER AND ASD DIAGNOSIS IN INTERVIEWS iii Table of Contents Title Page .. i Abstract .... ii Table of Contents .... iii List of Figures . iv Introduction ...... 1 Methods .......... 11 Results .... 18 Discussion ...... 25 Conclusion ...... 32 References ...... 33 Appendices . 43 EFFECTS OF GENDER AND ASD DIAGNOSIS IN INTERVIEWS iv List of Figures Figure 1: Perception of Hiring Recommendation ....19 Figure 2: Perception of Overall Performance .. 20 Figure 3: Perception of Absenteeism ........... 21 Figure 4: Perception of Bad Other Characteristics .... 24 Figure 5: Perception of Job Fit . 25 EFFECTS OF GENDER AND ASD DIAGNOSIS IN INTERVIEWS 1 Introduction General information Autism spectrum disorder (ASD) is a lifelong developmental disorder that is characterized by difficulties in social interaction and communication, as well as an assortment of distinctive and repetitive behaviours (Block, Block, & Halliday, 2006; Earl et al., 2017). Some individuals with ASD display fixations upon routines and sameness, while others may fixate on certain colors or numbers, or show extreme interest in certain topics (Bryson & Smith, 1998). ASDs are not always the most exact diagnoses, as each person will display a variety of differentiating characteristics that fall somewhere along a spectrum (Block et al., 2006), hence the spectrum disorder label (Newschaffer, Falb, & Gurney, 2005). Some of the more common behaviours associated with ASD include unusual preoccupations, compulsions, rituals, complex mannerisms, and idiosyncratic language (Ruzzano, Borsboom, & Geurts, 2014). Specifically, individuals with an ASD may avoid eye contact, lack ability to maintain normal conversations, have unusual behaviours such as rocking, hand tapping, and echoing other peoples words, as well as difficulty reading social cues and facial expressions (Wozniak, Leezenbaum, Northrup, West & Iverson, 2017). Additionally, individuals with ASD may show heightened sensory symptoms with increased sensitivity to sound, temperature, touch, and/or sight (Wozniak et al., 2017). According to recent studies in the United States 16.8 out of 1,000 children aged eight years old have been diagnosed with ASD (Baio et al., 2018). Prior to 1985, the prevalence was estimated to be around 4-5 per 10,000 individuals (Bryson & Smith, 1998), which indicates a 3000% increase in diagnosis in the last three decades. The exact cause of ASDs are yet to be discovered, although it is now known that multiple genes are involved, and that environmental factors such as parental age and teratogenic environmental exposure (e.g., lead, methyl mercury, EFFECTS OF GENDER AND ASD DIAGNOSIS IN INTERVIEWS 2 thalidomide, etc) can influence the expression of traits as the individual develops (Landrigran, 2010; Myers & Johnson, 2007). It has also been shown that relatives of those with ASD (specifically siblings) are more susceptible to also having an ASD (Yuen et al., 2015) and that several disorders (e.g., Fragile X syndrome, Phenylketonuria, Fetal alcohol syndrome, etc) have been linked to ASD (Myers & Johnson, 2007). Although it has been invalidated many times since it first emerged, the theory that vaccines cause ASD is still common (Baker, 2008; Dub, Vivion, & MacDonald, 2015). While the prevalence of ASD diagnoses are increasing rapidly (Matson & Kozlowski, 2011), there is some controversy about whether the increase in diagnoses is due to a true increase in prevalence or merely a reflection of changes in the diagnostic criteria for autism (Barbaresi, Colligan, Weaver & Katusic, 2009; Bryson & Smith, 1998). In recent years there have been changes to the diagnostic criteria of autism; instead of separate diagnoses with subtle differences between them, a larger diagnosis (Autism Spectrum Disorder) is used (Gulati et al., 2019). According to the Diagnostic and Statistical Manual of Mental Disorders (DSM-5; American Psychiatric Association [APA], 2013), in order for an individual to be diagnosed with an ASD, they need to display a lack of abilities across regarding social interaction and communication, restrictive repetitive behaviour, and impairment of daily functional activity, and will be diagnosed upon the severity of their symptoms (APA, 2013). When these changes in diagnostic criteria have been compared, it has been found that specificity (correctly identify those without the disorder) is high but sensitivity (correctly identify those with the disorder) is low, as one study showed that 22% of individuals diagnosed with either Aspergers or autistic disorder (previous diagnoses) did not meet new criteria required for ASD diagnosis (Murphy et al., 2018). These inconsistencies are alarming as even though these individuals have ASD traits, they are of insufficient severity to obtain an official diagnosis and therefore cannot receive the same assistance as diagnosed ASD individuals. Murphy et al.s 2018 study found no evidence to EFFECTS OF GENDER AND ASD DIAGNOSIS IN INTERVIEWS 3 suggest that any particular demographic (i.e., age, race, gender) is more likely to be affected by the change in diagnostic criteria than any other. There is no known genetic test that can be performed to diagnose an individual with ASD, and physicians instead rely on tests and observational techniques to determine a diagnosis (Filipek et al., 1999). When ASD symptoms first become apparent, they tend to appear in one of two ways; as early onset (symptoms appearing before 12 months and becoming more noticeable with time) or as regressive, where the child develops normally until around 24 months when ASD traits begin appearing (Ozonoff, Heung, Byrd, Hansen & Hertz-Picciotto, 2008). Even though symptoms of ASD can appear before age three (Filipek et al., 1999), the variability of ASD means that there is no exact age where a child receives a diagnosis as the childs environment plays a large role in which traits are noticeably expressed (Wozniak et al., 2017). Variability of trait expression is not the only reason why a diagnosis may be delayed however, with some others including: parental concerns about stereotypical labels being applied to the child or the possibility of a diagnosis causing family distress, as well as some parents hoping the ASD symptoms in the child will disappear in time (Filipek et al., 1999). Parents are the largest influencer of when a child receives a diagnosis, and factors such as socioeconomic status, symptom severity, and parental concern about symptoms in particular all influence how early a child receives a diagnosis (Daniels & Mandell, 2014). It is far more beneficial for the individual to receive an early diagnosis, however, as support can be provided for the child and their family as well as allowing the early implementation of education and treatment (Filipek et al., 1999). The benefits of early intervention of ASD were shown in a 2010 study by Itzchak and Zachor, specifically in regard to adaptive skills and cognitive gains. It was found that cognitive gains were increased not only by early intervention, but also by the level of maternal education, EFFECTS OF GENDER AND ASD DIAGNOSIS IN INTERVIEWS 4 and that other factors, such as ASD severity and language ability influenced the success of early intervention in children (Itzchak & Zachor, 2010). Gender differences in ASD Interestingly, ASD is diagnosed much more often in males than females, at an estimated 3:1 ratio (Loomes, Hull, & Mandy, 2017). The controversy of whether it is ASD occurrence or diagnostic rates that are increasing can be extended to ASD in males and females specifically does it actually occur more often in males or is it merely diagnosed more often? If it was actually the case that ASD occurs more often in males, a potential explanation could be that there are many male dominated genetic disorders caused by anomalies on the X chromosome (X-linked), of which ASD could be connected (Myers & Johnson, 2007; Rutter, 2005). As ASD also occurs in females, this is clearly not the only causal factor, but it could be a contributing one. One theory that has been used to explain the vast behavioural differences associated with ASD is Baron-Cohen, Knickmeyer & Belmontes (2005) extreme male brain (EMB) theory. This suggests that male brains are more specialized for systemizing while females are more specialized for empathizing. For instance, males are usually better at tests relating to mental rotation (Shepard & Metzler, 1971), spatial navigation (Kimura, 1999), and targeting (Watson & Kimura, 1991), while females tend to be better at emotional recognition (McClure, 2000), social sensitivity (Baron-Cohen, ORiordan, Stone, Jones & Plaisted, 1999), and verbal fluency (Hyde & Linn, 1988). The EMB theory then goes to state that the behavioural traits of ASD are caused by individuals possessing an extreme male brain pattern decreased empathizing and increased systemizing regardless of their gender (Baron-Cohen et al., 2005). Unfortunately, early ASD diagnosis is often difficult for females as there are concerns that the current diagnostic process is biased towards males (Kirkovski et al., 2013). The bias is caused predominantly by the vast majority of research and subsequent literature about ASD EFFECTS OF GENDER AND ASD DIAGNOSIS IN INTERVIEWS 5 involving its presentation in males (Kirkovski, Enticott, & Fitzgerald, 2013). This leads to females requiring more severe ASD symptoms to get the same recognition as their male counterparts, which leads to diagnoses occurring later in life (Bargiela, Steward, & Mandy, 2016). Additionally, ASD traits tend to present differently between males and females (Mandy et al., 2018). One possible explanation for this is the female compensation hypothesis, which asserts that females learn to adapt to social situations better than males. This means that the distinctive behavioural traits associated with ASD are not noticed as often as they are more biased towards male ASD traits (Mandy, Pellicano, Pourcain, Skuse & Heron, 2018). Similar to how females adapt to social situations, social camouflaging is a technique used by many individuals with ASD and has been documented in a variety of studies and involves the disguising of distinctive ASD behaviours (Dean, Harwood, & Kasari, 2017; Lai et al., 2017; Ratto et al., 2017). This camouflaging involves imitating social interactions and behaviours and learning to adapt to social situations that would come intuitively to non-ASD individuals (Lai et al., 2017). Additionally, as social camouflaging can be seen in individuals with ASD of both genders, rather than specifically females, it is best conceptualized as a coping mechanism that females utilize more rather than a specific attribute of one gender (Lai et al., 2017) Discrimination against individuals with ASD The attempt at social camouflaging is not always fully effective, however, as many children with ASD experience victimization and segregation from their peers (Little, 2002). One study found that children with ASD were four times more likely to experience bullying (Little, 2002). As children with ASD have elevated difficulties with normal peer interaction and social customs, they struggle to create and sustain friendships with their peers. This ultimately increases the likelihood of being subject to victimization, or of victimizing other children EFFECTS OF GENDER AND ASD DIAGNOSIS IN INTERVIEWS 6 (Cappadocia, Weiss, & Pepler, 2011; Sterzing, Shattuck, Narendorf, Wagner & Cooper, 2012). Removing children with ASD from the standard classroom and putting them in special education classes could reduce the risk of victimization (Zablotsky, Bradshaw, Anderson & Law, 2014). However, this could also hinder the children from developing vital social interaction skills that will be needed later in life (Picci & Scherf, 2015). A lack of skill in social interaction can have a much larger impact upon adult interactions than childhood ones. Rather than causing ostracization or bullying, this deficit could influence a person's chances of keeping a job, or being hired in the first place, as it has been demonstrated throughout multiple studies that first impressions in job interviews are vital (e.g., Barrick, Shaffer & DeGrassi, 2009; Doughtery, Turban, & Callender, 1994; Weiss, & Feldman, 2006). The same social camouflaging techniques displayed in children with ASD can be seen in adults trying to cope with workplace expectations in an attempt to avoid discrimination (Hull et al., 2017). Unfortunately, the extended use of social camouflaging techniques can have a negative impact on an individuals mental health by increasing the likelihood of depression and decreasing feelings of acceptance (Cage, Di Monaco, & Newell, 2018). It has been found that the highest number of discrimination charges are filed by young ASD individuals, which is unlike other disability groups from whom charges are usually filed by older individuals (Van Wieren, Reid & McMahon, 2008). This is potentially because they lack experience functioning in the workplace, and as such would benefit from being exposed to a work environment prior to being expected to function in one (Van Wieren, Reid, & McMahon, 2008). Transition programs that focus on preparing high school students with ASD for employment have been shown to have enormous success in helping integrate youth with ASD into mainstream employment. Wehman et al. (2014) showed 87.5% of a treatment group for ASD individuals just having finished high school obtained employment, compared to 6.25% EFFECTS OF GENDER AND ASD DIAGNOSIS IN INTERVIEWS 7 employment for the control group. Transition programs are not the only way to help individuals with ASD gain and maintain employment. Studies have shown that factors such as assistive technology, multidisciplinary collaboration, supported employment, autism awareness training, and environmental modifications and accommodations were all effective in increasing the likelihood of successful employment of individuals with ASD (Chen, Leader, Sung & Leahy, 2015; Hedley et al., 2017; Mller, Schuler, Burton & Yates, 2003). Even though individuals with ASD face difficulties in the workforce, females with ASD may struggle more than males. When studying the experiences and needs of adults with ASD in the workforce, it was found that 60% of women with ASD opposed to 40% of men with ASD, stated that social interaction was one of the top three difficulties in the workforce, and that 86% of unemployed women with ASD wished they were employed part-time, compared to 51% of men with ASD (Baldwin & Costley, 2016). Likewise, even though women with ASD tend to have better communication skills than men with ASD (i.e., maintaining eye contact, using appropriate body language and conversational skills), they still identify communication, social interaction, and stress as the biggest challenges they face at work (Hayward, McVilly, & Stokes, 2018). Specifically, a lack in communication skills such as reading between the lines in conversation, analyzing verbal information, and misunderstanding instructions were common (Hayward, McVilly, & Stokes, 2018; Mller et al., 2003). In particular, navigating through hiring processes pose many difficulties for all individuals with ASD (Mai, 2018). The theory of social distance (Akerlof, 1997) asserts that employers often ignore the difference between social decisions and conventional economic decision-making in favor of hiring applicants who follow similar social codes, even though those codes might be unrelated to work performance (Bursell, 2007). One study has used this theory to explain why Swedish employees might favor applicants with Swedish sounding names over EFFECTS OF GENDER AND ASD DIAGNOSIS IN INTERVIEWS 8 foreign sounding ones, even though the qualifications were identical (Bursell, 2007). The Swedish employers were obviously familiar with the Swedish culture, its values and behaviours, and so when making a hiring decision, they favored the applicants that they were more familiar with, could understand more, and share more experiences with. The same idea can be extended to why individuals with ASD might face discrimination in the workplace as employers prefer applicants with the same social codes as themselves, which individuals with ASD lack. First impressions are important tools in human interaction, and once made, they are difficult to change completely (Bhanji & Beer, 2013). These judgements are often made based on very little information (Ambady & Rosenthal, 1992), and behaviours that could be explained by one thing (e.g., an autism diagnosis) are often mistaken as another (e.g., rude behaviour) because of the lack of information (Sasson & Morrison, 2019). In an effort to better understand and decrease hiring discrimination, some studies have explored improving the interview skills of individuals with ASD and have shown positive results (Smith et al., 2014). Aspects of these studies included disclosure of diagnosis, specific training and instruction on how to present themselves, and running through simulations of possible questions and interview scenarios to better prepare responses (Smith et al., 2014; Whetzel, 2013). Many individuals with ASD are hesitant as to whether to disclose their ASD diagnosis to possible employers out of fear they may face discrimination because of it (Mai, 2018); in fact, for some individuals with ASD, disclosing their diagnosis was perceived to be as complex a decision as coming out is for LGBT (Lesbian Gay Bisexual Transsexual) people (Davidson & Henderson, 2010). Due to these fears, it was found that if an individual could conceal their disorder without negative repercussions, many would choose to do so rather than risk the possible consequences of disclosing (Davidson & Henderson, 2010). Nevertheless, a study of individuals with multiple sclerosis showed that even though the disclosure of their diagnosis was seen as very high risk, it actually increased their chancing of remaining employed after three years (Kirk-Brown, Van Dijik, Simmons, Bourne & EFFECTS OF GENDER AND ASD DIAGNOSIS IN INTERVIEWS 9 Cooper, 2014).. This was possibly because they were able to receive the support they needed from their employees and work environment, and so were helped rather than hindered by their disclosure (Kirk-Brown et al., 2014). Additionally, a recent study has shown that first impressions of individuals with ASD can be markedly improved if the observer is made aware of a diagnosis (Sasson & Morrison, 2019). As such, the current study aims to extend upon these findings in regard to specific hiring situations as well as investigate if gender is a factor. Focusing on hiring situations is especially important because it is an area where social skills tend to be heavily relied upon, and also an area where individuals with ASD struggle to fit societal standards. As such, any further information on how individuals with ASD can be assisted in hiring situations is both helpful and necessary. The underpinning theory of the current study is Akerlofs (1997) theory of social distance. As mentioned previously, Akerlof theorizes that for many potential employers, social decisions will take precedence over economic decisions when considering applicants that employers will choose the applicant who has similar social codes to themselves without factoring in work performance or experience (Akerlof, 1997; Bursell, 2007). This theory is important because it provides a possible explanation as to why individuals with ASD may be discriminated against in hiring situations. As individuals with ASD do not display normal social behaviours, the interviewer may prefer another applicant who displays similar social codes. If the ASD diagnosis is disclosed; however, then it would provide an explanation for the social differences and possibly combat the instinctual distance being created. While the Americans with Disabilities Act (ADA) ensures that an individual will never be forced to disclose a diagnosis until special recommendations are required (EEOC, 2002), some people might prefer to disclose prior to being legally required to do so, especially if they think accommodations could help them in the interview itself. Another theory was important to the development of the current study, as it facilitated the predicted results. This theory is the shifting standards model by Biernat & EFFECTS OF GENDER AND ASD DIAGNOSIS IN INTERVIEWS 10 Fuegen (2001), which states that when people are judged in stereotypical manners, they are judged using within-category reference points. This means that when an individual is doing something stereotypically they should be good at, they will be judged more strictly than someone else who is not stereotypically as good. The purpose of this study was to determine whether people view individuals with Autism Spectrum Disorder (ASD) more negatively if the ASD diagnosis has not been disclosed, and whether gender plays a significant role in these perceptions. This is important because there are countless situations in life in which explanations for another persons actions are not given and as such people must make first impression judgements on the limited information available. However, when additional information is presented, initial judgements can be adjusted and allowances are made if it is believed there is adequate justification for it. In this study, participants were asked to read through interview notes and assess the interviewee, then ultimately report whether the interviewee should be hired. There were four versions of the interview notes and all were identical except for whether the interviewee was male or female, and whether they had stated a diagnosis of ASD. Additionally, it has been established women face more discrimination than men in the workforce, both with and without an ASD diagnosis (Abrams, 1989; Bobbit-Zeher, 2011), and this study built upon that knowledge to determine whether the declaration of a diagnosis improved the way the ASD individuals were viewed. Therefore, this study had three primary hypotheses. Firstly, that gender and ASD diagnosis would interact to form a significant effect for the factors analysed. Secondly, that people who read the same resume and job interview notes will view women with ASD more negatively than men with ASD. As it has been established, women struggle against discrimination in the workplace more directly than men (Bobbit-Zeher, 2011), and so it was expected that women with ASD were viewed more negatively than men with ASD. The third EFFECTS OF GENDER AND ASD DIAGNOSIS IN INTERVIEWS 11 hypothesis focused more on the overall influence of the ASD diagnosis rather than the influence of gender, and was that people would make allowances for individuals with ASD and view them more positively than those without. Methods Participants Participants were recruited using Amazons Mechanical Turk (MTurk), which is an online service that crowdsources to provide participants for research studies, particularly in the social sciences (Amazon Mechanical Turk, Inc, 2018). It was decided to use MTurk as it was a better alternative to using a convenience sample of purely college students that are unlikely to have experience with hiring individuals. Using a service such as MTurk that accesses participants from a wide range of sources and ages would help create more generalizable results. There were no exclusion criteria regarding the participants, each of participants received monetary compensation ($0.50) for their participation, and the study itself was conducted using Qualtrics. A minimum sample size of 128 (to reach a power of 0.8, an effect size of 0.25, and an alpha level of .05) was determined using the G*Power calculator (Faul et al., 2009; see Appendix A). There were a total of 556 responses, however 440 were removed for various reasons: including, failing manipulation checks, failing all the attention checks, and if the time taken to complete the survey was two standard deviations above the mean (M=629 seconds, SD=442 seconds). This left a total of 116 participants in the final data. Participants ranged in age from 2273 years (M=36.32; SD=10.94) and there were 40 female participants and 76 male, of which the majority (63.8%) identified as white. Design and Procedure EFFECTS OF GENDER AND ASD DIAGNOSIS IN INTERVIEWS 12 The study was conducted online using MTurk and Qualtrics, so there was no direct contact between the participants and the researchers. Participants were presented with an informed consent document that briefed them about what the study would entail. Participants were told that they would be presented with a resume (Appendix B) and job interview notes (Appendix C), and then would be asked to answer questions regarding the personality and capability of the interviewee to assess their competence for the job. After they consented, they continued to the next screen where they filled out basic demographic information including age, gender, and race, and then continued to the next screen where they read the resume and interview notes. Qualtrics was set up to randomly assign participants after they entered their gender; this helped separate male/female/other participants evenly between each condition. Participants were instructed to read the subsequent information carefully as there would be questions about the content afterwards. The current study had two factors (gender and ASD disclosure) with two levels each. Thus, there were 4 total conditions, and each were identical other than two of the job interview notes were for a male (John Smith) and two were for a female (Jane Smith), and one of each gender included a sentence that stated the interviewee had been diagnosed with ASD (see Appendices B & C). Participants were randomly assigned to one of these four possible groups. The job interview notes were created using Block et al. (2006) and Baron-Cohens (2001) studies as a reference when deciding which ASD traits to include. A written transcript of the job interview was chosen rather than a video so that all four conditions could be exactly the same (excluding gender and ASD diagnosis) and so that possible confounds (i.e., race, clothing, individual preference of presentation, voice/tone, etc) could be avoided. The ASD traits had to be obvious enough that they could be picked up on by the reader, but subtle enough so that the participant could not very easily identify the individual as having ASD. It was decided that the job interview notes would include some of the more well-known ASD characteristics, (i.e., literal EFFECTS OF GENDER AND ASD DIAGNOSIS IN INTERVIEWS 13 answers, not making eye contact, stimming, and lack of ability making small talk). These are all common symptoms of ASD (Block et al., 2006), and are not so unusual that they cannot be otherwise explained. After reading the resume and interview notes, participants answered 40 survey questions scored with several versions of Likert-type scales (See Appendices D & E). After answering these questions, the participant moved on to a new page where they answered 3 open-ended questions about the job interview notes that served as attention checks and 1 open-ended question that served as a manipulation check (see Appendix F). They were unable to return to the notes while answering these questions. In the final section, the participant answered whether they believed the person had a developmental or social disorder of any type, and if so, which on? As the conditions where the interviewee disclosed their diagnosis included the fact that ASD was a social developmental disorder, this question also served as a manipulation check. Participants then moved onto the next page where they were asked whether they had ever received an ASD diagnosis, or known anyone else that had received one. Participants were instructed to provide as little or as much information as they were comfortable and this question served to gather more information about the participants exposure to ASD behaviours prior to taking the study. Participants then continued to a page thanking them for their participation. There was no debriefing of the true purpose of the study, as there was no risk of harm to the participant and no deceit in the introduction to the study. The study took an average of 10.48 minutes to complete with a standard deviation of 7.37 minutes. Measures Two pre-existing scales were used to create the survey questions used in this study; a subscale from Baron-Cohens (2001) Autism-Spectrum Quotient (AQ) which measures social EFFECTS OF GENDER AND ASD DIAGNOSIS IN INTERVIEWS 14 skills, and scales from Cunningham & Macans 2007 study which measures job capability. These scales include a hiring recommendation question, perceived qualification, absenteeism, turnover, feminine stereotypes, a good employee measure, job fit, and a number of other job-relevant personality characteristics. Social skill. The first 10 questions were drawn from a subscale in Baron-Cohens (2001) AQ that specifically measures social skill (e.g., He/she would prefer to do things with others rather than on their own). The AQ scale has been shown to have good test-retest reliability (r=0.7, p=0.02), as well as reasonable construct validity (=.77) (Baron-Cohen, 2001). The questions were adapted so that they were phrased to assess another person, rather than the individual answering them. The 10 items included in the social skill subscale can be found in Appendix D. These questions were answered on a 4-point Likert-type scale, ranging from 1 (Definitely Agree) to 4 (Definitely Disagree) and were combined to create the measure ASD Total. Job capability. Eighteen questions were taken from Cunningham & Macans (2007) study examining perceptions of pregnant women interviewing for jobs. This study used measures to generate a comprehensive understanding of how the participants perceived the interviewees job capability to be. The subscales of the measures in this study included a hiring recommendation, perceived qualifications, absenteeism, turnover, feminine stereotypes, good employee characteristics, and other job relevant characteristics. Hiring recommendation. The hiring recommendation was answered on a five-point scale (see Appendix E), ranging from 5 (Yes, I would definitely hire this person. This person is an extremely good candidate.) to 1 (No, I would definitely not hire this person. This person is not a good candidate.). Qualification. Interviewee qualifications were measured using two items which were rated on a 5-point Likert scale ranging from 1 (Strongly Disagree) to 5 (Strongly Agree) and then EFFECTS OF GENDER AND ASD DIAGNOSIS IN INTERVIEWS 15 averaged together. These items were I would evaluate this applicants qualification for this position favorably, and I feel this candidate would be well suited for the job (=.83). Overall performance. Interviewee overall performance was a rating of how well the participant thought the interviewee did overall, and was answered on a 5-point Likert-type scale ranging from 1 (Extremely Poor) and 5 (Extremely Good). The next five sections of the survey were all preceded by the question How characteristic of the applicant do you believe each of the following to be, and used the same scale response format of a 5-point Likert-type scale, ranging from 1 (Not Characteristic) to 5 (Very Characteristic). Absenteeism. To measure absenteeism, two items (e.g., likely to miss work and likely to need time off) were asked using the 5-point scale mentioned above and the scores were averaged together (=.84). Turnover. The item used to measure turnover was likely to quit, and was rated using the scale mentioned above. Feminine stereotypes. To measure the participants perceptions of femininity, four adjectives were listed, to which the participant had to rate with the scale mentioned above and the scores were averaged together. These characteristics were: Feminine, Affectionate, Gentle, and Nurturing (=.80). Good Employee. There were seven items used to rate perceptions of a good employee that the participant rated with the scale mentioned above and the scores were averaged together. The characteristics assigned to this measure were: demonstrates good work ethic, hardworking, self-reliant, successful, self-sufficient, self-confident, and ambitious (=.84). Other characteristics. To measure perceptions of personality traits, there were eleven additional items included that the participant rated using the scale mentioned above. These were: Committed, Dependable, Competent, Mature, Healthy, Aggressive, Intelligent, Physically EFFECTS OF GENDER AND ASD DIAGNOSIS IN INTERVIEWS 16 Limited, Verbal Communication, and Leadership Ability. These personality traits were split into two categories to be analyzed: good other characteristics (i.e., committed, dependable, competent, mature, healthy, intelligent, verbal communication, and leadership ability) and bad other characteristics (i.e., aggressive and physically limited). The scores of the traits in each category were averaged together to create final ratings for good other and bad other characteristics. Job fit. The last question was a single one asking the participant to rate how well they thought the candidate fits with job, and was rated on a 5-point Likert-type scale ranging from 1 (Low Fit) to 5 (High Fit). The above items and their corresponding scales can be found in full in Appendix E. As mentioned previously, there were 3 attention checks and 2 manipulation checks included in this study (Appendix F). The 2 manipulation checks were used to ensure that the participant paid enough attention that they recognised the gender of the participant and whether there had been a diagnosis stated while they answered the questions. Preceding all other questions in this study were basic demographic questions that asked about the participants age, gender, and race. As mentioned, the ages of participants ranged from 22 to 73, with a mean of 36.3 and standard deviation of 10.9. The majority of participants identified as male (65.5%), and no participants identified as any gender other than male or female. The participants mostly identified as White (63.8%), followed by Asian (20.7%), then Black (9.5%), Other (3.4%) and American Indian or Alaska Native (2.6%). These demographic questions were chosen so that more information could be provided about the background of the participants in this study, and so that the results can be generalized. Analyses EFFECTS OF GENDER AND ASD DIAGNOSIS IN INTERVIEWS 17 A 2x2 ANOVA analysis using SPSS (Statistical Package for the Social Sciences) was applied to examine the interaction effects between gender and ASD diagnosis disclosure. Main effects were also studied for the measures ASD Total (i.e., the average of the results found using Baron-Cohens 2001 study), Hiring Recommendation, Qualification, Absenteeism, Feminine Stereotypes, Good Employee, Good Other Characteristics (i.e., the positive other characteristics used in Cunningham & Macans 2007 study: Committed, Dependable, Competent, Mature, Healthy, Intelligent, Verbal Communication, and Leadership Ability) and Bad Other Charateristics (i.e., the negative other characteristics used in Cunningham & Macans 2007 study: Aggressive and Physically Limited). The measure ASD Total was used to provide information about how the participant recognised ASD symptoms in the interviewee. The results from the measure Hiring Recommendation was the main focus of the study as it answered, ultimately, whether the participant would hire the interviewee regardless of other perceptions. The results from the other measures was used to provide specific information regarding perceptions of males and females with or without an ASD diagnosis. As the presence of a diagnosis was one of two vital aspects of this study, it was critical that participants recognised and remembered whether there was an ASD diagnosis when answering the questions. If the participants that were in the condition where the interviewee was diagnosed with ASD, and did not confirm that they saw the diagnosis, they were removed from analysis as their data did not necessarily represent responses sensitive to the interviewees ASD diagnosis. Likewise, as gender was another important factor in this study, if a participant failed to correctly identify the gender of the interviewee their data was removed from the analysis. One of the attention checks served to support the gender manipulation check, as it asked about the name of the interviewee. The name given wasnt important, however, if it obviously was not of the same gender as that given then the participant was removed from analysis (i.e., participant identifies interviewee as male but says their name is Mary). The other two attention checks were EFFECTS OF GENDER AND ASD DIAGNOSIS IN INTERVIEWS 18 used to help remove participants who may be skipping through the questions and not giving valid answers. If a participant failed to answer or gave unrelated responses to all three of the attention checks then their data was not included in the analysis. Additionally, as it was mentioned before, participants were removed from analyses if the time taken to complete the study was two standard deviations above or below the mean. Prior to calculating these cut off figures, one participant was removed as their time was an extreme outlier. Results There were 53 participants who stated they did not have an ASD diagnosis, or know anyone that did, 22 participants who stated they either had an ASD diagnosis or knew someone that did, and 41 participants who did not answer the question. This means that only 19% of participants admitted to having prior knowledge of ASD and its characteristics, which is something to keep in mind when looking at results. A series of between-subjects, 2 (ASD Diagnosis Disclosure: disclosed versus undisclosed) X 2 (Interviewee Gender: male versus female) ANOVAs were conducted to examine a variety of personality and job capability traits after reviewing notes from a job interview. ASD Total ASD Total scores were created by the participant being asked to respond to how much they agreed with a variety of questions designed to measure ASD characteristics and social skill; a full list of these questions can be found in Appendix D. There was no significant interaction between ASD diagnosis and gender regarding the variable ASD Total (F(1,112)=.638, p=.426). There was no main effect found between gender (F(1,112)=3.44, p=.066), with the male condition M=.69, SD=.23, female condition M=.77, SD=.20. There was also no main effect found in ASD diagnosis (F(1,112)=1.93, p=.167), with the non-ASD diagnosis condition M=.69, SD=.20, ASD diagnosis condition M=.75, SD=.24. All relevant means can be found in Appendix H. EFFECTS OF GENDER AND ASD DIAGNOSIS IN INTERVIEWS 19 Hiring recommendation The effects of ASD diagnosis and gender can be seen in Figure 1. There was no significant interaction between ASD diagnosis and gender regarding the variable hiring recommendation (F(1, 112)=.574, p=.450); however, there was a significant main effect observed for gender (F(1,112)=6.13, p=.015, d=0.45), male condition M=3.69, SD=1.04, female condition M=3.18, SD=1.23. There was no main effect found in ASD diagnosis (F(1,112)=.574, p=.45), non-ASD diagnosis condition M=3.59, SD=1.22, ASD diagnosis condition M=3.42, SD=1.06. As can be seen, male interviewees more often received an affirmative answer when the participant was asked would you hire this individual? All relevant means can be found in Appendix H. Hiring Recommendation Male Female 5 4.5 4 MEAN 3.5 3.88 3.69 3.18 3.55 3.18 3.18 3 2.5 2 1.5 1 0.5 0 Total Without ASD Diagnosis With ASD diagnosis Figure 1. Perception of Hiring Recommendation Qualification There was no significant interaction between ASD diagnosis and gender regarding the variable Qualification (F(1,111)=.454, p=.502). This measure included the averaged scores from the participant being asked whether they would evaluate this applicants qualification for this position favorably and whether they feel this candidate would be well suited for the job. EFFECTS OF GENDER AND ASD DIAGNOSIS IN INTERVIEWS 20 There was no main effect found between gender (F(1,111)=.594, p=.443), with the male condition M=2.48, SD=1.06, female condition M=2.65, SD=1.07. There was also no main effect found in ASD diagnosis (F(1,111)=.117, p=.733), with the non-ASD diagnosis condition M=2.60, SD=1.13, ASD diagnosis condition M=2.49, SD=1.01. All relevant means can be found in Appendix H. Overall Performance The effects of ASD diagnosis and gender can be seen in Figure 2. There was no significant interaction between ASD diagnosis and gender regarding the variable Overall Performance (F(1,112)=.639, p=.426). There was a main effect found for gender (F(1,112)=7.50, p=.007, d=0.48), male condition M=3.44, SD=.933, female condition M=3.00, SD=.915. There was also a main effect found for ASD diagnosis (F(1,112),=5.63, p=.019, d=0.45), non-ASD diagnosis condition M=3.50, SD=.927, ASD diagnosis condition M=3.08, SD=.929. As shown, male interviewees were scored higher than females when asked how would you rate their overall performance?, and individuals without ASD diagnosis were scored higher than those with ASD diagnosis. All relevant means can be found in Appendix H. Overall Performance Male Female 5 4.5 3.75 4 MEAN 3.5 3.14 3 3.14 3 3.2 2.86 2.5 2 1.5 1 0.5 0 Total Without ASD Diagnosis Figure 2. Perception of Overall Performance With ASD diagnosis EFFECTS OF GENDER AND ASD DIAGNOSIS IN INTERVIEWS 21 Absenteeism The effects of ASD diagnosis and gender can be seen in Figure 3. There was no significant interaction between ASD diagnosis and gender regarding the variable Absenteeism (F(1,112)=.820, p=.367). There was a main effect found for gender (F(1,112)=5.39, p=.022, d=0.40), male condition M=2.14, SD=1.30, female condition M=1.66, SD=1.07. There was also a main effect found for ASD diagnosis (F(1,112),=7.37, p=.008, d=0.47), non-ASD diagnosis condition M=2.23, SD=1.34, ASD diagnosis condition M=1.66, SD=1.07. This measure included the averaged scores from the participant being asked how likely the interviewee was to miss work and need time off. As seen, male interviewees were perceived to be less characteristic to be absent, as were those interviewees who did not disclose their diagnosis. All relevant means can be found in Appendix H. Absenteeism Male Female 5 4.5 4 MEAN 3.5 3 2.5 2 2.59 2.14 1.66 1.86 1.5 1.78 1.46 1 0.5 0 Total Without ASD Diagnosis With ASD diagnosis Figure 3. Perception of Absenteeism Feminine Characteristics There was no significant interaction between ASD diagnosis and gender regarding the variable Feminine Characteristics (F(1,112)=1.08, p=.300). This measure was defined by the averaged scores from participant being asked how characteristic it was of the interviewee to be EFFECTS OF GENDER AND ASD DIAGNOSIS IN INTERVIEWS 22 feminine, affectionate, gentle, and nurturing. There was no main effect found between gender (F(1,112)=.029, p=.864), with the male condition M=2.48, SD=1.02, female condition M=2.48, SD=1.00. There was also no main effect found in ASD diagnosis (F(1,112)=3.50, p=.0.64), with the non-ASD diagnosis condition M=2.69, SD=1.00, ASD diagnosis condition M=2.29, SD=.990. All relevant means can be found in Appendix H. Good Employee There was no significant interaction between ASD diagnosis and gender regarding the variable Good Employee (F(1,112)=.056, p=.813). This measure was the result of the averaged scores from when the participants were asked how characteristic it was of the interviewee to be hardworking, self-reliant, successful, self-sufficient, self-confident, ambitious, and demonstrates good work ethic. There was no main effect found between gender (F(1,112)=.002, p=.964), with the male condition M=3.15, SD=.837, female condition M=3.51, SD=.945. There was also no main effect found in ASD diagnosis (F(1,112)=.081, p=.776), with the non-ASD diagnosis condition M=3.53, SD=.809, ASD diagnosis condition M=3.49, SD=.936. All relevant means can be found in Appendix H. Good Other Characteristics There was no significant interaction between ASD diagnosis and gender regarding the variable Good Other Characteristics (F(1,112)=0.69, p=.793). This measure was the result from the averaged scores when the participant was asked how characteristic it was of the interviewee to be committed, dependable, competent, healthy, intelligent, have leadership ability, verbal communication, and mature. There was no main effect found between gender (F(1,112)=.041, p=.839), with the male condition M=3.43, SD=.770, female condition M=3.41, SD=.729. There was also no main effect found in ASD diagnosis (F(1,112)=1.50, p=.223), with the non-ASD diagnosis condition M=3.51, SD=.705, ASD diagnosis condition M=3.34, SD=.787. All relevant means can be found in Appendix H. EFFECTS OF GENDER AND ASD DIAGNOSIS IN INTERVIEWS 23 Bad Other Characteristics The effects of ASD diagnosis and gender can be seen in Figure 4. There was no significant interaction between ASD diagnosis and gender regarding the variable Bad Other Characteristics (F(1,112)=1.05, p=.308). This measure was the result of the averaged scores when the participant was asked how characteristic it was of the interviewee to be aggressive, and physically limited. There was a main effect for gender (F(1,112)=5.27, p=.025, d=0.42), male condition M=2.35, SD=1.19, female condition M=1.93, SD=.767. There was no main effect found between ASD diagnosis (F(1,112)=2.92, p=0.09), non-ASD diagnosis condition M=2.39, SD=1.13, ASD diagnosis condition M=2.02, SD=.989. As shown, male interviewees were perceived to be more characteristic of displaying bad other characteristics than females. All relevant means can be found in Appendix H. 'Bad' Other Characteristics Male Female 5 4.5 4 MEAN 3.5 3 2.5 2 2.66 2.35 1.93 2 2.11 1.86 1.5 1 0.5 0 Total Without ASD Diagnosis With ASD diagnosis Figure 4. Perception of Bad Other Characteristics Job Fit The effects of ASD diagnosis and gender can be seen in Figure 5. A 2x2 ANOVA showed no significant interaction effect for ASD diagnosis and gender regarding the variable Job Fit (F(1,112)=3.77, p=.541). There was a main effect found for gender (F(1,112)=7.82, EFFECTS OF GENDER AND ASD DIAGNOSIS IN INTERVIEWS 24 p=.006, d=0.51), male condition M=3.47, SD=1.16, female condition M=2.82, SD=1.40. There was no main effect found for ASD diagnosis (F(1,112)=.980, p=.324), non-ASD diagnosis condition M=3.35, SD=1.28, ASD diagnosis condition M=3.11, SD=1.31. As can be seen, participants were more likely to provide a positive answer for male interviewees rather than female when asked how well they fit with the job. All relevant means can be found in Appendix H. Job Fit Male Female 5 4.5 4 MEAN 3.5 3 3.69 3.47 3.3 2.82 2.86 2.77 2.5 2 1.5 1 0.5 0 Total Without ASD Diagnosis With ASD diagnosis Figure 5. Perception of Job Fit Discussion There were two hypotheses being tested in the current study: first, that people who read the same resume and job interview notes would perceive men with ASD more positively than women with ASD, and second, that individuals with an ASD diagnosis of either gender were viewed more positively than those without a diagnosis. These hypotheses were not supported when looking at the interaction effect between gender and ASD diagnosis, however, multiple main effects showed significant results. EFFECTS OF GENDER AND ASD DIAGNOSIS IN INTERVIEWS 25 The primary goal of the current study was to better understand how a job candidates gender and the decision on whether to disclose having an ASD diagnosis may impact people interviewing for jobs. While no interaction effects were observed between gender and ASD diagnosis, some significant main effects were found. In line with previous literature, men were viewed more positively in the factors of Hiring Recommendation, Overall Performance, and Job Fit. Interestingly, even though males were seen to be rated higher in most of the positive categories, they were also rated higher in the negative factors of Absenteeism and Bad Other Characteristics (i.e. undesirable general characteristics; aggressive and physically limited). In summary, participants saw the male interviewees as both more likely to make good employees (they were viewed more positively across the board) and more likely to possess negative traits. The results from the Feminine Characteristics factor were of particular interest if the diagnostic bias of ASD in Kirkovski et al.s (2013) literature review is considered. It is a natural assumption that females would receive higher ratings by participants in the feminine characteristics factor, and yet even the total ratings of males and females (when non-diagnosis and diagnosis conditions were combined) did not see a difference in rating. A possible explanation for this finding could be that as the job interview notes in this study was designed to reflect the most common of ASD qualities, and these ASD qualities are possibly biased to portray male characteristics, it is feasible that the women who displayed such male characteristics were perceived more negatively. It was expected that women be more feminine than was portrayed, and as such scored lower on questions measuring their feminine characteristics. This is a prime example of the shifting standards model (Biernat & Fuegen, 2001), which was an underpinning theory for the predicted results of this study. This shifting standards model asserts that when a person is judging another in a stereotype-relevant manner, they will use within-category reference points (Biernat & Fuegen, EFFECTS OF GENDER AND ASD DIAGNOSIS IN INTERVIEWS 26 2001). For example, assuming the stereotype that men are better at fixing things than women, if a person was asked how well a women fixed a shelf, they may rate her as very good, but if a man fixed the shelf to the same level, then he may be rated as okay. This is because men are held to a higher standard than women due to a pre-existing stereotype. Therefore, it can be seen that in subjective rating scales, stereotypes can alter perceptions of abilities dependent on who is perceived to be greater within a held stereotype (Biernat & Fuegen, 2001). This theory is important for the current study as it provides a possible explanation as to why women might be rated more negatively than men in an interview like the one in this study (i.e., because they are stereotypically held to a higher level of perceived warmth and approachability). The job interview notes in this study were designed to reflect common ASD traits, and as awkwardness and distance in social situations is common to individuals with ASD, that was reflected in the job interview notes. As women are expected to be more feminine, affectionate, nurturing and gentle (the four characteristics that made up the Feminine Characteristics factor), the absence of such traits in the job interview would have been judged harsher by the participant when the interviewee was female rather than male. The second goal of the current study was to determine whether participants would make allowances for individuals with ASD, specifically whether they would view the interviewee more positively if they self-disclosed their ASD diagnosis. Those with an ASD diagnosis were noticeably viewed more negatively in the factor Overall Performance, although, those with an ASD diagnosis were viewed more positively (had lower ratings) in the factor Absenteeism. These findings suggest that participants viewed interviewees who self-disclosed their diagnosis less positively in their overall performance; however, they were seen more positively when considering possible absenteeism. A study by Sasson & Morrison (2019) showed that first impressions of individuals with a label of ASD were seen more positively than individuals with ASD who were not labelled. While this was consistent with the hypotheses of the current study, EFFECTS OF GENDER AND ASD DIAGNOSIS IN INTERVIEWS 27 it was not consistent with the findings. The only factor that the presence of a diagnosis was found to have a significant positive influence was Absenteeism. Additionally, Sasson & Morrison (2019) found that if the rater had previous knowledge of ASD, then the individual was seen even more positively. The final question of the current study was whether the participant themselves had been diagnosed with ASD, or knew anyone that was. The purpose of this question was to test the participants prior knowledge of ASD in a similar regard to Sasson & Morrisons (2019) study. In the current study, only 19.0% of participants disclosed that they either knew someone with ASD or had been diagnosed themselves; however, 35.3% of participants declined to answer the question so the true statistic could be higher. As Sasson & Morrisons (2019) findings found that more knowledge about ASD can be correlated with more positive views of individuals with ASD, it is possible that the participants apparent lack of knowledge in the current study could have contributed to the results. Limitations There are several limitations to this study which could have influenced the results. The first of these limitations was the data collection method. Out of 556 total responses, only 116 were useable data. Many responses had to be dropped due to incompletion or null answers, which made gathering the data difficult. There are concerns that members of MTurk are less attentive to instructions than when other methods of gathering participants are used, however, some studies refute these concerns (Berinsky, Huber, & Lenz, 2012; Hauser & Schwarz, 2016). As such a large number of participants were removed from this study due to incomplete answers, it could be considered that this be due to overly restrictive attention checks. For a participant to fail the attention checks in this study, they had to not answer at all, or give an answer completely different to the correct response. There were several I dont remember and not sure responses EFFECTS OF GENDER AND ASD DIAGNOSIS IN INTERVIEWS 28 that were accepted, as it indicated the participant had thought about it and taken the time to respond. Likewise, when asked what the interviewees name was, if the participant gave the interviewers name instead their responses was accepted. With the above information on how an attention check was considered a fail, and as a participant had to fail all three attention checks to be removed from the study, it is unlikely that the attention checks were too restrictive. After the first batch of responses (and high percentage of incompletes), it was thought that the amount of monetary compensation for participation was possibly too low to incentivize completion and participation of the study. The participants were paid $0.50 for their participation in this study, and as it took an average of 10.5 minutes to complete the study, the participants were effectively working for $3.00 p/hour not even half of minimum wage. Additionally, as there was such a large number of participants who were dropped from the study, it was difficult to keep the numbers in each condition relatively even; with 32 participants in condition 1 (Male, no ASD diagnosis), 40 participants in condition 2 (Male, ASD diagnosis), and 22 participants each in conditions 3 (Female, no ASD diagnosis) and 4 (Female, ASD diagnosis). Another limitation to the study could have been the method of displaying ASD characteristics. Using a written transcript instead of other visual methods (e.g., video) could have contributed to the participant missing some of the ASD characteristics that were trying to be conveyed. Additionally, a written text is far less personable than a video and as such may reduce the full impact that a video could make. However, while the effect of the initial interview could have been stronger in video form, it is less likely that all of the conditions would have been identical except for the variables that were being manipulated and so could have been influenced by unknown confounds. This is less likely as the budget for this study was limited and so it is unlikely that a video could have been made and replicated well enough between conditions to be identical. EFFECTS OF GENDER AND ASD DIAGNOSIS IN INTERVIEWS 29 A third limitation could be the choice of job that the interviewee was applying for. Junior IT administrator was chosen because it was believed that current societal perceptions see it as a field with low levels of gender bias. Upon further research, however, it can be seen that women earn computer science degrees at a far lesser rate than other STEM fields (Cheryan, Ziegler & Montoya, 2017). While it is unlikely to find a completely gender-neutral profession, more research could have been done prior to the creation of this study to find one that was more balanced. The reason why IT administrator was chosen was because, when compared to some other professions, it isnt based heavily on customer service and so it would be less likely that participants would take issue with the social skill deficits depicted in the interview notes. The idea was that if the participants properly considered the job, the low social skill requirement of the profession would counterbalance any possible gender discrimination that were also included. Another possible limitation of the current study could be that the study population was made up on individuals who were most likely unused to making hiring decisions. Studies have shown that MTurk participants can be generalized to the US population with some accuracy (Berinsky, Huber & Lenz, 2012), and as the vast majority of US citizens havent been trained to make hiring decisions, it is unlikely that the MTurk participants have been either. However, it is beneficial to the study that MTurk participants have been shown to be more representative of the general population than most convenience samples (Berinsky, Huber & Lenz, 2010). For this study to get most accurate results, a population sample of employers and individuals trained in making hiring decisions would be ideal. As this study was completed with limited funds and resources, the convenience sample would be college students and so the use of MTurk enabled there to be a higher likelihood of gathering older participants who did have experience with hiring decisions, rather than purely college students. EFFECTS OF GENDER AND ASD DIAGNOSIS IN INTERVIEWS 30 Additionally, there were no measures taken in the study that made sure the participant paid careful attention to the resume and job interview notes. An example of these measures could be a timing mechanism on the job interview notes to ensure the participants did not skip to the next page too quickly. While some participants may just wait the time out and still not pay attention, it would have been a proactive measure to try ensure that participants were carefully reading the information and not skipping through. Lastly, an additional limitation to this study is that a few analyses suggested by readers could not be completed due to situational circumstances. It was planned to run Chi-square analyses on the manipulation checks, as well as assumption testing to ensure normality. Unfortunately, due to the recent COVID-19 outbreak and the subsequent world-wide lockdowns and border closures, these analyses were not able to be completed prior to returning to home countries and everything becoming remote and online. It is acknowledged that these analyses would have been beneficial to the overall validity of the study, however, current circumstances have made it difficult to do so. Future Directions As mentioned previously, future directions for this field of study could possibly include using a video as a better method of portrayal of ASD symptoms, rather than a written transcript. Another possible future direction that this research could take would be to look more closely at the differences between males and females who have been diagnosed with ASD. Likewise, further studies could investigate whether ASD traits decreased the perceived femininity of women with ASD, impacting hirability in roles where femininity may be desired (e.g., customer service positions). While this study was looking at the interaction between gender and ASD diagnosis, future research could possibly focus on either just ASD or gender, not both. Looking at one of these factors instead of an interaction between them could possibly bring forth interesting EFFECTS OF GENDER AND ASD DIAGNOSIS IN INTERVIEWS 31 findings that were missed in this study. Additionally, as there were some concerns about whether the job type used in the study (i.e., IT industry) may have influenced the results due to gender bias, future research could do a similar study using a different profession. Likewise, future studies could also assist in managing the concerns regarding the traits and attitudes of individuals gathered by MTurk by gathering participants who are used to making hiring decisions (i.e., employers). Conclusion Overall, while the current study did not reveal any significant interaction effects between gender and ASD diagnosis in an interview situation and the hypotheses were not supported, it was still able to highlight a handful of promising future research directions through the appearance of significant main effects within the data. The limitations of this study can help future researchers explore new ways of studying how individuals with ASD, and women in particular, are disadvantaged in hiring situations and the results were able to raise questions that future researchers could consider investigating (e.g., whether ASD characteristics reduces perception of femininity in women with ASD). The results of this study also have interesting implications into how men and women are perceived in job interviews, as men were seen to be viewed more positively when considering hiring recommendations, overall performance, and job fit, whilst concurrently being seen as possessing more bad characteristics than women and more likely to be absent. This implication is interesting because it means that participants were still more likely to hire males even though they also thought they possess more negative characteristics than females. Furthermore, the results also had implications regarding the disclosure of an ASD diagnosis in interviews, as it was seen that while this may have caused the individuals to be viewed more negatively in some respects (i.e. overall performance), ultimately it did not appear to have an impact on hiring recommendation. EFFECTS OF GENDER AND ASD DIAGNOSIS IN INTERVIEWS 32 EFFECTS OF GENDER AND ASD DIAGNOSIS IN INTERVIEWS 33 References Abrams, K. (1989). 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Seeking to benefit an IT department with complex technical knowledge and excellent organisation skills. Highlights Hardware configurations Software and hardware installations Phone and online support Advanced TCP/IP knowledge LAN connectivity Applications troubleshooting Extensive windows experience Problem diagnosis Good presentation ability Excellent time manager Diligent and detail-orientation Professional Experience IT Help Desk at University of Townsville (2015-18) Utilise troubleshooting techniques in support work stations, networked printers, network hub/switches, routers, and circuits Responsible for providing desktop and network support for students and professors Provides both email, phone, and face-to-face support Keep track of inventory of equipment around campus Routinely install and configure software Remotely connect and diagnose PC/hardware problems Clerk at Townsville library (2013-16) Assisted groups and individuals in locating materials Performed routine descriptive cataloguing of new book, journal, audio, and video acquisitions on the library computer system Verified bibliographic information on order requests Maintained book stacks to perfect order Inspected returned books for damage, verified due-dates, and computed and received fines Education Bachelor of Science: Computer Science EFFECTS OF GENDER AND ASD DIAGNOSIS IN INTERVIEWS University of Townsville - Class of 2019 Graduated with a 3.8 GPA Certifications Cisco Certified Network Associate (CCNA) 45 EFFECTS OF GENDER AND ASD DIAGNOSIS IN INTERVIEWS 46 Appendix C Job Interview Notes Date: 17th December, 2018 Interviewed by: Mike Brooks Applicant name: John/Jane Smith* Position requested: Junior IT Administrator Date available: Begin in February Interview notes: Interviewer: Hello Mr/Ms.* Smith, thank you for coming in today. How are you? Applicant: Hello, I am good. Thank you. I: I trust the trip here went okay? A: Yes, it was fine. I: Alright, well I have looked over your resume, and it looks great, I just have some follow up questions for you today. I'll try to make things quick so we can get on with our days. Notes: Interviewee did not make eye contact during greeting, however, did shake Mr. Brooks' hand. Interviewee is well dressed in professional clothing, clean pressed. I: Okay so firstly, are you currently employed elsewhere? If so, why are you considering leaving? A: I mow my neighbours lawn every Sunday. I am not considering leaving that job. I: That's a pretty serious job you've got there, haha. Think you will be able to juggle both at once? A: It will be fine. N: Sits straight however taps leg repeatedly with hand and still wont make eye contact. I: This is an entry level position, but tell me more about your employment history - your resume mentions you worked in a library and an IT Help Desk? A: Yes, I worked in a library as a clerk for 3 years and 4 months. I liked working there. I liked the quiet and making sure the books were organised correctly, the filing system was intricate and interesting, and the librarians were very friendly. I have also worked at a cafe as a dish hand for 3 months. I did not like working there because it was loud and the chefs were always shouting. They didn't seem to care if I cleaned the plates properly, and got angry when I insisted on doing so. I have also worked at the IT help desk of my university for 3 years and 6 months years. I liked working there because I got to fix things around the campus, and I got to work with computers. N: Unrelated work experience about cafe job that is not pertinent to this interview. I: Tell me about a time you have faced a problem on the job, and how you resolved it. EFFECTS OF GENDER AND ASD DIAGNOSIS IN INTERVIEWS 47 A: Can you clarify what kind of problem? I: A time when you dealt with an issue you weren't sure how to fix, or an angry customer? A: I do not know how to fix angry customers. When customers were angry they usually asked to speak to my manager so I would ask them to wait, and I would get my manager. I: How did you deal with the angry customer before the manager got there? A: I would explain to them what the solution to their issue was and sometimes it helped but other times they got even angrier. I: Why did they get angrier? A: I don't know. I don't think they understood what I was explaining to them. I: Can you give me an example? A: One time a professor at the university came into the IT helpdesk and had trouble downloading Microsoft word due to account issues. I explained to him that we do not endorse or support Microsoft word, and suggest using Google Docs online. He did not seem to understand and grew frustrated that I could not help him, even though I explained that I was not able to as it is against policy. I suggested that he contact Microsoft office support, but this only seemed to make him more frustrated. In the end, I got my manager and he was able to handle the situation. N: Lack of ability to handle troubled clients individually? I: What do you consider to be your strengths? A: I like math, and computers, and am good with data. I like learning new things and I am good at it. I am also good at understanding problems and finding solutions to them quickly. I really like puzzles, and I'm good at figuring things out. I also have very good time management and organisation skills, and good attention to detail. I: What do you consider to be your weaknesses? A: I feel anxiety in stressful situations, and if I feel pressured I cannot perform at my full capacity. I: How have you overcome this in the past? A: I take 5 deep breaths and count to ten until I feel better. I: And this helps you work better? A: Yes. N: Applicant has identified personal issue and solution - self awareness I: How well do you work in a team? A: I am sufficient in working in a team. I have completed several group projects in my classes throughout the course of my degree. I like hearing other people's input and ideas on the project, however I do not it like when people do not do their fair share of work. I have worked as part of a team at the IT help desk. I: Why would you fit well in this position? A: I am qualified for the position with my degree in computer science, and am very interested in working in the IT department. I have worked in the IT department in my university throughout EFFECTS OF GENDER AND ASD DIAGNOSIS IN INTERVIEWS 48 my degree so I have experience in this regard. Even though I will now be dealing with working adults rather than students and professors, I believe the skills will transfer over well. I: What are your professional goals in the next 5 years? A: I would like to keep working in the IT department here. N: No defined career goals I: Okay, I think I've got everything I need on that end. Before we end for the day, tell me a little about yourself - any hobbies? What do you do in your free time? A: I already told you that I really like puzzles, I do them in my free time. I: I saw you attended University of Townsville, my friend's daughter goes there - did you like it? A: It is a good school and met all of my requirements throughout the course of my degree. I: It's not easy to get into is it? You have to have a pretty good SAT score right? A: Yes, I had an above average SAT and ACT score. I: And with a GPA of 3.8 I guess you did pretty well in school then A: Yes, I did. I: Well I think that is everything we need to know, is there anything you would like to add before we conclude this interview? A: Yes, actually. I have been diagnosed with Autism Spectrum Disorder. This is a social developmental disorder that causes me to act differently than you might expect in certain social situations. If you would like more information about this, I would be happy to provide it.* I: Thank you for your time, we will be in touch regarding this interview. A: You're welcome. Have a pleasant day. Further comments: Did not maintain eye contact, but instead let eyes wander around the room constantly Very knowledgeable about computers Confident demeanour when talking about strengths and interests, less confidence is displayed when not answering a direct question. Not good at making small talk or talking about themselves Doesn't seem to have much leadership experience Constantly tapped their leg with their hand throughout the entire interview Seemed distracted by the air purifier in the room and kept glancing at it EFFECTS OF GENDER AND ASD DIAGNOSIS IN INTERVIEWS 49 Appendix D Autism-Spectrum Quotient (AQ) scale (Baron-Cohen, 2001) 1. He/she would prefer to do things with others rather than on their own 11. He/she would find social situations easy 13. He/she would rather go to a library than a party 15. He/she would find themselves drawn more strongly to people than to things 22. He/she would find it hard to make new friends 36. He/she would find it easy to work out what someone is thinking or feeling just by looking at their face 44. He/she would enjoy social occasions 45. He/she would find it difficult to work out peoples intentions 47. He/she would enjoy meeting new people 48. He/she would be a good diplomat 4 point Likert-type scale 1 (Definitely Agree) 2 (Slightly Agree) 3 (Slightly Disagree) 4 (Definitely Disagree) Items 1, 13, 22, and 45 score 1 point for Definitely Agree or Slightly Agree responses. Items 11, 15, 36, 44, 47, and 48 score 1 point for Definitely Disagree or Slightly Disagree responses. EFFECTS OF GENDER AND ASD DIAGNOSIS IN INTERVIEWS 50 Appendix E Cunningham and Macans (2007) job interview perception measures Hiring recommendation 5 - Yes, I would definitely hire this person. This person is an extremely good candidate. 4 - Yes, I would hire this person with a few reservations. 3 - Im not sure if I would hire this person. 2 - I dont think I would hire this person although I might consider taking a look at some additional information about them. 1 - No, I would definitely not hire this person. This person is not a good candidate. Qualification 1. I would evaluate this applicants qualification for this position favorably. 2. I feel this candidate would be well suited for the job. 5-point Likert scale 1 (Strongly Agree) Disagree) 2 (Agree) 3 (Neither agree or disagree) 4 (Disagree) 5 (Strongly Overall rating of performance. 5-point Likert-type scale 1 (Extremely poor) 2 (Poor) 3 (Average) 4 (Good) 5 (Extremely Good) The following items are all preceded by the question: How characteristic of the applicant do you believe the following to be Absenteeism 1. Likely to miss work. 2. Likely to need time off. Turnover 1. Likely to quit. Feminine stereotypes 1. Feminine. 2. Affectionate. 3. Gentle. 4. Nurturing. Good Employee 1. Demonstrates good work ethic. 2. Hardworking. 3. Self-reliant. 4. Successful. 5. Self-sufficient. 6. Self-confident. 7. Ambitious. EFFECTS OF GENDER AND ASD DIAGNOSIS IN INTERVIEWS 51 Other characteristics 1. Leadership ability. 2. Committed. 3. Dependable. 4. Competent. 5. Mature. 6. Healthy. 7. Aggressive. 8. Intelligent. 9. Physically limited. 10. Verbal communication. 11. Leadership ability. 5-point Likert-type scale 1 (not characteristic) 2 (sometimes characteristic) 3 (Neither) 5 (very characteristic) 4 (often characteristic) Job fit 1. Fits with job. 5-point Likert-type scale 1 (Low Fit) 2 (Somewhat Fit) 3 (Neither) 4 (Mostly Fit) 5 (High Fit) EFFECTS OF GENDER AND ASD DIAGNOSIS IN INTERVIEWS 52 Appendix F Attention checks + Manipulation check 1. What university did the interviewee attend? 2. What was the name of the interviewee? 3. What gender does the interviewee identify as? 4. What score do you estimate the interviewee received on their SAT? Open ended Manipulation check 1. Does this person have a developmental disorder of any type, if so, which one? Feel free to expand on your answer. Open ended Further information 1. This question is optional to respond to. If you choose to not respond, please move onto the next page. Have you ever been diagnosed with Autism Spectrum Disorder, or do you know anyone who has received a diagnosis? You may provide as much or as little information as you are comfortable with. Open ended EFFECTS OF GENDER AND ASD DIAGNOSIS IN INTERVIEWS Appendix G 53 EFFECTS OF GENDER AND ASD DIAGNOSIS IN INTERVIEWS 54 Appendix H Means and Standard Deviations of All Factors Male total Female total Non-ASD diagnosis ASD diagnosis M SD N M SD N M SD N M SD N ASD total (out of 1) 0.69 .23 72 0.77 .20 44 .69 .20 54 .75 .24 62 Hiring recommendation (out of 5) 3.69 1.04 72 3.18 1.23 44 3.59 1.22 54 3.42 1.06 62 Qualification (out of 5) 2.48 1.06 71 2.65 1.07 44 2.60 1.13 54 2.49 1.01 61 Overall Performance (out of 5) 3.44 .933 72 3.00 .915 44 3.50 .927 54 3.08 .929 62 Absenteeism (out of 5) 2.14 1.30 72 1.66 1.07 44 2.23 1.34 54 1.66 1.07 62 Feminine Characteristics (out of 5) 2.48 1.02 72 2.48 1.00 44 2.69 1.00 54 2.29 .990 62 Good Employee (out of 5) 3.51 .837 72 3.51 .945 44 3.53 .809 54 3.49 .936 62 Good Other Characteristics (out of 5) 3.43 .770 72 3.41 .729 44 3.51 .705 54 3.34 .787 62 Bad Other Characteristics (out of 5) 2.35 1.19 72 1.93 .767 44 2.39 1.13 54 2.02 .989 62 Job Fit (out of 5) 3.47 1.16 72 2.82 1.40 44 3.35 1.28 54 3.11 1.31 62 Male without ASD diagnosis Male with ASD diagnosis Female without ASD diagnosis Female with ASD diagnosis M SD N M SD N M SD N M SD N ASD total (out of 1) 0.64 .21 32 0.73 .24 40 0.75 .15 22 0.78 .24 22 Hiring recommendation (out of 5) 3.88 1.04 32 3.55 1.04 40 3.18 1.37 22 3.18 1.10 22 Qualification (out of 5) 2.59 1.17 32 2.39 .976 39 2.61 1.09 22 2.68 1.06 22 3.75 .842 32 3.20 .939 40 3.14 .941 22 2.86 .889 22 2.59 1.35 32 1.78 1.15 40 1.86 1.23 22 1.46 .872 22 2.79 1.06 32 2.23 .931 40 2.56 .913 22 2.40 1.10 22 3.52 8.20 32 3.51 .861 40 3.55 .811 22 3.46 1.08 22 3.50 .753 32 3.37 .787 40 3.52 .646 22 3.30 .804 22 2.66 1.33 32 2.11 1.02 40 2.00 .577 22 1.86 .928 22 3.69 1.06 32 3.30 1.22 40 2.86 1.42 22 2.77 1.41 22 Overall Performance (out of 5) Absenteeism (out of 5) Feminine Characteristics (out of 5) Good Employee (out of 5) Good Other Characteristics (out of 5) Bad Other Characteristics (out of 5) Job Fit (out of 5) EFFECTS OF GENDER AND ASD DIAGNOSIS IN INTERVIEWS Appendix I 55 EFFECTS OF GENDER AND ASD DIAGNOSIS IN INTERVIEWS 56 EFFECTS OF GENDER AND ASD DIAGNOSIS IN INTERVIEWS 57 EFFECTS OF GENDER AND ASD DIAGNOSIS IN INTERVIEWS Appendix J 58  ...

... The Effect of Fish-Extracted Collagen on the Growth of Malignant Cells By Katherine May Honors Project Thesis Submitted in Partial Fulfillment Of the Requirements for a Baccalaureate Degree With Distinction In the Ron and Laura Strain Honors College of THE UNIVERSITY OF INDIANAPOLIS May 2020 Faculty Advisor: Dean A. Wiseman, Ph.D. Executive Director of Honors: James B. Williams, Ph.D. Katherine May 2020. Copyright in this work rests solely with the author. Please ensure that any reproduction or re-use is done in accordance with relevant national copyright protection. ii Abstract Collagen, the protein that makes up the majority of extracellular matrix structure, is essential in cell communication and proliferation. Research has found that collagen is also responsible for promoting many types of cancer growth because it both provides the infrastructure needed for the cells to expand, and it lessens the need for cell-cell interactions, therefore promoting cell motility. Clinically, collagen is used to treat severe burn victims as it assists in regenerating skinthis therapy typically involves bovine collagen or synthetic collagen. While these forms of collagen are readily available to clinicians, they are expensive, and it would be extremely useful to identify lower-cost alternatives. Recently, it has been discovered that tilapia, a fish popular for dietary consumption, and is now farmed all over the world, possesses high concentrations of collagen in its skin that can be easily extracted. Our hypothesis is that tilapia collagen could serve as a substitute to collagen derived from other sources in cell culture. By examining how malignant cells respond to tilapia collagen, we can determine if adding collagen to the media and/or as a coating on the surface of the tissue culture flasks in which these cells are grown on, creates a more authentic environment for the cells to grow in, better mimicking what cells would encounter in vivo. Firstly, we employ a cellproliferation assay, comparing the impact that tilapia-derived collagen has on cell proliferation rate, with the findings providing insight into both cell growth and wound healing. We also utilized colony-forming assays to determine whether cancer cells respond differently to cancer drugs in the presence of collagen. This would give insight into the feasibility of using tilapia collagen in further cancer research without significant changes to traditional cell culture. iii Table of Contents Abstract ........................................................................................................................................... ii Acknowledgements ........................................................................................................................ iv List of Tables .................................................................................................................................. v List of Figures ................................................................................................................................ vi Statement of Purpose .................................................................................................................... vii Introduction ..................................................................................................................................... 1 Creating a better in vitro cell culture model system.................................................................... 5 Tilapia-extracted collagen to create a better model system ........................................................ 6 Cancer Chemotherapy ................................................................................................................. 6 Farnesol as a Cancer Drug .......................................................................................................... 8 Methods and Procedures ................................................................................................................. 9 Colony-Forming Assay ............................................................................................................... 9 MTS Assay ................................................................................................................................ 10 Statistical Analysis .................................................................................................................... 11 Results ........................................................................................................................................... 12 Analysis/Conclusion ..................................................................................................................... 17 Reflection ...................................................................................................................................... 21 Bibliography ................................................................................................................................. 22 iv Acknowledgements I would like to thank the Strain Honors College for making my research possible. Without their support, I would not have been able to conduct the research or have the resources that I needed available. I would also like to thank Dr. Dean Wiseman for his guidance and support throughout the entire project. v List of Tables Table 1: Data from the Colony Forming Assay 12 Table 2: Data from MTS Assay 14 vi List of Figures Figure 1: Colony Forming Assay Graph and Plate Example 13 Figure 2: Graph of data collected from MTS Assay 15 Figure 3: Linear Slope Index of MTS Assay 16 vii Statement of Purpose The purpose of this study was to determine if tilapia-derived collagen has a promoting effect on the growth of breast cancer cells in vitro, with the goal of determining the feasibility of use as a substitute to more expensive cell culture reagents. In addition, this new potential cell culture reagent needed assessment to determine if and how tilapia collagen affects the behavior of this malignant cell culture model system. To those ends, in this study we first tested and compared the growth of these cells in different concentrations of collagen and ran a colonyforming assay. We also tested to see if the presence of collagen influences cell growth and/or drug resistance within MCF7 breast cancer cells via MTS colorimetric cell proliferation assay. 1 Introduction The basis of this project was to observe how added tilapia collagen in cell cultures promotes cell proliferation and growth in malignant cancer cells. Previous research has shown that type 1 collagen, found in high concentrations in cytoplasm, promotes cell regrowth and proliferation (3). Initially used for burn victims, doctors would apply collagen-rich bandages, from bovine, equine, or porcine sources, or skin grafts to promote skin regrowth (1). In countries with less access to these resources, intact tilapia skin was used as an alternative because it is rich in collagen, inexpensive, and abundant in those countries (1). The initial idea to use tilapia skin occurred in 2017 in Brazil, when it was discovered that tilapia skin could, indeed, be used as bandages for burn victims (1). The lack of accessibility to both human skin and the typical animal skin that is used to treat burns led to this discovery. Typically, doctors would use bandages and creams to dress wounds, but this method was difficult and inefficient as the bandages would dry out and had to be changed every day. An idea to use tilapia skin was centered around the fact that it can hold moisture better than gauze can, and has a high collagen content that is thought to promote keratinocyte and skin fibroblast cell regrowth. Collagen bandages offer more therapeutic benefits than typical wound dressings because they not only prevent infection, but also promote regrowth and provide more substantial protection of the wound from the environment than traditional bandages and dressings (2). Collagen creates a barrier that is impermeable to bacteria, keeping out further complications like bacterial infection. One study involving clinical assessment of open wounds such as ulcers, postoperative wounds, and infection sites, suggests that using collagen bandages in replacement of typical wound dressings such as silver sulfadiazine or nadifloxacin, reduces loss of fluids and 2 could potentially help the patient avoid skin grafting (2). While the study showed that both groups with or without collagen had good healing results, the group treated with collagen had earlier cell motility, and overall a less painful recovery. This reinforces the idea that collagen is beneficial in wound treatment and healing, and supports the idea that further research into collagen and its properties on cell growth of all kinds is merited. When researchers tested tilapia skin to determine collagen content, they were shocked at the amount of both collagen 1 and collagen 3 they found present in tilapia skin. The presence of this collagen is significant because these types of collagen have been previously shown to promote healing through cell regeneration (3). Other researchers have explored the idea of using tilapia in medicine (4,5,6). Not only can tilapia skin be used as a bandage, but collagen contained within tilapia skin has shown to have many other applications, including uses in the food industry, cosmetics, and as pharmaceutical drug coatings (6). The original extraction process was developed to extract gelatina denatured form of collagenfrom salmon (4). A more efficient method was developed for tilapia collagen extraction (7), which creates a product that is closer to the product we ultimately decided to use in our experiments. Due to constraints in our ability to extract tilapia directly and efficiently, we elected to purchase 100% tilapia extracted collagen for use in our experiments. One interesting advantage tilapia collagen has over bovine collagen is that the latter poses an issue to certain vegetarians and Hindus, given that consumption of cows and items derived from cows is against their beliefs. By replacing this bovine collagen with tilapia collagen, a large variety of products are now available for these groups of people that would otherwise not be. Thus, this new source of collagen will impact both the medical field as well as the food industry in a manner that is generally acceptable to a majority of people around the world. Once tilapia 3 collagen was established to be biologically safe (7), these prospective ideas became more plausible. Coupled with the ability to extract collagen (4,5) the potential uses of this protein source are many. When testing gelatin created from fish collagen, researchers found that collagen from fish sources can enhance cell adhesion, cell growth, and wound healing in HaCaT cells (a keratinocyte cell line from adult human skin) that had been exposed to H2O2-induced cellular damage (5). Cell adhesion is necessary in all cells, but especially the most aggressively malignant cell cultures because it serves as a growth signal to cells, promoting proliferation, migration, and differentiation. Extremely malignant cell types, on the other hand, either secrete their own growth-promoting cell signals (autocrine stimulation), or the signal receptor pathways have mutated to the point that they no longer need external signaling in order to grow and proliferate. By increasing the ability of relatively normal, non-malignant HaCaT cells to do this, it created a more realistic model system in terms of similarity to actual skin tissue that could give researchers a more specific insight into how these cells think and function in terms of wound healing. Collagen is a normally insoluble fibrous protein found in the extracellular matrix (ECM) of cells and makes up 25% of the total protein mass found in vertebrates (4). It promotes cell growth because it provides structural support and helps maintain tissue homeostasis by creating the scaffolding that allows the cell to have the appropriate space to carry out necessary functions (3). Previous research has also found that collagen contains a high amount of arginine-glycineaspartic acid amino acids, which research has shown also promotes cell adhesion and proliferation (6). There are currently 27 identified types of collagen, but most research focuses on type 1 because it is the most abundant in the human body. 4 The ability of collagen to promote cell growth is transferrable to cancer cells. Studies utilizing a model system for lung metastasis show that tumor growth was negatively affected when collagen 1 was depleted from the cellular environment (8). Moreover, as cancer cells spread, collagen fibers encountered by the cells would strengthen and remodel to better fit the metabolic needs of the cancer cells. So, from the idea that depleting collagen levels would decrease cell growth, comes the idea that increasing collagen levels would promote cell growth (3). It was once believed that collagen acted as a barrier, prohibiting cancer from spreading as rapidly, but as it turns out, collagen can also promote cancer cell growth by changing the microenvironment (3). Collagen protein fibers typically comprise up to 90% of the extracellular matrix (ECM), which is where cancer tumor cells initially invade as they progress through the bodyin fact, the definition of malignancy is the point when cancer cells move beyond the tissue of origin into other tissues, and the ECM (9). This is important because the ECM is the location where signaling that is involved in proliferation, migration, invasion and other important functions of the cells takes place (10). Because the ECM is so rich in collagen, when cancer cells invade, the contents of the ECM promote the spread of cancer by essentially building pathways for the cancer to follow and promoting cell adhesion as well. In addition, collagen is involved in sustaining a phenomenon termed epithelial-mesenchymal transition (EMT) which is the controlling of different proteins to promote or obstruct cell-cell contacts (10). When collagen is present in normal amounts and cancer encounters the ECM, EMT promotes the downregulation of epithelial proteins and an upregulation of mesenchymal proteins. This in effect causes the loss of cell-cell contact and therefore leads to more cellular motility, and ability to invade additional 5 tissues. Essentially, collagen helps increase the ability of the cancer cells to move around and therefore spread (10). Creating a better in vitro cell culture model system Two-dimensional (2D) models of cells are traditional for research; some research has indicated that a three-dimensional cell culture model, an artificially created environment in which cells are permitted to grow or interact with their surroundings in all three dimensions, more closely mimics conditions inside the body. By growing the cells in a three-dimensional pattern, cells can more readily transfer nutrients, communicate, and allow ECM interactions that cannot normally take place in 2D models (14). However, using 3D models in research has its own set of disadvantages: they are expensive to create and hard to observe. To keep expenses low, researchers often fall back on the less accurate 2D models for cell culture, at the risk of cells not being able to communicate and proliferate as readily, and not effectively mimicking in vivo physiological conditions. Collagen is a very fibrous substance that acts as scaffolding for the cells that it encounters. This extra support allows the cells to grow on different microscopic planes within the plate, rather than flat on the surface. Adding collagenand in this case tilapia-extracted collagento promote the growth of cells in culture could potentially serve as an effective compromise, boosting the proliferation rate and making a lower-cost, 2D model cell culture system more analogous to conditions found in actual cancerous tumors. To understand how cancer works, Weinberg contends that we must first understand a minimum of six biological processes he terms hallmarks of cancer(9). These hallmarks appear to be biological commonalities across all cancer cells, or barriers that cells must overcome in 6 order to develop into full-blown metastatic cancer and help us understand and develop therapeutic and preventive clinical strategies against them. These hallmarks include: sustaining proliferative signaling; evading growth suppressing signals; activating invasion and metastasis; enabling DNA replicative immortality (normal cells seem to reach a limit in terms of number of cell divisions, termed the Hayflick Limit); inducing growth of new blood vessels (termed angiogenesis); and resisting cell death (15). Most traditional chemotherapeutic agents to date are used to exploit how cancer activates invasion and metastasis and sustains proliferation signals. It is important however, to keep in mind the other four hallmarks for therapy, as they might provide an explanation for why the cancer cells act in specific ways. Tilapia-extracted collagen to create a better model system The discovery of collagen-rich tilapia skin has led to many advancements in ideas and products throughout the world. What started as a basic idea to satisfy a lack of resources, has exploded into hundreds of journals and research articles outlining other ways this collagen can be useful to us. By extending this idea further into the realm of cancer research, if successful, could be advantageous. Creating a better model system at a cheaper cost that more closely replicates the physiological conditions of cancer cells in the body, will open the door for future research to be done and create endless research and clinical possibilities, particularly at smaller universities where financial constraints are significant. Cancer Chemotherapy Chemotherapy is one of the most common methods used to treat, and hopefully cure, cancer in a patient. It works by using chemical compounds to target specific metabolic vulnerabilities in cancer cells. This would include DNA replication and metabolic targets that 7 impact different phases of the cell division cycle and kills the cells. Alternatively, some chemotherapeutic agents are used with the goal of shutting off certain types of intracellular signals that induce cells to grow and divide, or to block certain normal processes in cells and tissues which have been hijacked by cancer cellssuch as triggering new growth of blood vessels. Because cancer cells are dividing at a rapid rate and are highly active metabolically, cancer cells tend to be more vulnerable than normal cellsthis difference is implied in what is known as a therapeutic index (11), where chemotherapeutic drugs can successfully affect cancerous cells at doses below that which causes toxicity and other significant adverse side effects in patients (12). Chemotherapy drugs include DNA alkylating agents, antimetabolites, anti-tumor antibiotics, topoisomerase inhibitors, and receptor inhibitors (12). The negative side of using these drugs in therapy is that they can frequently affect healthy cells, especially cell populations which are rapidly dividing, such as bone marrow (which makes red and white blood cells), hair, and the digestive tract. And so, patients typically see some fairly severe side effects. On the other hand, using chemotherapy drugs or other anti-cancer drugs are useful in cancer research, because the effects observed in cancer cells under research conditions can be helpful to predict outcomes in the patient. Perhaps just as importantly, even less than highly effective drugs used in the research lab can provide important information about the biology of cancer cells, as well as provide insight into potential specific targets for which a more effective compound might be developed (13). To that end, it is vitally important that when creating a new model system for growing cellsbe them cancer cells or other types of cells intended for therapeutic use, to note how cells react in various environments and how closely this relates to the in vivo conditions within 8 actual patients. Creating biologically authentic environments in vitro, including a collagen-rich environment that we are interested in, can tell a researcher a lot about what could happen to cells in vivo if we either change the environment inside a patient, or subject the patient to chemotherapy. If the environment created does not accurately replicate the in vivo biological system, then the data that is collected will likely not be as predictive as it could be. Farnesol as a Cancer Drug The anti-cancer drug that we used in this study was farnesol. This orphan drug (as categorized by the FDA), that has remained mostly undeveloped commercially due to lack of profitability, is found mostly in nature within the essential oils of plants. Farnesol is a relatively new anti-cancer drug, and therefore its mechanism of attack is not understood completely. It has many uses, including anti-inflammatory effects and alleviating allergic asthma and edema, though the most important property of farnesol for our study is its anti-cancer effects. Farnesol has an antiproliferative effect on cells due to its ability to induce apoptosis, and/or upregulate regulatory proteins which delay or halt the cell cycle altogether (17). We used various concentrations of this drug in order to observe the potential of some cells living after applying the drug. In one study, scientists observed the effect of farnesol and other anti-cancer drugs on pancreatic cancer cells to determine when the drug plays a role on the cell cycle. They determined that farnesol, and the other drugs, have a G0/G1 phase cell cycle arrest on cells that correlated with an increase in cyclin kinase inhibitor proteins (18). This is important to note because if the drug effects the cells in the early phases of proliferation, like the G0 or G1 phase, it could have a stronger effect on cells and kill more of the living cells. These phases are crucial because during this time, the cells are preparing for DNA replication and are vulnerable. 9 Methods and Procedures Colony-Forming Assay This procedure is based on a protocol developed by Shoemaker, et al (19). For this set of experiments, we used Premium Marine Collagen Peptides by Zammex to pre-treat tissue culture plants onto which we would apply a low density (~1-5 cells/cm2). The only ingredient in the collagen was from tilapia non-GMO Type I and III collagen. This simplified the procedure for extracting the collagen from tilapia ourselves and allowed us to work solely with the cells. We used this collagen to create different concentrations of collagen mixed with water. The three concentrations we decided to use were 0.5 mg/ml, 1.0 mg/ml, and 2.0 mg/ml. We created these concentrations by measuring out a designated amount of collagen, adding it to a 50mL flask and then adding the appropriate amount of water. To make sure the collagen was evenly distributed and dissolved, we left them on a laboratory rocker over night as a constant speed and temperature. Next, we applied 1mL of the 0.5 mg/ml mixture to a sterile culture dish. We repeated this step twice for each concentration for a total of 6 plates. Next, we left them in the sterile hood over night for the water to evaporate, leaving only the collagen. We also plated two culture dishes with just 1mL of distilled water as a control. After the dishes were dried, approximately 500,000 MCF-7 cells were plated in a volume of 3 ml of standard Dulbeccos Modified Eagle Medium (DMEM). Cultures were incubated in humidified 37C incubator with an atmosphere of 5% CO2 in air, and control plates were monitored for growth using an inverted microscope. At the time of maximum colony formation, (7 to 21 days in culture), final colony counts were obtained with a Bausch and Lomb Omnicon (FAS II) image analysis system. We performed our counting procedure on day 9 of the cells 10 being incubated, based on a plateau of growth observed. Objects presenting a circular profile minimum approximate diameter of 60 um were scored as colonies by the system. For quality control purposes, control plates were also counted on the day of plating (Day 0) or the following day (Day 1). The colony forming assay tests the ability of cells to grow into colonies. It essentially gives us information about the viability of a cell and its potential for continued growth and reproduction. We chose this assay to see if there were more viable cells and better cell replication present on plates with or without collagen present. MTS Assay The next assay that we performed was the MTS Assay. We started by creating our collagen mixtures again of 0.5 mg/ml, 1.0 mg/ml, and 2.0 mg/ml, and allow them to mix at room temperature overnight. Then we plated a 96-well plate with 24 wells of control, which just consisted of purified water, and 24 wells of each concentration of collagen. We allowed these to dry in aseptic conditions overnight, and then plated our cells the next day. After 24 hours, we exchanged the media in each well and added fresh DMEM to the cells. After allowing an additional 24 hours of growth, we then added DMEM with our cancer drug (farnesol) in various concentrations (0, 20, 40, and 80 M) to the cells. We chose to use farnesol because of our accessibility to it and its known effect on cancer cells. After exposing the cancer drug to the cells for 24 hours, we examined cell viability using an MTS assay to determine how many cells were still living. Following the manufacturers recommended protocol, briefly, we added 20 M/well of the MTS reagent and incubated the plates for 3 hours at 37C. After the incubation period, we measured the absorbance of the collagen treated verses untreated cells using a plate reader at OD= 490 nm. 11 We chose to perform an MTS assay to see if collagen promoted drug resistance and/ or increased growth following the cancer drug treatment. Adding farnesol is analogous to a wounding event, therefore observing the number of living cells following the treatment can tell us a lot about how the presence of collagen influenced the results. Statistical Analysis Samples were tabulated and subjected to analysis for statistical significance using a twotailed unpaired Students t-Test (21). Samples with a p-value less than, or equal to 0.05 were considered statistically significant. Those that were considered statistically significant were marked with an asterisk on the graph. 12 Results The colony forming assay produced results shown in Figure 1, and Table 1 below. Each blue dot on the plates shown at the top of Figure 1 is a viable colony and the values listed in the table below come from averaging all the data across trays with the same plated collagen concentration. Any colonies smaller than approximately 0.25 mm were omitted. We deidentified and labeled the trays alphabetically so that there was no biased when counting the viable cells. We then later determined the concentration associated with each tray to record the data. Table 1: Raw data from the Colony Forming Assay . 13 Figure 1. Effect of tilapia collagen on MCF-7 cell proliferation and migration. Approximately 5 x 105 MCF-7 breast cancer cells were plated on 35mm tissue culture plates pretreated with 0-2mg/ml tilapia collagen solution in ddH2O. After 9 days of incubation, colonies were counted, samples were deidentified with letters, as shown at the top of the figure, and counted. Each plate contained a different concentration of collagen. The graph represents the average number of colonies on each plate (n=8) Standard deviation. * p0.05 vs. untreated. 14 The next analysis that we performed was the MTS assay. After calculating the viability of the cells following the addition of the cancer drug, we came up with the following graph to exhibit the data. Table 2. Raw data from MTS assay 15 16 17 Analysis/Conclusion The results we collected from the colony-forming assay indicate that there is a progrowth and migration effect from addition of tilapia collagen for MCF7 cells, but also that too much collagen prohibits cell growth. There is a statistical significance between the untreated plate and the plate treated with 0.5 mg/ml of collagen. This indicates that having the collagen present likely created more favorable conditions for cells to grow and adhere to the plate. When we increase the collagen concentration to 1.0 mg/ml, there is another slight increase in the number of cells present, but it is not statistically significant relative to 0.5 mg/ml. On the other hand, when we increased the concentration to 2.0 mg/ml, there appears to be an inhibiting effect, given that there were fewer cell colonies present than the untreated plate. It is possible that the cells were overcrowded by the amount of collagen on the plate and therefore couldnt spread and grow as rapidly and efficiently as the cells on the other plates. Another possibility is that excess collagen blocked other signals that the cells need to grow. The data from the MTS assay presents a similar finding. When analyzing samples that did not receive any farnesol, the results support what we noticed with the colony-forming assay; Cells grew more abundantly in 1 mg/ml collagen plates, and the 2 mg/ml collagen caused an inhibiting effect. As we look at cells grown in the 1 mg/ml plates, they did statistically significantly better at surviving/growing than the untreated cells following the farnesol treatment. Furthermore, when we compare the number of cells present following the 10 M farnesol treatment for cells grown in 0.5 mg/ml and 1 mg/ml we also observe a large increase in the cells plated with 1 mg/ml collagen. This clearly supports our hypothesis that collagen influences the growth following a wounding event such as our anti-cancer treatment. The cells grown in the 18 optimal concentration of collagen exhibited better wound healing and growth factors than other plates. On the other hand, in spite of the improved growth characteristics of collagen, and some statistically different responses to the anti-cancer drug farnesol (Figure 2.), when we subjected the dose-response curves to a linear slope analysis (Figure 3.), we find that the average curve slope versus drug dose does change, but not to the point where we would consider the difference in slope to be of significant concern in future experiments. Any time that dependent variables are changed in an experiment, there is always the risk than an untoward effect will occur. This is especially of concern regarding in vitro drug efficacy studies prior to entering an in vivo or clinical model system. Such effect may be pharmacokinetic, such as theoretically our treatment may act as an absorbent, preventing some of the drug from encountering the cell. A more insidious potential effect is that the tilapia collagen affects the internal biochemistry of the cell, resulting in the synthesis or expression of molecules which confer resistance. By conducting this slope-index analysis, we conclude that adding tilapia collagen may better mimic the growth characteristics of malignant disease while at the same time not significantly impacting the cellular response to a drug that the cancer cells are known from previous studies to be sensitive to. The key finding from our experiments is that there is a balance, perhaps even dosedependent phenomenon in the amount of collagen present between pro-growth and anti-growth effect in MCF7 cells. When there is too much collagen present, it causes a decrease in the proliferation of the cells. As we transition to look at how the cells respond to the cancer drug in different environments, we see clearly that each plate is affected by the cancer drug. However, 19 when 1 mg/ml of collagen was present, there were more viable cells following the cancer drug treatment. These findings give us insight into creating a better, more clinically-relevant model system for studying cancer cells in vitro, which is tremendously more efficient and less expensive than in vivo, much less clinical alternatives. Moreover, if our early, in vitro models are more in alignment with the other systems, then any significant findings in vitro will be more likely to be successful in vivo and in clinical trials, avoiding unnecessary false positive situations. If an institution uses collagen on their plates, the cells will be more robust and easier to study. We find from this study that 1.0 mg/ml is the ideal concentration of collagen to be effective from the three concentrations we observed. Another interesting thing to note is that when there is a lot of collagen present, the cells seem to do poorly, as noted in the colony forming assay. However, when we look at the MTS Assay, and how the cells in the 2.0 mg/ml plate reacted to the cancer drug in reference to the untreated plate reacting to the cancer drug, the plate with collagen had more surviving cells. This proves that the presence of any amount of collagen influences the cells growth. In relation to wound healing, this experiment proves more definitively that collagen has a huge impact on the growth of cells. While we were not able to perform a wound-healing assay, we can still draw from our data that cells perform better when collagen is present. Specifically, in reference to the MTS assay, we saw that there were more viable cells following the farnesol treatment when there was any amount of collagen present with the cells. Ultimately, the collagen either promoted drug resistance or the collagen helped with regrowth of surviving cells after the treatment. This is advantageous to doctors that specialize in burns because it reiterates the 20 important fact that collagen promotes cell proliferation and a patient can potentially see a faster recovery if collagen is directly involved in their healing process. In total, I would say that collagen is very influential in the growth of cells as mentioned in the introduction of this article. Many scientists have concluded that it can promote growth of keratinocytes to benefit wound healing and we have shown that it also influences cancer cells in a similar way. This study showed that there is an optimal concentration of collagen that promotes the most cell growth which will be beneficial moving forward. Knowing that 1.0 mg/ml had the best response among the cells allows for further tests to be done while allowing that variable to be held constant. Moving forward, I think it is important to test how other cancer drugs effect the growth of cells when collagen is present. While we can draw conclusions about what we think might happen to cancer cells within a person, we do not know for certain the various ways in which collagen effects a person. It could be important for future researchers to look at the effects of a collagen-rich diet versus heavy usage of collagen-rich products in relation to a persons cells. Growing cells on a collagen coated plate cannot be directly compared to the complex ways in which the human body uses collagen, but gaining a well-rounded understanding of how cancer cells think and act in different environments is helpful when treating patients that have cancer. While this study might not have a direct link to what cancer cells experience in vivo, it does tell us a lot about the environment cancer cells prefer and how they respond to changing environments. Moving forward, I think it would be helpful to also test different types of cancer cells to see how they react in the various environments. Gathering as much information as possible on the growth dynamics of cancer cells could help in determining the best course of action used to treat them in the future. 21 Reflection Overall, we were able to collect good data on the cells that we grew. We had a lot of difficulties in the beginning with our cells dying when we pulled them out of the liquid nitrogen tank. We also had a lot of issues with mold forming within our cells from the incubator. While we managed to overcome these difficulties, it did force us to change the scope of the project slightly. Unfortunately, we did not get to perform a wound-healing assay which wouldve helped us see if the collagen promoted the cells growing back together following a wounding event. We also were not able to get skin cells to use in the study. The project took a turn towards cancer cells because that is all we were able to grow, but we managed to gather some great data about how resilient the cells are and how collagen effects their growth. Working on this project was a great experience for me as a student because it promoted critical thinking and gave me an opportunity to apply my classwork. I really enjoyed developing the research plan and then watching it come to fruition. Having to adapt to the various issues that we encountered taught me a lot about the struggles of research and gave me a deeper appreciation for this process. 22 Bibliography 1. Sussman N. Why this Brazilian city uses tilapia fish skin to treat burn victims. PBS. 2017 Mar 3 [accessed 2019 Apr 14]. 2. Singh O, Gupta S, Soni M, Moses S, Shukla S, Mathur R. Collagen dressing versus conventional dressings in burn and chronic wounds: A retrospective study. Journal of Cutaneous and Aesthetic Surgery. 2011;4(1):12. doi:10.4103/0974-2077.79180 3. 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Yamamoto K, Igawa K, Sugimoto K, Yoshizawa Y, Yanagiguchi K, Ikeda T, Yamada S, Hayashi Y. Biological Safety of Fish (Tilapia) Collagen. BioMed Research International. 2014;2014:19. doi:10.1155/2014/630757 8. Zhang L, Wang Y, Xia T, Yu Q, Zhang Q, Yang Y, Cun X, Lu L, Gao H, Zhang Z, et al. Suppression for lung metastasis by depletion of collagen I and lysyl oxidase via losartan 23 assisted with paclitaxel-loaded pH-sensitive liposomes in breast cancer. Drug Delivery. 2016;23(8):29702979. doi:10.3109/10717544.2015.1132798 9. Hanahan D, Weinberg RA. Hallmarks of Cancer: The Next Generation. Cell. 2011;144(5):646674. doi:10.1016/j.cell.2011.02.013 10. Venning FA, Wullkopf L, Erler JT. Targeting ECM disrupts cancer progression. Frontiers in Oncology. 2015;5. 11. Gustafson DL, Page RL. Cancer Chemotherapy. Withrow and MacEwens Small Animal Clinical Oncology. 2013:157179. doi:10.1016/b978-1-4377-2362-5.00011-6 12. How Chemotherapy Drugs Work. American Cancer Society. 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... Spermiogenesis Within the Testes of the Tokay Gecko, Gekko gecko By Diana Mancuso Honors Project Manuscript In the Ron and Laura Strain Honors College of THE UNIVERSITY OF INDIANAPOLIS March 2020 Faculty Advisor: Kevin M. Gribbins, Ph.D. Executive Director of Honors: James B. Williams, Ph.D. Citation Style: The Council of Science Editors(CSE) 1 Abstract The Tokay Gecko, Gekko gecko, is a common house gecko that is native to Asia, has a widespread distribution across its native range, and also is invasive in Florida. The purpose of this research is to elucidate the ultrastructural changes that occur to spermatids during the process of spermiogenesis within the Tokay Gecko using transmission electron microscopy. Spermiogenesis is the final stage of spermatogenesis and is a reproductive process that mature spermatids undergo to become mature spermatozoa. This research is important because it will extend the knowledge of spermiogenesis within the Gekkonidae family, which previously had only one species account of spermiogenesis. This type of morphological data may also be valuable in future studies that incorporate the ultrastructural data gathered here for life history, phylogenetics, or histopathological research within reptiles. Testicular tissues from three G. gecko were collected from captive bred adult males at the University of Arkansas, fixed in Trumps fixative, and sent to Dr. Kevin Gribbins research lab at the University of Indianapolis. The tissues were processed for transmission electron microscopy. Images and the ultrastructural description of spermiogenesis in the Tokay Gecko were then compared to that of the Mediterranean Gecko, Hemidactylus trucicus, as well as other lizards in order to observe and compare similarities and difference between spermatid ultrastructure as they proceed through spermiogenesis. 2 Table of Contents Acknowledgements... iii List of figures..... iv Statement of purpose..... 1 Introduction... 2 Method/ Procedure..... 4 Results.... 5 Analysis/Conclusion.. 14 Reflection.... 19 References/ Bibliography.. 21 Appendices.. 24 CITI Training..... 24 Safety Data Sheets .. 25 3 Acknowledgements I would like to thank my advisor, Dr. Kevin Gribbins, for his mentorship while conducting this research. I would also like to thank the University of Indianapolis and the Honors College for the financial support on this project. 4 List of Figures Figure 1. Early stages of spermiogenesis: the round spermatid and early elongation stages within G. gecko... 5 Figure 2. Middle stages for elongation of the spermatid within G. gecko.... 7 Figure 3. Late stages of elongation of spermiogenesis within G. gecko . 9 Figure 4. Transmission electron microscopy of the mature spermatid just before spermiation within G. gecko.... 11 1 Statement of Purpose The process of spermiogenesis within reptiles is an understudied area of research. Spermiogenesis is the maturation of spermatids to create mature spermatozoa. Spermiogenesis is the last stage in spermatogenesis and can often be signified by the swelling of the Golgi complex and the development of the acrosome vesicle (Rheubert et al. 2016). In some species, the initial stages of spermiogenesis occur when the spermatids are joined by cytoplasmic bridges, allowing for cytoplasmic communication between spermatids during the developmental process (Rheubert et al. 2011). Spermiogenesis also often varies from species to species. Due to this and the lack of research on the subject, it is often hard to agree upon what parts of the process are commonly represented within lizard species. Within the more than 5600 species of lizards (Pincheira-Donoso et al. 2013), roughly around a dozen species have complete ultrastructural data on spermiogenesis (Gribbins and Rheubert 2015); hence, the purpose of this project is to do a full histological analysis of spermiogenesis in the Tokay Gecko, Gekko gecko. Thus, this study will provide needed data in a family of lizard where only one species (Hemidactylus turcicus; Rheubert et al. 2011) has current information on the morphological development of spermatids as they progress through spermiogenesis. Furthermore, the Geckos are thought to be a stem taxon within Squamata (Pyron et al. 2013) and thus spermiogenic data from these species could aid in our understanding of the ancestral characters involved in the evolution of sperm characters in lizards. 2 Introduction Over the last decade, there has been an increase in studies that provide completed ultrastructural analysis on spermiogenesis within lizards, which may be of future phylogenetic significance (Jamieson et al. 1996; Oliver et al. 1996; Vieira et al. 2005; Rheubert et al. 2010; Gribbins and Rheubert et al. 2011; Rhuebert et al. 2016). This type of research benefits the field of herpetology because of the constant shifts within taxonomic relationships and the level of difficulty in determining evolutionary kinship between species of reptiles, including lizards. Even small studies analyzing morphological or molecular information about a species could significantly alter its placement within a phylogenic tree. This ever-changing phylogenic comparison makes the study of sperm maturation and the morphology of spermatozoa important because it has been shown that there is more variation within spermatids and spermatozoa within reptiles and lizards than that of other amniotic taxa (Jamieson 1995; Gribbins et al. 2013a). By studying spermiogenesis in multiple species of lizards, a large character matrix of comparable ultrastructural features can be created that allows for similarity and differences to be observed between the spermatids of different species. These character traits may allow for future phylogenetic analysis if larger numbers of families or several species within a genus have comparable data on spermatid development (Gribbins and Rheubert 2015). Currently, there are 13 studies that have full ultrastructural descriptions of spermatid development during spermiogenesis in lizards (Gribbins and Rheubert 2015). These studies 3 include the Common Lizard (Zootoca vivipara; Courtens and Depeiges 1985), the Oscillated Skink (Chalcides ocellatus; Carcupino et al. 1989), the Amazon Lava Lizard (Tropidurus torquatus; Verira et al. 2001), the Green Iguana (Iguana iguana; Ferreira and Dolder 2002), the Ground Skink (Scincella lateralis; Gribbins et al. 2007), the Jamaican Gray Anole (Anolis lineatopus; Rheubert et al. 2010), the Mediterranean Gecko, (Hemidactylus turcicus; Rheubert et al. 2011), the Bunchgrass Lizard (Sceloporus bicanthalis; Rheubert et al. 2012), the Imbricate Alligator Lizard (Barisia Imbricata; Gribbins et al. 2013a), the Rosebelly Lizard (Sceloporus variabilis; Gribbins et al. 2013b), the Eastern Fence Lizard (Sceloporus undulatus; Rheubert et al. 2016), the Sandfish Skink (Scincus scincus; Ahmed et al. 2017), and the Prairie Lizard (Sceloperus consibrinus; Rheubert et al. 2017). Of these studies listed, only one study has been done in the Gekkonidae family, the Mediterranean Gecko (Rheubert et al. 2011). Since the Tokay Gecko falls in the Gekkonidae family, the hypothesis of this study is that most spermatid morphology should be homologous between these two species compared to that of other lizards. However, it is also hypothesized that some variation should be recognizable within the development of the acrosome complex and the flagellum of these two geckos similar to that seen in the genus Sceloporus. In S. variabilis, spermatids differ in several characteristics when compared to the sibling species S. undulatus and S. bicanthalis. In S. variabilis, mitochondria were found separating the Golgi body where the acrosome was developing. Myelin figures in acrosome development were common in S. undulatus and S. bicanthalis, but rare in S. variabilis (Rheubert et al. 2012; Gribbins et al. 2013b; Rheubert et al. 2016). The differences within the acrosome between species of Sceloporus, as well as differences within the midpiece number of mitochondria (Gribbins and Rheubert 2015), provided us with specific areas of spermatid 4 morphology that were the focus in this study. As mentioned earlier, these differences are of importance to further understand the morphological relationship of geckos and other lizards, as geckos are thought to be a stem taxon within Squamata (Pyron et al. 2013). The hypothesis tested here was that a majority of characters are conserved between G. gecko and H. turcicus, with occasional character differences observed during acrosome and flagellar development. Methods The sample size for this study was three captive bred Tokay Geckos from Dr. Stanely Trauth at the University of Arkansas. The testicular tissues were collected and sent to the University of Indianapolis and were first post fixed in osmium tetroxide for two hours. The tissues were then rinsed with DI water, dehydrated in a series of graded ethanol solutions (70%-100%), and cleared with propylene oxide. The propylene oxide was subsequently replaced with a 1:1 and then a 1:2 solution of propylene oxide and epoxy resin from the Spurrs Low Viscosity Kit. Next, the tissues were incubated in pure plastic for 24 hours and placed in beam capsules with freshly mixed Spurrs resin, then hardened and cured at 60 C for 48 hours in a vacuum oven. Once the testicular tissues were embedded in hard plastic, the blocks were cut with dry glass knives on a microtome to get two-micron thick sections that were stained with 1% toluidine blue and 3% basic fuschin. Blocks of tissue that showed the right stages of spermatogenesis (spermiogenically active) were then cut (90nm sections) using a diamond knife and ultramicrotome to be viewed under the Zeiss 109EM transmission electron microscope. The spermatids were photographed from these thin sections and compared 5 morphologically using Photoshop CS 7 to that known within the Mediterranean Gecko (Rheubert et al. 2011) and within other lizards. Results: Figure 1: Early stages of spermiogenesis: the round spermatid (A,B) and early elongation stages (C,D) within G. gecko. Acrosome vesicle (Av); Cell membrane (Cm); Acrosome granule (Ag); Subacrosomal space (Sas); Subacrosome granule (Sag); Nucleus (Nu); Golgi Apparatus (Ga). 6 In Gekko gecko, spermiogenesis begins with the formation of the acrosome vesicle (Fig. 1, Av) and the acrosome granule (Fig. 1, Ag) near the apical surface of the nucleus (Fig. 1, Nu). It has been suggested that the acrosome vesicle and granule form from the transport vesicles from the Golgi apparatus (Fig. 1B, Ga) (Gribbins and Rheubert 2015). The acrosome vesicle and its granule grow in size and begin to move towards the nucleus where it creates a deep nuclear acrosome complex and forms the mature round spermatid. The subacrosome granule is observed during the mid-round spermatid stage. The acrosome vesicle then flattens the apex of the nucleus of the early elongating spermatid, leaving the acrosome granule intact (Fig. 1E), and begins to compartmentalize it into the two main subdivisions of the acrosome complex, the acrosome vesicle and the subacrosomal space. 7 Figure 2: Middle stages for elongation of the spermatid within G. gecko. Acrosome medulla (Acm); Acrosome preforatorium (Pe); Perforatorial base plate (Pbp); Acrosome lucent ridge (Alr); Nucleus (Nu); Manchette (Ma); Flagellum (Fl), Nuclear fossa (Nf); Pericentriolar material (Pm); Distal centriole (Dc); Subacrosome cone (Sac); Acrosome cortex (Acc). 8 The manchette (Fig. 2 A and B, Ma), consisting of a series of microtubules, surrounds the circumference of the nucleus (Fig. 2, Nu), and is thought to help aid the elongation of the nucleus. The nucleus begins to elongate into a rod-like cylinder and becomes more electron dense (filamentous condensation of chromatin) as it continues through elongation (Fig. 2, 3), with the acrosome complex on its apical surface. As the nucleus elongates (Fig. 2B, Nu), the flagellum (Fig. 2B, Fl) begins to attach itself to the nuclear fossa (Fig. 2B, Nf) on the base of the nucleus with the aid of the pericentriolar material (Fig. 2B, Pm). While the nucleus is elongating, the acrosome complex continues to compartmentalize and matures into several different structures. The most apical structure is the acrosome cortex (Fig. 2C, Acc), followed by the electron dense acrosome medulla (Fig. 2C, Acm). At this point in elongation, the perforatorial base plate (Fig. 2C, Pbp) is the deeply stained small circular structure at the apical edge of the acrosome lucent ridge (Fig. 2C, Alr) and just above the subacrosome cone (Fig. 2C, Sac). As seen in figure 3B, closer to the end of elongation, the acrosome preforatorium (Fig. 2A and 3B, Pe), will also become present. 9 Figure 3: Late stages of elongation of spermiogenesis within G. gecko. Manchette (Ma); Mitochondria (Mi); Nucleus (Nu); Acrosome medulla (Acm); Acrosome preforatorium (Pe); Acrosome cortex (Acc); Perforatorial base plate (Pbp); Acrosome lucent ridge (Alr); Epinuclear lucent zone (Elz); Subacrosome cone (Sac); Nuclear rostrum (Nr); Nuclear fossa (Nf); Proximal centriole (Pc); Distal centriole (Dc); Dense bodies (Db); Annulus (An); Fibrous sheath (Fs); Principle piece (Pp). 10 Towards the end of elongation, the nucleus becomes more electron dense (Fig. 3, Nu), which can be noted by its intense black color. In the later stages of elongation, more structures within the acrosome complex will mature until all the structures of completed spermatozoa are present. As shown in an earlier stage of elongation, the acrosome cortex (Fig. 3B, Acc) rests at the most apical and lateral points or shoulders of the acrosome. Just beneath the acrosome cortex, is the acrosome medulla (Fig. 2B, Acm), which is deep to the cortex. Within the medulla is the acrosome preforatorium and opaque area containing rods of actin (Fig. 2B, Pe). The perforatorial base plate (Fig. 2B, Pbp) is just below the preforatorium, and above acrosome lucent ridge (Fig. 2B , Alr), which continues down past the nuclear rostrum (Fig. 2B , Nr). The nuclear rostrum is the most superior part of the nucleus (Fig. 2B , Nu) and is just below the epinuclear lucent zone (Fig. 2B , Elz) and is surrounded by the subacrosome cone (Fig. 2B, Sac) resting below the acrosome lucent ridge. During the process of elongation of the nucleus, the flagellum also begins to elongate into its matured state. The nuclear fossa (Fig. 3C, Nf) resides at the base of the nucleus (Fig. 3C, N). The midpiece is the portion of the flagella that has the mitochondria (Fig. 3C, Mi) on its outer edges. Each mitochondria is separated by the round electron dense body (Fig. 3C, Db), with the last, more irregular body called the annulus (Fig. 3C, An). Within the midpiece, is the proximal centriole (Fig. 3C, Pc), located closer to the nuclear fossa and the distal centriole (Fig. 3C, Dc), located closer to the annulus. The principle piece (Fig. 3C, Pp) is the portion of the flagellum below the midpiece and can be identified by its fibrous sheath (Fig. 3C, Fs). 11 Figure 4: Transmission electron microscopy of the mature spermatid just before spermiation within Gekko gecko. Acrosome medulla (Acm); Acrosome cortex (Acc); Perforatorium (Pe); Perforatorial base plate (Pbp); Epinuclear lucent zone (Elz); Acrosome lucent ridge (Alr); Subacrosome cone (Sac); Nuclear rostrum (Nr); Nucleus (Nu); Manchette (Ma); Nuclear fossa (Nf); Distal centriole (Dc); Proximal Centriole (Pc); Lacuna (La); Axoneme (Ax); Fibrous sheath (Fs); Peripheral fibers (Pf); Mitochondria (Mi), Cell membrane (Cm). 12 Once the process of elongation is complete, the mature spermatid is ready for spermiation from the seminiferous epithelium (Fig. 4). The mature spermatid consists of three main parts, the acrosome complex, the nucleus, and the flagellum. The outermost portion of the acrosome complex, the acrosome cortex (Fig. 4A, B, and C, Acc), spans the majority of the acrosome vesicle. Within the acrosome cortex, there are two visibly distinct halves of the complex, which are separated by the most apical region of the acrosome lucent ridge (Fig. 4). In longitudinal section, the apex of the acrosome complex above the acrosome lucent ridge is the acrosome medulla (Fig. 4A, Acm), with the acrosome preforatorium (Fig. 4A, Pe) within. The perforatorial base plate (Fig. 4, Pbp) is just beneath the acrosome preforatorium and rests just above the epinuclear lucent zone (Fig. 4, Elz), which sits on the apical, central, portion of the nuclear rostrum (Fig 4, Nr). At the point where the nuclear rostrum ends, the epinuclear lucent zone (Fig. 4B, Elz) is the central structure at the apex of the rostrum. Lateral to the epinuclear lucent zone is the subacrosome cone (Fig. 4B, Sac), the acrosome lucent ridge (Fig. 4B, Alr), and then the acrosome cortex (Fig. 4B, Acc). Further down the acrosome complex, the nuclear rostrum (Fig. 4C, Nf) is the central portion of cross sections. Outside the rostrum is the subacrosome cone (Fig. 4C, Sac), the acrosome lucent ridge (Fig. 4C, Alr), and then the acrosome cortex/shoulders (Fig. 4C, Acc). Unlike the acrosome complex, the nucleus of the spermatozoa does not have much specialization and is the same throughout. Apart from the lacunae (Fig. 4D, La), which does not span the entirety of the nucleus, the nucleus (Fig. 4D, Nu) is the central portion of the nuclear region, with only the manchette (Fig. 4D, Ma) surrounding it. The flagellum starts where the nucleus ends, at the nuclear fossa (Fig. 4, Nf), and like the acrosome complex, the flagellum has 13 several different features the further distally you travel. The connecting piece is closest to the nuclear fossa and includes the proximal and distal centrioles. The proximal centriole rests in the upper portion of the connecting piece and the distal centriole (Fig. 4, Dc) resides centrally in the lower portion of the connecting piece. Just below the connecting piece is the midpiece (Fig. 4E). The midpiece is lined by irregular, more trapezoidal, shaped mitochondria (Fig. 4E, Mi). Dense bodies, which could be seen during early elongation are absent in the mature spermatid. Within the midpiece, there is a fibrous sheath (Fig. 4E and F, Fs) which contains a central axoneme (Fig. 4E, Ax), as well as the continuation of the peripheral fibers three and eight (Fig. 4E, Pf). The fibrous sheath begins at tier two of the mitochondria of the midpiece. However, peripheral fibers three and eight terminate within the midpiece and are not present in cross sections of the principle piece (Fig. 4F). The fibrous sheath also continues past the terminal end of the midpiece, marked by the annulus (Fig. 3C, An) down into the next part of the flagellum, the principle piece (Fig. 4F). Like the midpiece, the proximal piece still contains a central axoneme (Fig. 4F, Ax) with a 9 + 2 arrangement of microtubules. Just outside the fibrous sheath is the cell membrane (Fig. 4F, Cm). The terminal piece of the flagellum, the endpiece (Fig. 4G), lacks a fibrous sheath and only contains the axoneme (Fig. 4G, Ax) and the cell membrane (Fig. 4G, Cm). 14 Analysis/Conclusion As mentioned by most researchers on this subject, the process of spermiogenesis in squamates is moderately conserved (Jamison 1999; Gribbins 2011; Gribbins and Rheubert 2011). The Tokay Gecko, G. gecko, is no exception, and allows for a comparative relationship to spermatid development within the Mediterranean gecko, H. turcicus (Rheubert et al. 2011), a lizard within the same family, Gekkonidae, as well as those in other saurian families. Gekkonidae is typically considered one of the basal or stem taxon with squamates and thus should have many characters that are similar to that of other lizards and thus homologous and sympleisomorphic (ancestral) (Pyron et al. 2013). The characters from this study (described below) will aid our understanding of phylogenetic relationships among lizards using spermiogenic characters as our data aids in our understanding of which characteristics in sperm development are most likely ancestral. Spermiogenesis begins with the formation of the acrosome vesicle aided by transport vesicles from the Golgi apparatus, which is very common in squamates (Clark 1967; Da CruzLandim and Da Druz-Hofling 1977; Carcupino et al. 1989; Butler and Gabri 1984; Dehlawi et al. 1992; Ferreira and Dolder 2002, 2003; Gribbins et al. 2007, 2013, 2015; Rhebert et al. 2011, 2012, 2016). Not surprisingly, acrosome development starts near the Golgi in G. gecko. The acrosome granule also forms during this time, before the vesicle comes in contact with the nuclear surface. In many cases the acrosome vesicle forms close to the apical surface of the nucleus. The acrosome vesicle then indents the apical nucleus as it makes contact with the nuclear surface, as described in Zootoca vivipara (Courtens and Depeiges 1985), S. lateralis (Gribbins et al. 2007), and C. ocellatus (Carcupino et al. 1989). The acrosome vesicle formed 15 close to the nuclear membrane and had a deep indention into the nucleus, while S. variabilis (Gribbins et al. 2013) acrosome vesicle formed further away from the membrane and had a very shallow nuclear indentation. Gekko gecko also had a deep indent but was more shallow than that of H. turcicus (Rheubert et al. 2011). This could potentially mean that the acrosome vesicle formed further away from the nuclear membrane in G. gecko. Also, like H. turcicus, the formation of the subacrosomal granule within the subascrosomal space occurs during the round spermatid stage in G. gecko. From our images, it appears, as stated by Gribbins and Rheubert (2011), that this granule becomes the basal plate of the perforatorium based on its location and staining intensity. Once the process of elongation starts, a manchette surrounds the nucleus. Some differences have been reported within the two different types of microtubules, longitudinal microtubules and circum-cylindrical tubules, which, apart from a few species, are moderately equal in numbers within the manchette (Gribbins and Rheubert 2015). Only one reptile has lacked a manchette entirely, A. lineatopus (Rheubert et al. 2010). As for G. gecko, the manchette appears to be less developed than the manchette of H. turcicus (Rheubert et al. 2011). As expected, within G. gecko there appears to be equal numbers of both types of microtubules. The microtubules are known to aid in elongation (Gribbins and Rheubert, 2015). Sceloporus variabilis has well developed circum-cylinder microtubules and under-developed longitudinal microtubules, while S. bicanthalis has the opposite. In both cases nuclear elongation appears normal, but with S.variabilis, the mature spermatozoa seems to have more of a curve while in S. bicanthalis the spermatozoa seems to be straighter and more wide (Gribbins et al. 2006; 2013). With respect to these noted differences, it is possible that both 16 types of microtubules aid in elongation, the circum-cylinder microtubules allow for a more curved and thinner shape, while the longitudinal microtubules allow for a thicker, straighter shape. These suggestions must be made with caution as there are not many lizards with one type of microtubule dominant over the other. During elongation, it is uncommon for the spermatid nucleus to have lacunae, but it becomes more common as it reaches maturity. Gekko gecko has visible lacunae, but H. turcicus (Rheubert et al. 2011) does not, a character difference that is worth noting between the two species. Lacunae are common after elongation, as shown in Scincella laterale (Gribbins et al. 2006), and Sceloporus consobrinus (Gribbins and Rheubert 2015). Although not shown in all species lizards and other reptiles, it is possible that the lacunae is there and was not sectioned or missed by researchers. This is possible because the lacunae does not span the entirety of the nucleus (Gribbins and Rheubert 2015). Further into the process of elongation, the acrosome complex continues to compartmentalize and become several parts, while the flagellum also develops into several regions. The acrosome complex is very similar in most previous studies of spermiogenesis within reptiles, but the developing and mature flagellum tends to vary more greatly. The proximal centriole, part of the connecting piece, is where the flagellum connects to the nuclear fossa (Gribbins and Rheubert 2015). This is common in all species of lizard including H. turcicus (Rheubert et al. 2011), S. undulatus (Rheubert et al. 2016), and S. scincus (Ahmed et al. 2017). Although this is common in lizards and other reptiles, the attachment of the flagellum has also been noted to occur in the early stages of elongation in at least three species: S. variabilis (Gribbins et al. 2013), S. bicanthalis (Rheubert et al. 2012), and S. laterale (Gribbins et al. 2007). 17 Other studies suggest the proximal centriole appears during the round spermatid stage of development (Gribbins and Rheubert 2011). Since the proximal and distal centrioles are responsible for flagellar development and the flagellum shows up in almost all amniotes studied to date, then it is safe to hypothesize that the centrioles align themselves with the nuclear fossa during the cytoplasmic shift (Gribbins and Rheubert 2015) that occurs during the late round spermatid stage. In lizards, the midpiece is often shown to have only four to six tiers of mitochondria (Gribbins and Rheubert 2015). In G. gecko, there are typically five to six tiers of mitochondria. Although the number of mitochondria in G. gecko is a common trait that coincides with most lizards, it is significantly different from what was seen in H. turcicus. In H. turcicus, Rheubert et al. (2011) discovered that the midpiece was longer than most lizards, and had a midpiece twelve mitochondrial tiers deep, which is more commonly observed in snakes. This is uncommon in lizards, and since it is not the case for G. gecko, it adds to the possibility that the elongated midpiece is a possible automorphy for H. turcicus (Rheubert et al. 2011; Gribbins and Rheubert 2015). However, caution must be maintained as only two species of geckos to this point have complete data on spermiogenesis. Further studies might show that this elongated midpiece could be a synapomorphy of closely related sibling species of Hemidactylus. There are dense bodies associated with these mitochondria during the mid-elongation stage, but these bodies tend to disappear from the midpiece at the maturation point of the elongating spermatid. This is an interesting characteristic that has not been described before in lizards and requires further investigation in saurians. 18 Apart from the differences in length of the midpiece, other differences within the midpiece come from the surrounding mitochondria. In G. gecko, the mitochondria in cross section are trapezoidal in shape. This is less common but has also been seen in Iguana iguana (Viera et al. 2004). The more common shape of the mitochondria is more round or ovular. This can be seen in H. turcicus (Rheubert et al. 2011), Crotaphytus bicintores (Scheltinga et al. 2001) and S. bicanthalis (Gribbins et al. 2012). The principle piece and the end piece are very similar within squamates. All squamates studied to date, including G. gecko, have a fibrous sheath around flagellar axonemes, starting just within the midpiece and this is considered to be a synapomorphy of squamates (Jamieson 1999). The fibrous sheath then ends where the end piece starts and leaves the end piece with just a cell membrane surrounding the axonemes. The fibrous sheath begins at mitochondria tier two in G. gecko. This is common, as almost all lizards have their fibrous sheath begin at either tier two or three of the mitochondria, except for S. lateralis (Gribbins et al. 2007), which began at tier one. In contrast to G. gecko, the fibrous sheath of H. turcicus begins at tier three (Rheubert et al. 2011). This is the first study recorded regarding spermiogenesis in the genus Gekko, and the second in the Gekkonidae family. As hypothesized, there are many similarities between the Tokay gecko, G. gecko, and the Mediterranean gecko, H. turcicus, as well as with other reptiles and lizards. Some of these shared characters include the formation of the acrosome vesicle with the Golgi apparatus, the deep indent of the acrosome vesicle into the nucleus to form a round spermatid, and the presence of the fibrous sheath in the midpiece and the principle piece. Although Geckos are thought to be a stem taxon within Squamata (Pyron et al. 2013), 19 there appears to be no shared characteristics for sperm development specifically between G. gecko and H. turcicus to support this theory. Also, as expected, there were some differences found. These differences include the length of the midpiece, the shape of the mitochondria in the midpiece, the mitochondrial tier in which the fibrous sheath began, and the lacunae in the nucleus. The most interesting difference found between G. gecko and other lizards is the disappearance of dense bodies in the mature midpiece. This has not been recorded in any other lizard to date and is a possible automorphy of G. gecko, or a synapomorphy of this genus of Gekko, as there is no other spermiogenesis data of species within the Gekko genus. Reflection Before the start of this project, I had very little experience in the field of histology, and even less in regard to spermiogenesis. With that being said, this project was a very big learning process for me. While studying the subject, I gained a great deal of practice in reading research articles, which I used to avoid at all costs. On top of researching the subject in general, I had to learn several new techniques concerning the field of histology. These skills included using a microtome, transferring the samples to a slide without wrinkling them (which was definitely the hardest part), and to stain the samples. Shortly after beginning this project, I took Dr. Gribbins histology class, which was very helpful in teaching me about the more general parts of histology. Not only did the histology class help me with my honors project, but my honors project helped me with the histological techniques and information gathered from that class. In class, we had partner research projects 20 and the skills learned from my honors project allowed me to excel in using the equipment needed in working on the research in this class. On top of being helpful in class, the skills I learned during this project are also of professional importance. After I graduate, if any jobs in histology are available, I will now know how to use the equipment, or will, at least, be able to easily learn to use similar equipment and analyze histological results. Also, after a gap year, I am still planning on applying to vet school, and histology is a subject in veterinary school. Hopefully when, and not if, I get accepted I will have a head start in this particular subject after completing this project. One of the biggest challenges I faced, apart from the normal procrastination of a college student, was analyzing the results. After labeling all of the images, I had to analyze what I found and compare them to the other species of reptiles with spermiogenesis research. This was difficult because there were many similarities between the species apart from the occasional difference. It was difficult to go through all of the papers and try and find those differences, because they werent always mentioned in other research papers. I also had to decide which of these features were of importance, and which were not. With all of this being said, I would not call myself an expert on this subject now, but I definitely know a lot more than I did, and I enjoyed learning about this subject and working on the project. 21 References: Ahamed, M., Aldokhi, O.A., Alenezy E.S. 2017. Ultrastructure Differentiation of Spermiogenesis in Scincus scincus (Scincidae, Reptilia). Saudi Journal of Biological Sciences. 24: 711-1721. Butler, R.D., Gabri, M.S. 1984. Structure and development of the sperm head in the lizard Podarcis (Lacterta) taurica. Journal of Ultrastructure Research. 88: 261-274 Carcupino, M., Corso, G., Pala, M. 1989. Spermiogenesis in Chalcides ocellatus tiligugu (Gmelin) (Squamata, Scincidae): An electron microscope study. Bolletino Di Zoologia, 56(2): 119-124. Clark, A.Q. 1967. Some Aspects of Spermiogenesis in a Lizard. American Journal of Anatomy. 121: 369400. Courtens, J.L., Depeiges, A. 1985. Spermiogenesis in Lacerta vivipara. Journal of Ultrastructure Research. 90: 203-220. Da Cruz-Landim, C., Da Cruz-Hofling, M.A. 1997. Electron Microscope Study of Lizard Spermiogenesis in Tropidurus torquatus (Lacertilia). CaryolodiaI. 30: 151-162 Dehlawi, G.Y., Ismail, M.F., Hamdi, S.A. and Jamjoom, M.B. 1992. Ultrastructure of spermiogenesis of a Saurian reptile. The sperm head differentiation in Agama adramitana. Archives of Andrology. 28: 223-234. Ferreira, A., Dolder, H. 2002. Ultrastructural Analysis of Spermiogenesis in Iguana iguana (Reptilia: Sauria: Iguanidae). European Journal of Morphology. 40(2): 89-99. Ferreira, A., Dolder, H. 2003. Sperm ultrastructure and spermiogenesis in the lizard, Tropidurus itambre. Biocell. 27: 353-362. Gribbins, K.M., Mills, E M., Sever, D.M. 2007. Ultrastructural examination of spermiogenesis within the testis of the ground skink, Scincella laterale (Squamata, Sauria, Scincidae). Journal of Morphology. 268(2): 181-192. Gribbins, K., Rheubert, J. 2011. The Ophidian Testis, Spermatogenesis and Mature Spermatozoa. Reproductive Biology and Phylogeny of Snakes Reproductive Biology and Phylogeny. 183-264. Gribbins, K.M. 2011. Reptilian spermatogenesis: A histological and ultrastructural perspective. Spermatogenesis. 1: 250-269. 22 Gribbins, K.M., Rheubert, J.L., Touzinsky, K., Hanover, J., Matchett, C.L., Granados-Gonzlez, G., Hernndez-Gallegos, O. 2013. Spermiogenesis in the imbricate alligator lizard, Barisia imbricate (Reptilia, Squamata, Anguidae). Journal of Morphology. 274(6): 603-614. Gribbins, K.M., Matchett, C.L., Delbello, K.A., Rheubert, J., Villagrn-Santacruz, M., Granados-Gonzlez, G., Hernndez-Gallegos, O. 2013. The ultrastructure of spermatid development during spermiogenesis within the rosebelly lizard, Sceloporus variabilis (Reptilia, Squamata, Phrynosomatidae). Journal of Morphology. 275(3): 258-268. Gribbins, K.M., Rheubert, J.L. 2015. The Architecture of the Testis, Spermatogenesis, and Mature Spermatozoa. Reproductive Biology and Phylogeny of Lizards and Tuatara. 341-401. Jamieson B.G.M. 1995. The Ultrastructure of Spermatozoa of the Squamata (Reptilia) with phylogenetic considerations. Advances in spermatozoal phylogeny and taxonomy. 166: 359383. Jamieson, B.G., Oliver, S.C., Scheltinga, D. M. 1996. The Ultrastructure of the Spermatozoa of Squamata-I. Scincidae, Gekkonidae and Pygopodidae (Reptilia). Acta Zoologica. 77(1): 85-100. Jamieson, B.G.M. 1999. Spermatozoal phylogeny of the Vertebrata. The Male Gamete. From Basic Science to Clinical Applications. Cache River Press, Clearwater, FL. 303-331. Oliver, S.C., Jamieson, B G.M., Scheltinga, D.M. 1996. The Ultrastructure of the Spermatozoa of Squamata II. Agamidae, Varanidae, Colubridae, Elapidae and Boidae (Reptilia). Herpetologica. 52: 216-241. Pyron, R.A., Burbrink, F.T., Wiens, J.J. 2013. A phylogeny and revised classification of Squamata, including 4161 species of lizards and snakes. BMC Evolutionary Biology. 13(93). Pincheria-Donoso, D., Bauer, A.M., Meiri, S., Uetz, P. 2013 Global Taxonomic Diversity of Living Reptiles. PLoS ONE. 8(3): e59741. Rheubert, J.L., Wilson, B.S., Wolf, K.W., & Gribbins, K.M. 2010. Ultrastructural study of spermiogenesis in the Jamaican Gray Anole, Anolis lineatopus (Reptilia: Polychrotidae). Acta Zoologica. 91(4): 484-494. Rheubert, J.L., Siegel, D.S., Venable, K.J., Sever, D.M., Gribbins, K.M. 2011. Ultrastructural description of spermiogenesis within the Mediterranean Gecko, Hemidactylus turcicus (Squamata: Gekkonidae). Micron. 42(7): 680-690. Rheubert, J., Touzinsky, K., Hernndez-Gallegos, O., Granados-Gonzlez, G., Gribbins, K. 2012. Ontogenic development of spermatids during spermiogenesis in the high-altitude bunchgrass lizard (Sceloporus bicanthalis). Spermatogenesis. 2(2): 94-103. 23 Rheubert, J.L., Sever, D.M., Siegel, D.S., Gribbins, K.M. 2016. Ultrastructural analysis of spermiogenesis in the Eastern Fence Lizard, Sceloporus undulatus (Squamata: Phrynosomatidae). Micron. 81: 16-22. Rheubert, J., Messak, J.A., Siegel, D.S., Gribbins, K.M., Trauth,S.E., Sever, S.M. 2017. Inter- and intraspecific variation in sperm morphology of Sceloporus consobrinus and Sceloporus undulatus (Squamata: Phrynosomatidae). Biological Journal of the Linnean Society. 121(2): 355 364. Russell, L.D., Hikim, S.A.P., Ettlin, R.A., Legg, E.D. 1990. Histological and Histopathological Evaluation of the Testes. Cache River Press, Clearwater Fl. 211-275. Scheltinga, D.M., Jamieson, B.G.M., Espinoza, R.E.,Orrell, K.S. 2001. Descriptions of the mature spermatozoa of the lizards Crotapthytus bicinctores, Gambelia wislizenii (Crotaphytidae), and Anolis carolinesis (Polychrotidae) (Reptilia, Squamata, Iguania). Journal of Morphology. 247: 160-171. Vieira, G.H., Colli, G., Bo, S., Wiederhecker, H. 2001. Spermiogenesis and testicular cycle of the lizard Tropidurus torquatus (Squamata, Tropiduridae) in the Cerrado of central Brazil. AmphibiaReptilia. 22(2): 217-233. Vieira, G. H., Colli, G.R., Bao, S.N. 2005. Phylogenetic relationships of corytophanid lizards (Iguania, Squamata, Reptilia) based on partitioned and total evidence analyses of sperm morphology, gross morphology, and DNA data. Zoologica Scripta. 34(6): 605-625. 24 Appendix: CITI Training: 25 Safety Data Sheets: Trumps Fixative 26 27 Osmium Tetroxide 28 29 30 Sodium Cacodylate 31 32 33 Ethanol 34 Propylene Oxide 35 36 37 38 Epoxy Resin 39  ...

... The Effects of Gender on Perceptions of Nurses Addicted to Opioids By Mark Anthony Jones II Honors Project Thesis Submitted in Partial Fulfillment Of the Requirements for a Baccalaureate Degree With Distinction In the Ron and Laura Strain Honors College of THE UNIVERSITY OF INDIANAPOLIS May 2020 Faculty Advisor: Melissa J. Loria, Ph.D Executive Director of Honors: James B. Williams, Ph.D. Mark Anthony Jones II, 2020. Copyright in this work rests solely with the author. Please ensure that any reproduction or re-use is done in accordance with relevant national copyright protection. 2 Abstract The purpose of this study is to understand the perceptions of future healthcare workers and perceptions of the general public on nurses who become addicted to opioids while working. Participants in the study read one of two vignettes that tell the same story but have different main characters (one male nurse and one female nurse) who become addicted to opioids while at work. The perceptions of the reader were then measured to determine the level of stigma regarding addiction, perception of addiction, and perception of the addicted nurse. A sample size of 32 future healthcare workers participated in Part A and 93 individuals from the general community completed Part B. A series of independent sample t-tests were run to analyze differences between groups based upon their responses to the survey questions. The results of Part A of the study revealed that there were significant differences between conditions on the Stigma Scale and Factor 4 of The Perceptions of Nursing Impairment Inventory (PNII) among future nurses. Part B of the study revealed that there were no significant differences observed between conditions when testing the general population. These findings suggest that among nursing students the gender of the nurse that engaged in opioid diversion influenced the level of stigma and awareness of peers who engage in opioid diversion that participants felt towards the nurse in the vignette. 3 Table of Contents Acknowledgements .....4 Introduction, Intellectual Context, Framework and Theory......5 Review of Literature.7 Methods......17 Results....20 Discussion..26 Conclusion.29 Reflection/Future Directions..30 References List...33 Appendices.40 4 Acknowledgements I would like to express my very great appreciation to The Ron and Laura Strain Honors College for the additional funding during the implementation of this research work. Their willingness to contribute so generously has been very much appreciated throughout the process. I would also like to express my very great appreciation to Dr. James Williams, Anthony Walters and Jennifer Kennedy for their edits on the proposal and this manuscript. Their continued willingness to help and support this project has been very much appreciated throughout this process. 5 Introduction, Intellectual Context, Framework and Theory The trend of drug addiction and the negative stigma, or mark of shame held by a group of individuals, associated with it has affected many within the United States and sparked a national health crisis (Merriam-Webster, n.d.). The abuse of various substances, such as marijuana and cocaine, has increased among the general population, with the largest increase of substance abuse represented by opioid drug abuse. (Substance Abuse and Mental Health Services Administration (SAMHSA), 2018). Opioids are defined as a natural, synthetic, or semi-synthetic chemical that interacts with opioid receptors on nerve cells in the body and brain, and reduce the intensity of pain signals and feelings of pain (Centers for Disease and Control, 2020.) A drastic escalation in access to opioids in the market has manifested through an approximate 30% rise in opioid-related visits to the Emergency Department spanning between 2016 to 2018 (VivoloKantor, Seth, Gladden, Mattson, Baldwin, Kite-Powell & Coletta, 2018). The trend of drug addiction affects healthcare professionals in the same manner as the general population, however, the onset differs (Kunyk, 2015). The need to understand and treat addictive behaviors in healthcare professionals has become increasingly relevant related to the rise in opioid availability (SAMHSA, 2018). As healthcare professionals continue to report addictive tendencies, direct negative impacts on patient care have ensued (Tanga, 2011). In response to these trends, hospitals have begun to implement new methods of controlling addiction and developed many recovery programs aimed to benefit addicted healthcare professionals (American Society of Addiction Medicine, 2011). As healthcare professionals and the general population battle addiction, a growth in negative perceptions has followed. The application of addiction-related labels prevents many individuals from seeking the help that they desire (Wakeman & Rich, 2016). Patients designated 6 as addicts are less likely to seek resources that benefit their direct care and rehabilitation (Wakeman & Rich, 2016). The increase of this stigma is recognized in particularly vulnerable populations and has spread to include altered perceptions of healthcare professionals (Mittal, Drummond, Blevins, Curran, Corrigan & Sullivan, 2013). As these stigmas are alleviated and eventually eliminated, healthcare institutions can begin to implement early identification strategies and new rehabilitation strategies to break down barriers between perception and reality (Monroe, Vandoren, Smith, Cole, & Kenaga, 2011). There is research aligned with the view that there may be increased substance abuse disorders among healthcare workers, however much of the research is outdated and exists in far fewer numbers compared to general population drug abuse research. This lack of current research necessitates the development of new data collection strategies. Vignettes, factual or fictional stories for research participants to read and consider, are a useful tool to elicit data, such as specific thoughts, feelings, and emotions from individuals as they relate to the research question. In order to understand specific perceptions of healthcare professionals and the general population on opioid addiction, two unique vignettes have been created: Vignette A and Vignette B. Distinct participants in the study from the healthcare field and the general population will be presented one of the two vignettes and then asked to respond to questions. The survey questions aim to test the exposure participants have to drug abuse as well as stigmas associated with drug abuse. Review of Literature Demographics of Addiction Substance abuse has expanded to manifest as a national health concern across the country. Substance Abuse Disorder is an addiction to a specific substance that leads to 7 dependence (American Psychiatric Association, 2013). Overall addiction trends among the general population within the United States must be considered with a specific focus of addiction placed specifically on prescription opioids and the nonmedical use of opioids. Furthermore, gender differences that exist within the current trends of addiction will be examined and analyzed. The rate of addiction within the United States has grown substantially over the years. Among individuals 12 and older, substance use has risen from 51% in 1999 to approximately 59% in 2015 (SAMHSA, 2018; Kantor, Rehm, Haas, Chan, & Giovannucci, 2015). It has been reported that in 2008-09, approximately 10.88 million individuals were addicted to marijuana; this number increased to approximately 14.5 million individuals in 2016-17, accounting for a 33.27% increase in marijuana abuse (SAMHSA, 2018). Additionally, approximately 2.01 million individuals were addicted to cocaine in 2008-09 and rose to approximately 2.03 million individuals in 2016-17; this accounts for a 0.99% increase in cocaine abuse (SAMHSA, 2018). Notably, the abuse of opioid pain relievers in 2008-09 was approximately 8.50 million individuals and that number rose to approximately 11.50 million individuals in 2016-17; this growth accounts for a 35.29% increase in opioid abuse during that period (SAMHSA, 2018). As the rate of substance abuse increases within the United States, many researchers have shifted their focus on understanding the various elements that influence addiction. Substance abuse currently affects approximately 1 in 10 Americans, or about 10.6% of the total population aged 12 and older (Ahrnsbrak, Bose, Hedden, Lipari & Park-Lee, 2016). Similarly, addiction rates among nurses mirrors the general population (Kunyk, 2015). The overall use of substances has increased among the general population; however, the greatest increase has been tied to the prescription and nonprescription abuse of opioids (Saha, 8 Kerridge, Goldstein, Chou, Zhang, Jung, Pickering Grant, 2016). Non-Medical Prescription Opioid Use (NMPOU) and Non-Medical Prescription Opioid Use Disorders (NMPOUD) have risen dramatically from 2002-2012; NMPOU had risen by 161% and NMPOUD had risen by 125% (Saha et al, 2016). The number of opioids being prescribed in hospitals rose related to increases in dosages, as well as increases in advocacy for opioid-related pain management (Saha et al., 2016). The influx of opioid use does not come without consequence. The total number of opioid-related deaths in the United States in 2014 accounted for 61% of all overdose-related deaths that year, or approximately 28,647 (Rudd, Seth, David & Scholl, 2016). In 2015, the number of opioid-related overdoses increased to 33,091, constituting approximately 63% of all 52,404 overdose-related deaths that year (Rudd et al., 2016). Opioid use has risen among the general population and the differences that exist between male and female addiction is critical. As of 2016, it has been reported that 5.2% of males and 4.0% of females abuse opioids within the United States (SAMHSA, 2016). The rate of opioid use ranks slightly higher in Indiana than the national average, with a 5.7% prevalence rate for males and 4.5% prevalence rate for females (SAMHSA, 2016). Both nationally and locally, males are reported to have a slightly higher incidence of opioid misuse than females. When examining the addictive natures of males and females, the chemical dependence concerning long-term addiction must be considered. It has been discovered that females are more likely to exhibit long-term use and escalation of drug use (Becker, McClellan, & Reed, 2016). Further, females are reported to have increased sensitivity and long-term addiction to the use of drugs and response to stress (Bobzean, DeNobrega, & Perrotti, 2014). It has been shown that increased levels of estrogen in the body made it more susceptible to producing a larger reward. Thus, if drugs are introduced into a system with a high amount of estrogen, the individual will experience a larger reward and 9 the likelihood of addiction will increase (Bobzean, DeNorbrega, & Perrotti, 2014). Stigma and negative attitudes towards women can make seeking treatment difficult for those who are caught in the addiction cycle (Becker, McClellan & Reed, 2016). Furthermore, a review of the national labor statistics finds that female registered nurses outnumber male registered nurses 9:1 (Angeles, 2018). Gender differences among the general population are important to recognize. It is also of great importance to note that the growing population of healthcare workers is not immune to addictive behavior. Prevalence in Healthcare Drug addiction has negatively affected healthcare workers and the general population alike, but how addiction manifests is unique to the healthcare setting. First, it is important to establish the prevalence of addiction among healthcare professionals, as well as the increase in the availability of opioids within the American healthcare system. This increase in opioid abuse among healthcare professionals leads to negative impacts on direct patient care. In response, institutions are pressured to develop new methods to control substances and create recovery programs for healthcare professionals with addiction. Despite their expertise in human health, healthcare professionals are not immune to addiction on the national, state, or local level. The prevalence of substance abuse among physicians and nurses has been documented: ranging from 1999 to 2018, approximately 99,367 physicians and nurses nationwide received a reprimand for substance and alcohol abuse ("Drug and Alcohol," 2018). Between 1999 and 2017, there were more than 2,450 disciplinary actions taken by the Indiana state medical and nursing disciplinary boards. Indiana ranks below 15 other states that report a higher incidence of adverse actions taken ("Drug and Alcohol," 2018). All healthcare professionals are affected by addiction, but nurses are reportedly five times more 10 likely than physicians to abuse opioids on the job, largely because of their direct access to medications (Drug and Alcohol, 2018). This is further explained by the fact that nurses who work in the hospital generally outnumber physicians 3:1 ("Drug and Alcohol," 2018). Despite the prevalence of reported cases, hospitals nationwide are not required by law to report nurses who have committed an offense, which can skew the results of data that is currently available (Drug and Alcohol, 2018). To understand how the prevalence of addiction has increased among healthcare workers, it is important to note the rise of opioids in the American healthcare system. A comparison of the opioid prescriptions within the United States and Japan revealed that physicians in the United States were far more likely to prescribe opioids to satisfy and please their patients (Onishi, Kobayashi, Dexter, Marino, Maeno & Devo, 2017). Physicians who practice in the United States are more likely to prescribe opioids for both chronic and acute pain and do so at a rate higher than Japanese doctors, leading to an influx of opioids in the market (Onishi et al., 2017). Japan, however, only prescribes opioids for cancer pain, which allows for an informative comparison of opioid prescription practices and their subsequent effects in each country (Onishi et al., 2017). With the increase in opioid abuse among healthcare professionals, there is a direct negative impact on patient care (Tanga, 2011). Nurses who are addicted tend to be less alert to their patients needs and tend to spend most of their time away from the unit (Tanga, 2011). When nurses are away from the unit, they fail to acknowledge the needs of their patients which can potentially reveal other underlying medical conditions or developments (Tanga, 2011). Nurses who use opioids in the clinical setting place their patient care and assessments on other coworkers, which leads to an increase in the workloads of other nurses (Tanga, 2011). 11 Drug diversion, or the illegal transfer of prescription medications to someone other than the patient they were prescribed to, has increased among hospital staff (Wood, 2015). With harm befalling patients, healthcare institutions have responded by developing new methods to help curb the growing rate of addiction. The interaction nurses have with medications within the hospital has been limited by the development of new medication dispensers (Vrabel, 2010). In addition, hospitals have begun to implement barcode scanning of medications to both limit medication errors and ensure that medication reaches the patient (Strudwick, Reisdorfer, Warnock, Kalia, Sulkers, Clark, & Booth, 2018). Nurses, however, still possess direct access to a patient's medication records, which means they are more aware of prescriptions and have a higher likelihood of being able to find a way to divert those medications (Ross, Berry, Smye & Goldner, 2017). As a result of this practice, nurses are under scrutiny and face backlash and repercussions for engaging in any type of drug diversion that may benefit their own drug seeking behaviors. (American Society of Addiction Medicine, 2011). Since these rates of drug diversion still exist, administrators and staff are being proactive in monitoring for drug diversion (Vrabel, 2010). Healthcare institutions have also responded to the opioid crisis among its workers by developing programs to better treat their addiction. Many institutions and state boards of nursing are adopting alternative-to-discipline approaches to handle cases when nurses become addicted (Worley, 2017). These types of approaches are better aimed to get nurses the help they need in recovery and then reintroduce them back into the workforce (Worley, 2017). Many of these programs offered are funded by the state boards of nursing and include a three- to five-year commitment to the program to avoid discreditation by the board (Worley, 2017). Additionally, it has been reported that 81% of nurses would still work with a coworker who enters recovery and 12 76% of those nurses said they do not think nurses should be subjected to disciplinary actions for addiction (Cook, 2013). Recovery programs and other help with addiction continues to be refined to better serve nurses who become addicts, but the process is limited in growth and popularity because many nurses do not utilize the available resource (Juergens, 2019). Many of the negative approaches to these programs are related to the stigma that becomes associated with addiction and the subsequent fear that members of the general public will not be accepting of them (Wakeman & Rich, 2016). Stigma and Addiction The growth of opioid use has allowed for many negative labels to be applied and as such has affected the way addicted Americans can seek care. Stigma is defined as a mark of shame or discredit and often refers to a set of negative or unfair beliefs that a group has about something, typically associated with mental illness (Merriam-Webster, n.d.) Many patients who have been given a label of addiction are unable to seek the necessary care and the resources they desire. Additionally, an inability to reach out for aid has been noted in veterans and is associated with an increase in addiction and mental illness. Likewise, this type of attitude affects healthcare workers who attempt to seek help for any addictive behaviors. By working to eliminate these labels, more focus can be placed on creating awareness and decreasing the stigmatization of addiction. A negative view of addiction nationwide has affected the way many patients seek medical attention. Stigma is often a factor that prevents individuals from seeking treatment options beneficial to their well-being (Wakeman & Rich, 2016). Reports have revealed that 44% of Americans find opioid addiction to be the result of a lack of will-power and discipline (Associated Press National Opinion Research Center, 2018). The application of these negative 13 views has affected addicted individuals with a history of intravenous (IV) use. Many of these patients seeking care are denied treatment to receive vital IV antibiotic therapy, with many hospitals citing the belief that they will use the IV catheter to inject illicit substances (Jewell, Weaver, Sgroi, Anderson, & Sayeed, 2016). Many users with this type of history will not seek treatment due to the stereotypes that have become associated with their addiction (Wakeman & Rich, 2016). An inability to reach out for help when addiction is prevalent makes it difficult for many addicted Americans to seek out necessary resources (Johnson, 2018). One example of this negative association was reported with Post-Traumatic Stress Disorder in veterans. Researchers discovered that many veterans were unlikely to seek help for their mental disorder because they did not want to discuss the illness or perceived attitudes they felt others held against them (Mittal et al, 2013). This type of self-stigma was affecting the way veterans were seeking treatment; they felt other veterans suffering from the same disorder best understood what they were experiencing (Mittal et al., 2013). The use of such labels as mental illness will reduce the ability of the user to seek help, especially when they are unable to gain access to the resources they need (Jewell et al., 2013). The negative association of stigma and mental illness affects the manner in which veterans seek treatment, but these same types of attitudes affect how healthcare professionals seek help and report suspected individuals as well. Individuals within the clinical setting hesitate to seek help because of the stigma and fear of what will happen to them in both their professional and personal lives (DesRoches, Rao, Fromson, Birnbaum, Iezzoni, Vogeli & Campbell, 2010). The number one reason that physicians failed to report impaired colleagues occurred when the label of an addiction was associated (DesRoches et al., 2010). It has been reported that 14 approximately 19% of healthcare workers failed to report an impaired colleague because they believed that someone else within their profession was handling the situation (DesRoches et al., 2010). Additionally, 12% of healthcare workers failed to report impaired colleagues for fear of retribution and 10% of physicians failed to report impaired colleagues because they felt it was not their responsibility (DesRoches et al., 2010). This lack of intuition to report impaired healthcare workers contributes to unsafe environments for their patients and is detrimental to their own ability to seek necessary resources (DesRoches et al., 2010) Since hospital workers are not immune to the negative associations of addiction, many institutions are working to eliminate the necessary associations of addiction among their staff. Education of staff is a vital step toward early identification of addictive behaviors in users (Worley, 2017). These early identification strategies are being implemented at the nursing school level and work directly with students who are or are suspected of being addicted (Monroe et al, 2011). As a result of these strategies, many hospitals can reform their policies to accept suspected users and break down the barriers that prevent them from seeking proper care (Wakeman & Rich, 2016). The types of labels used, such as addict, need to be eliminated to promote awareness and divert more attention to the addiction process while also decreasing the stigmatization that becomes associated with it (Goodyear, Haass-Koffler, & Chayanne, 2019). The Use of Vignettes and the Proposed Study A vignette is a carefully constructed description of a person, place, or event that is written in such a way that thoughts and feelings can be evoked within a reader (Atzmlle & Steiner, 2010). Additionally, the use of vignettes is a way to briefly stimulate the thoughts and feelings of the participants in a way that does not directly influence their opinion on a subject (Hughes & Huby, 2004). Because vignettes are used frequently in research, it is imperative to look at the 15 validity surrounding the use of vignettes in experimental methodological design. A vignette is constructed by asking specifically what resources, expert advice, and review was utilized to ensure that vignettes contained the validity necessary to meet the end goal: what thoughts and feelings are being elicited (Gould, 1996). To construct a vignette capable of evoking the necessary thoughts and feelings, all goals must be met in construction. By maintaining validity and a legitimate construction, the vignette becomes a valuable tool that can be used to invoke thoughts and feelings in a participant to ask questions surrounding nearly any area of interest (Hughes & Huby, 2004). To better understand how addiction has influenced the perception by both the general population and healthcare professionals, researchers could utilize vignettes. By utilizing these data collection methods, researchers can better understand where current perception lies. The use of vignettes in this proposed study will test the participants perceptions of drug use among nurses and stigma associated with it. Through the careful construction of two nearly identical vignettes, the reader will be presented with either a male nurse (Vignette A) or a female nurse (Vignette B) and be asked to respond to questions. Careful consideration was placed in the creation of the vignettes to meet the necessary goals of the proposed study. Stereotypes become associated with the difference between male and female nurses. Many non-hospitalized patients view the female nurse with more positive feminine qualities and view the male nurse with more negative qualities (Aranda, CastilloMayn & Montes-Berges, 2015). By utilizing the social dominance orientation, a scale testing feminine qualities in a population, it was reported that individuals scoring high on the scale were more likely to have greater stereotypes towards male and female nurses (Aranda, Castillo-Mayn & Montes-Berges, 2015). Therefore, the results of these studies further the need to understand 16 the view of the general population and other healthcare professionals hold against addicted nurses. The introduction of two unique populations allows for the creation of a study to focus on perceptions of two distinct groups. The proposed study aims to understand how the perceptions each population has concerning opioid addiction among nurses may vary depending on the gender of the nurse. Two versions (male and female subject) of a vignette will be utilized to tell the story of a nurse who has fallen victim to opioid addiction. The first vignette will involve John, a male nurse (see Appendix A) and the second vignette will involve Sarah, a female nurse (see Appendix B). Besides sex and name of the nurse, everything else in the vignette is identical. The participants in the proposed study will read one of the two vignettes and be asked to respond to multiple questions from four different surveys: The Exposure to Drug Users Index, The Stigma of Drug Users Scale, The Drug Use Stigmatization Scale, and The Perceptions of Nursing Impairment Inventory (PNII). The results of these surveys will be subject to a series of ttests which will test the results for significance. 17 Methods Participants The current study was composed of two parts: Part A measured perceptions of an addicted nurse featured in a vignette among undergraduate students at the University of Indianapolis School of Nursing and Part B was an identical study using participants who are not healthcare workers recruited through Amazons Mechanical Turk (mTurk), an online recruit-forwork service. The G*Power calculator indicated that a sample size of 102 participants should be recruited for each part of the study to reach a power of 0.8, with an effect size of 0.5, and a significance level set at .05 (see Appendix C). Materials Data was collected utilizing a quantitative experimental research design method to analyze how stereotypes associated with gender and nursing can influence individuals perceptions of male and female nurses who are struggling with addiction. Qualtrics, an online survey platform, was used to conduct the experiment and to collect and store raw data. Participants were able to access the study through a link that was included in an email they received (nursing students) or through mTurk (non-healthcare workers). The vignettes, written in 3rd person perspective, were created compiling the results of several articles written on the legal and ethical considerations associated with reporting nurses who are thought to be impaired while working (see Appendixes A and B). The following scales were utilized to measure the perception of participants: 1. The Exposure to Drug Users Index (see Appendix D) was developed to assess exposure to working or engaging with someone who has previously used an illicit drug (Palamar & Klang, 2011). The index allowed for the researcher to fill in what substance was being 18 referenced to. For the current experiments, non-medical opioid use was filled in. The measure allows participants to respond to each question with "Yes," "No," or "Not Sure" (Palamar & Klang, 2011). 2. The PNII (see Appendix E) was developed to analyze the perceptions of nurses and nursing supervisors on drug addiction in the workplace (Hendrix, Sabritt, McDaniel, & Field, 1987). Responses to each question were scored on a 4-point Likert-type scale ranging from 1 (strongly agree) to 4 (strongly disagree). Several distinct factors related to impairment and perception of illness were analyzed through the questions included in the PNII (Hendrix et al, 1987). The validity and reliability of this scale were determined through a pilot study in which a Cronbach Alpha of .82 was achieved. The scale was determined to fit the criteria needed to achieve an accurate response rate when implemented in a clinical setting (Hendrix et al, 1987). 3. The Stigma of Drug Users Scale (see Appendix F) has previously been used to assess clinicians views towards opioid drug users (Palamar & Klang, 2011). Responses to each question were scored on a 5-point Likert-type scale ranging from 1 (strongly agree) to 5 (strongly disagree). 4. The Drug Use Stigmatization Scale (see appendix G) was used to assess an individual's views on using opioids (Palamar & Klang, 2011). Responses to each question were scored on a 5-point Likert-type scale ranging from 1 (strongly agree) to 5 (strongly disagree). The Stigma of Drug Users Scale and Drug Use Stigmatization Scale have been tested for validity in assessing the relationship between stigma and stigmatization of drug users (Palamar & 19 Klang, 2011). Upon completion of testing, the scales were narrowed to their respective forms as they currently exist (Palamar & Klang, 2011) Design and Procedure Part A. All participants were told that the study was optional, completely anonymous and that they could quit the study at any point. On the first page of the Qualtrics survey, participants were asked to read the informed consent form. Consent was obtained when they clicked to continue, which allowed the participant to begin the study. Qualtrics then randomly assigned participants to read one of the two vignettes (one featuring a male nurse and the other featuring a female nurse) and then asked them to consider the vignette as they responded to several questions. The survey format was divided into two parts. Part I of the survey included a randomized combination of questions from the Stigma of Drug Users Scale, The Drug Use Stigmatization Scale and the Perceptions of Nursing Impairment Inventory. There were a handful of simple questions related to the vignette itself to serve as attention checks throughout Part I. This helped to ensure that the participant actually read the vignette and was paying attention to the questions. Part II collected information using the Exposure to Drug Users Index. Finally, participants who completed the survey were asked to submit their responses and were thanked for their time and participation. Participants through the University of Indianapolis School of Nursing then had the option to enter for a drawing (through a separate form, unlinked to their study responses) for one of five $50 Amazon gift cards. The entire survey took approximately 20-25 minutes to complete. Part B. Participants recruited through mTurk went through the same steps as the University of Indianapolis School of Nursing participants (see above). However, they were 20 recruited through mTurk and at the end of the survey were given a code to be paid $1.00 as compensation for completion of the survey. Results Part A: University of Indianapolis School of Nursing Participants Stigma Scale. An independent samples t-test was conducted to compare participants perceptions of drug use depending on whether a male nurse or a female nurse was the subject of the vignette. There was a significant difference between the Stigma Scale scores for the male nurse (M = 34.75, SD = 3.59) and the female nurse (M = 31.94, SD = 7.33) conditions; t(30) =1.38, p = 0.02. These results reveal that among nursing students, the vignette featuring the male nurse led to significantly higher stigma levels than the vignette featuring the female nurse. Drug Use Stigmatization Scale. An independent samples t-test was conducted to compare the perception of drug use stigmatization depending on whether a male nurse or a female nurse was the subject of a vignette. There was not a significant difference between the Drug Use Stigmatization Scale scores for the male nurse (M = 14.31, SD = 3.61) and the female nurse (M = 16.56, SD = 4.38) conditions; t(30) =-1.59, p = 0.49. These results suggest that the sex of the nurse in the vignette had no effect on the stigma attributed to drug use. Factor 1: Disciplinary Orientation. An independent samples t-test was conducted to compare the disciplinary orientation depending on whether a male nurse or a female nurse was the subject of a vignette. There was not a significant difference between the Factor 1: Disciplinary Orientation scores for the male nurse (M = 17.44, SD = 2.36) and the female nurse (M = 18.44, SD = 1.41) conditions; t(30) =-1.45, p = 0.07. These results suggest that the sex of the nurse in the vignette had no effect on the disciplinary orientation when the nurse was caught abusing drugs. 21 Factor 2: Orientation to Helping Responsibility. An independent samples t-test was conducted to compare the orientation to helping responsibly depending on whether a male nurse or a female nurse was the subject of a vignette. There was not a significant difference between the Factor 2: Orientation to Helping Responsibility scores for the male nurse (M = 16.00, SD = 1.51) and the female nurse (M = 16.25, SD = 1.44) conditions; t(29) =-0.47, p = 0.89. These results suggest that the sex of the nurse in the vignette had no effect on the orientation to helping responsibly when the nurse was caught abusing drugs. Factor 3: Distinctiveness to Nursing. An independent samples t-test was conducted to compare the distinctiveness to nursing depending on whether a male nurse or a female nurse was the subject of a vignette. There was not a significant difference between the Factor 2: Distinctiveness to Nursing scores for the male nurse (M = 10.38, SD = 1.09) and female the nurse (M = 11.19, SD = 1.38) conditions; t(30) =-1.85, p = 0.49. These results suggest that the sex of the nurse in the vignette had no effect on the distinctiveness to nursing when the nurse was caught abusing drugs. Factor 4: Orientation to the Need to Know. An independent samples t-test was conducted to compare the orientation to the need to know about drug abuse depending on whether a male nurse or a female nurse was the subject of a vignette. There was a significant difference between the Factor 4: Orientation to the Need to Know scores for the male nurse (M = 10.06, SD = 0.85) and the female nurse (M = 9.75, SD = 1.48) conditions; t(30) =0.73, p = 0.01. These results suggest that among future nurses, the vignette featuring the male nurse led to a significantly higher level of orientation to suspected drug abuse as compared to the vignette featuring the female nurse. This factor is a measure of the amount of awareness to peers who engage in diversion and other drug related behavior. 22 Factor 5: Treatability Orientation. An independent samples t-test was conducted to compare the treatability orientation depending on whether a male nurse or a female nurse was the subject of a vignette. There was not a significant difference between the Factor 5: Treatability Orientation scores for the male nurse (M = 8.81, SD = 0.66) and the female nurse (M = 8.63, SD = 1.09) conditions; t(30) =0.59, p = 0.09. These results suggest that the sex of the nurse in the vignette had no effect on the treatability orientation when the nurse was caught abusing drugs. Factor 6: Orientation to Nurses Ability to Help. An independent samples t-test was conducted to compare the orientation to the nurses ability to help depending on whether a male nurse or a female nurse was the subject of a vignette. There was not a significant difference between the Factor 6: Orientation to the Nurses Ability to Help scores for the male nurse (M = 11.00, SD = 1.37) and the female nurse (M = 10.44, SD = 1.26) conditions; t(30) =1.20, p = 0.98. These results suggest that the sex of the nurse in the vignette had no effect on the nurses ability to help when the nurse was caught abusing drugs. Factor 7: Perception of Prevalence. An independent samples t-test was conducted to compare the perception of prevalence depending on whether a male nurse or a female nurse was the subject of a vignette. There was not a significant difference between the Factor 7: Perception of Prevalence scores for the male nurse (M = 4.13, SD = 0.81) and the female nurse (M = 3.75, SD = 0.77) conditions; t(30) =1.34, p = 0.80. These results suggest that the sex of the nurse in the vignette had no effect on the perception of prevalence when the nurse was caught abusing drugs. Factor 8: Orientation to Impairment as Illness. An independent samples t-test was conducted to compare the orientation to impairment as an illness depending on whether a male nurse or a female nurse was the subject of a vignette. There was not a significant difference between the Factor 8: Orientation to Impairment as Illness scores for the male nurse (M = 4.33, 23 SD = 0.98) and the female nurse (M = 4.75, SD = 0.77) conditions; t(29) =-1.32, p = 0.40. These results suggest that the sex of the nurse in the vignette had no effect on the orientation to impairment as an illness when the nurse was caught abusing drugs. Factor 9: Perception of Recognizability. An independent samples t-test was conducted to compare the perception of recognizability depending on whether a male nurse or a female nurse was the subject of a vignette. There was not a significant difference between the Factor 9: Perception of Recognizability scores for the male nurse (M = 5.06, SD = 1.00) and the female nurse (M = 4.44, SD = 0.73) conditions; t(30) =2.02, p = 0.88. These results suggest that the sex of the nurse in the vignette had no effect on the perception of recognizability when the nurse was caught abusing drugs. Part B: mTurk Participants Stigma Scale. An independent samples t-test was conducted to compare participants perceptions of drug use depending on whether a male nurse or a female nurse was the subject of the vignette. There was not a significant difference between the Stigma Scale scores for the male nurse (M = 34.51, SD = 5.65) and the female nurse (M = 33.14, SD = 5.89) conditions; t(90) =1.13, p = 0.91. These results suggest that the sex of the nurse in the vignette does not have an effect on the stigma attributed to drug use. Drug Use Stigmatization Scale. An independent samples t-test was conducted to compare the perception of drug use depending on whether a male nurse or a female nurse was the subject of a vignette. There was not a significant difference between the Drug Use Stigmatization Scale scores for the male nurse (M = 16.07, SD = 5.97) and the female nurse (M = 15.53, SD = 4.78) conditions; t(91) =.48, p = 0.21. These results suggest that the sex of the nurse in the vignette had no effect on the stigma attributed to drug use. 24 Factor 1: Disciplinary Orientation. An independent samples t-test was conducted to compare the disciplinary orientation depending on whether a male nurse or a female nurse was the subject of a vignette. There was not a significant difference between the Factor 1: Disciplinary Orientation scores for the male nurse (M = 17.95, SD = 2.79) and the female nurse (M = 17.53, SD = 3.32) conditions; t(91) =0.66, p = 0.11. These results suggest that the sex of the nurse in the vignette had no effect on the disciplinary orientation when the nurse was caught abusing drugs. Factor 2: Orientation to Helping Responsibility. An independent samples t-test was conducted to compare the orientation to helping responsibly depending on whether a male nurse or a female nurse was the subject of a vignette. There was not a significant difference between the Factor 2: Orientation to Helping Responsibility scores for the male nurse (M = 15.57, SD = 1.92) and the female nurse (M = 15.96, SD = 1.84) conditions; t(91) =-1.00, p = 0.35. These results suggest that the sex of the nurse in the vignette had no effect on the orientation to helping responsibly when the nurse was caught abusing drugs. Factor 3: Distinctiveness to Nursing An independent samples t-test was conducted to compare the distinctiveness to nursing depending on whether a male nurse or a female nurse was the subject of a vignette. There was not a significant difference between the Factor 2: Distinctiveness to Nursing scores for the male nurse (M = 10.35, SD = 1.49) and female the nurse (M = 10.41, SD = 1.62) conditions; t(90) =-0.18, p = 0.66. These results suggest that the sex of the nurse in the vignette had no effect on the distinctiveness to nursing when the nurse was caught abusing drugs. Factor 4: Orientation to the Need to Know. An independent samples t-test was conducted to compare the orientation to the need to know depending on whether a male nurse or 25 a female nurse was the subject of a vignette. There was not a significant difference between the Factor 4: Orientation to the Need to Know scores for the male nurse (M = 9.26, SD = 1.20) and the female nurse (M = 9.35, SD = 1.15) conditions; t(90) =-0.37, p = 0.63. These results suggest that the sex of the nurse in the vignette does not have an effect on the orientation to the need to know when the nurse was caught abusing drugs. Factor 5: Treatability Orientation. An independent samples t-test was conducted to compare the treatability orientation depending on whether a male nurse or a female nurse was the subject of a vignette. There was not a significant difference between the Factor 5: Treatability Orientation scores for the male nurse (M = 8.39, SD = 1.10) and the female nurse (M = 8.39, SD = 0.98) conditions; t(91) =-0.01, p = 0.39. These results suggest that the sex of the nurse in the vignette had no effect on the treatability orientation when the nurse was caught abusing drugs. Factor 6: Orientation to Nurses Ability to Help. An independent samples t-test was conducted to compare the orientation to the nurses ability to help depending on whether a male nurse or a female nurse was the subject of a vignette. There was not a significant difference between the Factor 6: Orientation to the Nurses Ability to Help scores for the male nurse (M = 10.41, SD = 1.44) and the female nurse (M = 10.48, SD = 1.37) conditions; t(90) =-0.24, p = 0.68. These results suggest that the sex of the nurse in the vignette had no effect on the nurses ability to help when the nurse was caught abusing drugs. Factor 7: Perception of Prevalence. An independent samples t-test was conducted to compare the perception of prevalence depending on whether a male nurse or a female nurse was the subject of a vignette. There was not a significant difference between the Factor 7: Perception of Prevalence scores for the male nurse (M = 4.70, SD = 1.21) and the female nurse (M = 4.80, SD = 1.02) conditions; t(91) =-0.40, p = 0.13. These results suggest that the sex of the nurse in 26 the vignette had no effect on the perception of prevalence when the nurse was caught abusing drugs. Factor 8: Orientation to Impairment as Illness. An independent samples t-test was conducted to compare the orientation to impairment as an illness depending on whether a male nurse or a female nurse was the subject of a vignette. There was not a significant difference between the Factor 8: Orientation to Impairment as Illness scores for the male nurse (M = 5.29, SD = 0.97) and the female nurse (M = 4.96, SD = 0.82) conditions; t(91) =1.78, p = 0.08. These results suggest that the sex of the nurse in the vignette had no effect on the orientation to impairment as an illness when the nurse was caught abusing drugs. Factor 9: Perception of Recognizability. An independent samples t-test was conducted to compare the perception of recognizability depending on whether a male nurse or a female nurse was the subject of a vignette. There was not a significant difference between the Factor 9: Perception of Recognizability scores for the male nurse (M = 5.14, SD = 0.90) and the female nurse (M = 5.12, SD = 0.93) conditions; t(91) =0.07, p = 0.77. These results suggest that the sex of the nurse in the vignette had no effect on the perception of recognizability when the nurse was caught abusing drugs. Discussion The purpose of this study was to examine the impact that the gender of nurses may have on healthcare worker and non-healthcare worker perceptions of opioid abuse among nurses on the job. Two vignettes were created in which either a male or female nurse was the subject of opioid diversion. The study consisted of two parts: Part A examined the perception of nursing students enrolled at the University of Indianapolis School of Nursing while Part B examined the perception of non-healthcare workers in the general public through Amazons mTurk. 27 Part A of the current study revealed that among the University of Indianapolis School of Nursing students, there were some significant differences observed depending on whether the nurse in the vignette was male or female. Findings from the Stigma Scale suggest that the gender of the nurse that engaged in opioid diversion influenced the level of stigma participants felt towards the nurse in the vignette. Findings from Factor 4 of the PNII suggest that the gender of the nurse that engaged in diversion influenced the awareness of the peers to this behavior. While there were significant differences observed between conditions for the Stigma Scale and Factor 4 of the PNII among the University of Indianapolis nursing students, there were not enough participants to feel fully confident in these results. If it had been possible to recruit the appropriate number of participants, there is a chance that these results may have been more in line with what was observed among non-healthcare workers in Part B of the study. However, if this remained significant upon collection of the ideal number of participants, the results that were seen in Part A of the study may be explained by the age of the participants in the study and the types of exposure that they may have had to opioid addiction. The results that were seen with Part A of the study are surprising as they do not align with the recent research that has been aligned with gender stereotypes among younger generations. The University of Indianapolis School of Nursing Students are within a generation of younger minds where gender, equity, and role definition within society are prevalent (Schroth, 2019). Based on this study, the results of the study should have been more aligned with being open-minded and containing less stigma towards the gender of the nurse (Schroth, 2019). When the gender of the nurse was changed in relation to the situation, the stigma towards the nurse changed. This may be explained by the gender stereotypes that tend to be associated with various occupations. Male nurses are viewed as a low stereotyped profession (less males in the field) and females are 28 viewed in a highly stereotyped profession (more females in the field) (Dawar & Anand, 2018). Again, the results that were seen with Part A of the study do not align with what is being seen in current literature and research. The difference in findings may indicate that the smaller sample size is to blame for the results seen. Future exploration of male and female job stereotypes is worth conducting to determine whether the results are consistent with findings of this study and whether they are in line with current research. Based on the lack of significant results in Part B of the study, it seems that at the very least, non-healthcare workers appear to not be affected by the gender of the nurse who is diverting drugs. The results that were seen are not consistent with research that has been done in the past which has found that males were more likely to be reported for addictive tendencies or opioid misuse (Silver & Hur, 2020). The results that were seen with Part B suggest that drug diversion on the job may be a significant enough action that the sex of the individual does not make a difference in the perception of diversion among individuals. Further studies would need to reexamine this finding to determine if the results gathered from Part B are deviating from the current research or if there were limitations that were encountered. Limitations One of the limitations to the current study could be the population that was surveyed. The data collected through mTurk could be skewed as a result of mTurk workers attempting to make money by moving through surveys like these as quickly as possible, leading to a lack of full consideration of the vignette and the questions. In past research, mTurk participants have been shown to attempt to guess what the researcher wishes to find in the study and focuses less on the questions themselves (MacInnis, Boss, & Bourdage, 2020). Even so, many mTurk participants may view this position as more a part of their full-time job and care less about the actual results 29 (Almaatouq, Krafft, Dunham, Rand, & Pentland, 2020). Participants in this study were paid $1.00 for completion of the survey; however, a larger payment may have been needed for a to ensure quality responses. Workers through mTurk may need to be compensated or have procedures explained to a higher degree in an effort to reduce any sort of in-group bias that may exist when results are collected (Almaatouq, Krafft, Dunham, Rand, & Pentland, 2020). To expand these efforts, future researchers may need to look at the platform being used and to make the necessary adjustments to better encompass the results needed from the workers, especially when using mTurk for data collection. One the potential limitation with the results were the length of the survey in general. The total predicted time to complete was approximately 25-30 minutes. Such a significant time could deter the participant from remaining focused throughout the course of the study. Attention checks were included but they were broad, more specific checks may be needed to determine the focus of the participant throughout the entirety of the study. Another potential limitation was an influx of information that was given in the vignette. The vignette was not pilot tested, which indicated that there was no way to determine if the information provided was beneficial or harmful. Pilot testing the vignette would have revealed any flaws and limitations that could have hindered results when answering the survey questions. In addition, one of the survey questions from the stigma of drug users index was left off when copying over to the survey platform. While this was one question, there may have been an impact in the results of the survey answers. Conclusion The results of this study revealed significance in the stigma of drug use and the willingness to intervene when misuse was suspected among nursing students in Part A. The results from Part B found no significance and suggest that the perception related to drug use remains even when the 30 gender of the nurse is changed in the vignette. The access to medication within the hospital has decreased significantly in recent years. Exposure to medication and practices to divert medication largely remain shadowed in teaching current nurses and future nurses. Continuing to place a focus on diversion and access will ensure that these detrimental practices are decreased. By promoting rehabilitation and exposure, the next generation of healthcare providers can be educated and understand what will happen if engaged in such practices. Reflection and Future Directions One area that was changed during the study was the switch from using members within the Community Health Network to using members within the University of Indianapolis School of Nursing. This was a change that was approved through the Honors College with a submission of a justification for change document (see appendix H). It is important to note that Appendix H only includes the justification for the change, the methods section that was included in the original document is now reflected in the methods section of this manuscript. One of the biggest reasons for this change was how slow the process was in gaining approval to conduct the study through Community Health Network. Several colleagues at the University of Indianapolis had voiced their concerns and worry for completion of the project if we continued to seek Community employee responses. Since the University of Indianapolis had a large nursing school, it was determined that gathering data through them would be more sensible and therefore, the change was proposed and approved. One of the significant challenges that was faced when collecting data for this study was the limited number of participants that were recruited for Part A of the study. While 32 participants were recruited, it was difficult in spreading the word and recruiting the desired number of students to participate in the study. This may have been less of an issue if nursing 31 students were the population of interest from the start of the experiment. Throughout the conduction of the study, several emails were sent to the nursing students in the University of Indianapolis School of Nursing and several faculty members reached out who were willing to spread the word to their classes. Despite these continued efforts, we were still only able to recruit 32 participants. If more time to collect data was permitted, more participants could have been recruited which would have played an effect and could have potentially led to more areas of significance. A future project would need to be shifted to focus more on the impact that drug diversion and opioid addiction could have when caring for patients. A much greater focus should be placed on the amount of access and the wasting practices that are implemented in the hospital. Many times, nurses are exposed directly to the medication and have the moral obligation to waste the medication in the presence of another nurse, however, this may not always be the case. Hospital managers and nursing schools in general, should place a greater focus on ensuring that what is taught and what is implemented in practice are in line. One other future project can look at the amount of exposure nursing students have to treatment and reporting addiction within their institution and how that may affect perceptions of addiction. Nursing students and college students in general may receive input from their university on exposure to drug use and the education received regarding drug abuse. Many college campuses have received and developed tailored programs that focus on specific genders and how information is tailored and presented regarding drug abuse (Wolfson, Stinson & Poole, 2020). The types of programs that have been implement on college campuses work to decrease the negative side-effects of seeking treatment and better integrate gender and equality into making rational decisions (Wolfson, Stinson & Poole, 2020). These views and practices can 32 influence the manner in which the individual perceives the addict and how they appropriately respond to the possibility of intervening on a situation. Future vignettes used for this type of study may benefit from being pilot tested to determine the quality of the information and whether it would impact the decisions made when compared to the survey questions. Future studies in this area should continue to focus on the impact that diversion and addiction has on new and future nurses. Access to medication has become more limited, but the exposure within the healthcare system is still great. 33 References Almaatouq, A., Krafft, P., Dunham, P., Rand, D., & Pentland, A. (2020). Turkers of the world unite: Multilevel in-group bias among crowdworkers on Amazon Mechanical Turk. Social Psychological and Personality Science, 11(2), 151-159. doi:10.1177/194855061983002 Ahrnsbrak, R., Bose, J., Hedden, S. L., Lipari, R. N., & Park-Lee, E. (2017). Key substance use and mental health indicators in the United States: Results from the 2016 National Survey on Drug Use and Health. Retrieved from https://www.samhsa.gov/data/report/keysubstance-use-and-mental-health-indicators-united-states-results-2016-national-survey American Psychiatric Association. (2013). Diagnostic and statistical manual of mental disorders (5th ed.). 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(2019). Are you ready for Gen Z in the workplace? California Management Review, 00(0). 1-14. doi: 10.1177/0008125619841006 Silver, E. R., & Hur, C. (2020). Gender differences in prescription opioid use and misuse: Implications for mens health and the opioid epidemic. Preventive Medicine, 131. doi: https://doi.org/10.1016/j.ypmed.2019.105946 Strudwick, G., Reisdorfer, E., Warnock, C., Kalia, K., Sulkers, H., Clark, C., & Booth, R. (2018). Factors associated with barcode medication administration technology that contribute to patient safety. Journal of Nursing Care Quality, 33(1), 79-85. doi: 10.1097/NCQ. 0000000000000270 Tanga, H. Y. (2011). Nurse drug diversion and nursing leaders' responsibilities: Legal, regulatory, ethical, humanistic, and practical considerations. JONA,13(1), 13-16. Retrieved from https://pdfs.semanticscholar.org/45c7/f0d6ec9c26e3407a17eabadd42cf7c7afd48.pdf. 39 Vivolo-Kantor, A. M., Seth, P., Gladden, M., Mattson, C. L., Baldwin, G. T., Kite-Powell, A., Coletta, M. A. (2018) Vital signs: Trends in emergency department visits for suspected opioid overdoses United States. Morb Mortal Wkly Rep, 67, 279-285. doi: http://dx.doi.org/10.15585/mmwr.mm6709e1 Vrabel, R. (2010). Identifying and dealing with drug diversion. Health Management Technology, 31(12), 18-19. Retrieved from https://europepmc.org/abstract/med/21197899. Wakeman, S. E., & Rich, J. D. (2016). Barriers to post-acute care for patients on opioid agonist therapy; an example of systematic stigmatization of addiction. J Gen Intern Med, 32(1), 17-19. doi:10.1007/s11606-016-3799-7 Wolfson, L., Stinson, J., & Poole, N. (2020). Gender informed or gender ignored? Opportunities for gender transformative approaches in brief alcohol interventions on college campuses. International Journal of Environmental Research and Public Health, 17. 1-17. doi:10.3390/ijerph17020396 Wood, D. (2015). Drug diversion. Australian Prescriber, 38(5). 164-166. doi: 10.18773/austprescr.2015.058 Worley, J., (2017). Nurses with substance use disorders: Where we are and what needs to be done. Journal of Psychosocial Nursing, 55(12), 11-14. doi:10.3928/02793695-2017111302 40 Appendix A John is a nurse on a Medical-Surgical unit with a significant shortage of staff. This shortage has put a strain on nurses and has required him to pick up extra shifts and work overtime. The increasing demand for high-quality patient care combined with increased workload has created a high-stress environment. Johns coworkers have begun to take notice of his behavior. Specifically, they have noticed changes in Johns rounding accuracy and decreased quality of patient care in Johns day-to-day interactions with patients. Patient complaints have increased when interacting with John. He also seems to be away from the floor more often than usual. When confronted by coworkers about increased breaks, John says that he needs breaks from the high-stress environment. Recently, there has been a miscount of controlled substances in the automatic medication dispenser. At first, the miscount was subtle, but in recent weeks it has increased to several vials of an opioid analgesic every week. Floor managers have investigated the recent issues and have noticed a pattern of missing medication when John is working. The nursing supervisor has been made aware of the situation and is investigating diverted medication records. After several weeks of odd behavior, John is found acting strange and secretive in the bathroom by a coworker, who is aware of the diverted medication. When asked if everything is okay, John brushes them off and rushes out of the bathroom. The coworker, after noticing this behavior, is suspicious that John may be the one diverting and using the medication. A floor meeting is called to discuss the recently discovered diversion and the protocol for reporting an impaired colleague. Supervisors discuss the treatment options that are available for healthcare workers who are addicted and using controlled substances while working. They clarify the implications of licensing and reprimands if they report themselves before being discovered of diverting medication. Many programs are in place to aide an impaired nurse if they are diverting medication and addicted. A coworker who has noticed the erratic and strange behavior of John reports them to the nursing supervisor several days after the floor meeting. They are concerned about the safety of patients and were worried that if they failed to report their colleague that they would be reprimanded. John is called in for a meeting with the nursing supervisor and he confesses to diverting controlled opioid analgesics from the medication room for personal use. He admits that he has been addicted to opioid analgesics for the past year but has only recently begun to use while on the job. He is recommended to begin treatment immediately at an inpatient rehabilitation facility. Upon successful completion, he will be closely monitored and reinstated back into his role on the floor with a restriction to workload and the number of shifts worked. All underlined words will be adjusted depending on which condition the participant is assigned to. John and Sarah will be used as the male and female forenames, (Macrae et al., 2002). 41 Appendix B Sarah is a nurse on a Medical-Surgical unit with a significant shortage of staff. This shortage has put a strain on nurses and has required her to pick up extra shifts and work overtime. The increasing demand for high-quality patient care combined with increased workload has created a high-stress environment. Sarahs coworkers have begun to take notice of her behavior. Specifically, they have noticed changes in Sarahs rounding accuracy and decreased quality of patient care in Sarahs day-to-day interactions with patients. Patient complaints have increased when interacting with Sarah. She also seems to be away from the floor more often than usual. When confronted by coworkers about increased breaks, Sarah says that she needs breaks from the high-stress environment. Recently, there has been a miscount of controlled substances in the automatic medication dispenser. At first, the miscount was subtle, but in recent weeks it has increased to several vials of an opioid analgesic every week. Floor managers have investigated the recent issues and have noticed a pattern of missing medication when Sarah is working. The nursing supervisor has been made aware of the situation and is investigating diverted medication records. After several weeks of odd behavior, Sarah is found acting strange and secretive in the bathroom by a coworker, who is aware of the diverted medication. When asked if everything is okay, Sarah brushes them off and rushes out of the bathroom. The coworker, after noticing this behavior, is suspicious that Sarah may be the one diverting and using the medication. A floor meeting is called to discuss the recently discovered diversion and the protocol for reporting an impaired colleague. Supervisors discuss the treatment options that are available for healthcare workers who are addicted and using controlled substances while working. They clarify the implications of licensing and reprimands if they report themselves before being discovered of diverting medication. Many programs are in place to aide an impaired nurse if they are diverting medication and addicted. A coworker who has noticed the erratic and strange behavior of Sarah reports them to the nursing supervisor several days after the floor meeting. They are concerned about the safety of patients and were worried that if they failed to report their colleague that they would be reprimanded. Sarah is called in for a meeting with the nursing supervisor and he confesses to diverting controlled opioid analgesics from the medication room for personal use. She admits that she has been addicted to opioid analgesics for the past year but has only recently begun to use while on the job. She is recommended to begin treatment immediately at an inpatient rehabilitation facility. Upon successful completion, she will be closely monitored and reinstated back into her role on the floor with a restriction to workload and the number of shifts worked. 42 Appendix C 43 Appendix D Exposure to Drug Users Index (3-Point Likert Scale: Yes, No, Not Sure) 1. I have observed people who use opioids frequently. 2. I have worked with a person that uses opioids. 3. I have a friend who uses opioids. 4. I have been in class with a person that uses opioids. 5. I have a family member or relative who uses opioids. 6. I have lived with a person who uses opioids. 7. People in my neighborhood use opioids. 44 Appendix E PNII (4-Point Likert Scale: Strongly Disagree, Disagree, Agree, Strongly Agree) A. Factor 1: Disciplinary Orientation 1. As a rule, impaired nurses should not be allowed to work as registered nurses until they have successfully completed a treatment program. 2. When a nursing supervisor has concrete evidence that a nurse is impaired, the supervisor has a responsibility to suspend that individual pending investigation of the charges. 3. While receiving treatment, most impaired nurses are capable of continuing to work as registered nurses. Palamar, J., & Klang, M.V. (2011). Exposure to Drug Users Index. doi: 10.3109/10826084.2011.596606 4. When a nursing supervisor has concrete evidence that a nurse is impaired, the supervisor has a responsibility to dismiss that individual immediately and report the case to the State Board of Nursing. 5. In most cases, public safety should require that impaired nurses' licenses be revoked. 6. For purposes of public protection, the State Board of Nursing should continue to publish the names of all nurses found to be impaired. B. Factor 2: Orientation to Helping Responsibility Within the Profession 1. The State Board of Nursing's responsibility should include offering the impaired nurse referral to sources of assistance. 2. The State Board of Nursing should provide resources to support research on the prevention and treatment of impairment. 3. Major health care agencies should be required to provide employee assistance programs which could serve the impaired nurse. 4. When a nurse has reason to believe that a co-worker is impaired, he/she has a responsibility to help that person receive assistance. 5. The State Board of Nursing has a responsibility to provide nurses suspected of impairment with specific information about their legal and due process rights in all disciplinary procedures. C. Factor 3: Distinctiveness to Nursing. 1. In most cases, the problems of impaired nurses stem from difficulties which those individuals has already encountered before becoming nurses. 2. The problems of impaired nurses are often a reflection of stressful situations on the job. 3. Becoming impaired is something that could happen to any nurse. 4. Problems of impaired nurses are basically not very different than those of other individuals with substance abuse or emotional problems. D. Factor 4: Orientation to the Need to Know. 1. If a nurse is impaired and receiving treatment, it is important for his/her supervisor to be aware of that fact. 45 E. F. G. H. I. 2. If an impaired nurse is receiving treatment, it is important for his/her coworkers to be aware of that fact. 3. Nurses have an obligation to notify their supervisor when they suspect impairment in a coworker. Factor 5: Treatability Orientation. 1. Even after treatment it is unusual for an impaired nurse to be productive and trustworthy. 2. There is little that can be done to help nurses who are impaired. 3. In most cases, public safety can be assured by placing a probationary period on the license of the impaired nurse. Factor 6: Orientation to the Nurses Ability to Help. 1. The help needed by impaired nurses usually requires types of insight which only other nurses are likely to provide. 2. When made aware of a coworkers impairment, fellow nurses are usually able to offer assistance. 3. Most impaired nurses could be helped in a support group with other nurses. 4. When suspecting impairment in a coworker, the nurses first response should be to confront that individual. Factor 7: Perception of Prevalence. 1. Impairment is a widespread problem among nurses. 2. Impairment occurs less frequently in nursing than in other health-related fields. Factor 8: Orientation to Impairment as Illness. 1. Impaired nurses can best be understood as people who suffer from an illness. 2. Impairment is generally the result of a weakness in the nurses personality. Factor 9: Perception of Recognizability. 1. I could probably recognize an impaired nurse in the work setting by his/her behavior. 2. Impairment, when it occurs, is more likely to be reported in nursing than in other health-related fields. 46 Appendix F Stigma of Drug Users Scale (5- Point Likert Scale: Strongly Disagree, Disagree, Neutral, Agree, Strongly Agree) 1. Most people believe opioid users cannot be trusted. 2. Most people believe that opioid users are dangerous. 3. Most people would not accept an opioid user as a close friend. 4. Most people feel that opioid use is a sign of personal failure. 5. Most people will take a known opioid users opinions less seriously. 6. Most people think less of a person that uses opioids. 7. Most people would treat an opioid user just as they would treat anyone else. 8. Most employers will not hire a person uses opioids. 9. Most people would not accept an opioid user as a teacher of young children in a public school. Hendrix, M. J., Sabritt, D., McDaniel, A., & Field, B. (1987) Perceptions and attitudes toward nursing impairment. Research in Nursing & Health, 10, 323-333. doi:/10.1002/nur.4770100506 Palamar, J., & Klang, M.V. (2011). Stigma of Drug Users Scale. doi: 10.3109/10826084.2011.596606 47 Appendix G A. Drug Use Stigmatization Scale (5- Point Likert Scale: Strongly Disagree, Disagree, Neutral, Agree, Strongly Agree) 1. Using opioids is morally wrong. 2. Opioid users should go to prison. 3. Opioid users are weak minded. 4. Opioid users have no future. 5. Most opioid users are not well educated. 6. Opioid users are dishonest. 7. Opioid users make me angry. 48 Appendix H Note from Melissa Loria I have recently learned that working with Community Health to collect data from their employees has been a serious issue for students in the past. This coupled with the significant number of revisions this proposal required is leading us to request permission to collect data from nursing students at UIndy in place of Community employees. The mTurk portion of the Palamar, J., & Klang, M. V. (2011). Drug Use Stigmatization Scale. doi: 10.3109/10826084.2011.596606 study will remain the same. The original research question is almost entirely unaffected by this change, as using nursing students instead of healthcare professionals allows us to measure individuals perceptions who are being trained as healthcare professionals and who plan to be employed as healthcare professionals. Further, Mark has done an excellent job highlighting the similarities between healthcare workers and nursing students below. The biggest concern at this point is that Mark will not have enough time to collect the Community data if we run into issues that students have been limited by in the past. I am very concerned that if we do not make this adjustment for Mark, he will not be able to graduate with honors on time simply due to possibly hang-ups with Community Health and not because of anything hes done wrong. Marks Justification for Change The trend of drug addiction affects healthcare professionals and nursing students in the same manner as the general population (Emergency Nurses Association, 2017). The need to understand and treat the addictive behaviors in nursing students has become increasingly 49 relevant, especially as the rise in opioids and opioid use continues (SAMHSA, 2018). While treatment options are limited for registered nurses, they are even more limited for nursing students. Many nursing schools are zero-tolerance programs and when students are found to be addicted to substances, they are removed from the program (Worley, 2019). Students who are dismissed from one nursing program can then apply to another without disclosing the incident that occurred at the previous institution (Worley, 2019). This type of cycle can potentially compromise patient safety, but more importantly, the nursing student is not able to receive the help they may need for fear of punishment (Worley, 2019). As future healthcare professionals (nursing students) and the general population battle addiction, a growth in negative perceptions has followed. The application of addiction-related labels prevents many individuals from seeking the help that they desire (Wakeman & Rich, 2016). Patients designated as addicts are less likely to seek resources that benefit their direct care and rehabilitation (Wakeman & Rich, 2016). The increase of this stigma is recognized in particularly vulnerable populations and has spread to include altered perceptions of healthcare professionals (Mittal, Drummond, Blevins, Curran, Corrigan & Sullivan, 2013). As these stigmas are alleviated and eventually eliminated, many nursing schools can begin to look at other treatment options, without the outright dismissal of the student in general (Monroe, Vandoren, Smith, Cole, & Kenaga, 2011). The early identification and screening of drug use among nursing students is needed in order to prevent long-term career addiction and potential complications to patient safety (Monroe, Vandoren, Smith, Cole, & Kenaga, 2011). 50 Appendix I  ...

... The Effects of Highlighting Posttraumatic Growth After Trauma on Individuals Perceptions of Trauma Victims By Melissa Ann Johnston Honors Project Thesis Submitted in Partial Fulfillment Of the Requirements for a Baccalaureate Degree With Distinction In the Ron and Laura Strain Honors College of THE UNIVERSITY OF INDIANAPOLIS May 2020 Faculty Advisor: Melissa Loria, Ph.D. Executive Director of Honors: James B. Williams, Ph.D. Melissa Johnston 2020. Copyright in this work rests solely with the author. Please ensure that any reproduction or re-use is done in accordance with relevant national copyright protection. 1 Abstract Ninety percent of people have been affected by trauma, which is defined as an adverse event or events that change an individuals narrative about their life and the world. The outcomes of trauma can vary substantially. With a widespread focus on the long-term negative outcomes of trauma, few have heard about the positive outcomes after a traumatic incident seen in trauma victims, known as posttraumatic growth. This study aims to evaluate whether personal vignettes that focus either on the positive or the negative long-term outcomes of trauma can influence participants perceptions of trauma survivors. Unexpected results reveal that participants who read the vignette highlighting posttraumatic growth had increased stigmatization towards the character in the vignette in comparison to those who read solely about posttraumatic stress. Theories for what may reduce posttraumatic growth and increase posttraumatic stress and lack of resilience, making it less common in society and literature are discussed. Future directions and limitations are addressed. 2 Table of Contents 1. Introduction, Intellectual Context, Framework and Theory...Page 5 2. Method...Page 16 3. Results....Page 20 4. Discussion..Page 26 5. Conclusion..Page 29 6. Reflection & Future Directions..Page 29 7. References..Page 32 8. Appendices a. Appendix A: G Power Calculator...Page 42 b. Appendix B: Demographics....Page 43 c. Appendix C: Vignettes....Page 46 d. Appendix D: Survey Questions adapted from Link et al. (1999)Page 48 e. Appendix E: Infographic..Page 50 f. Appendix F: Informed Consent....Page 51 3 Acknowledgements I would like to thank the Ron and Strain Honors College from the University of Indianapolis for administering and supporting this project, as well as funding the payment to all participants for completing the study. I would also like to thank Dr. Melissa Loria for agreeing to be a great advisor and helping me through all the setbacks this project has faced and still creating an amazing study with me. Also, a large acknowledgment and expression of gratitude is due for Dr. James Williams for his committed devotion to his honors students and his willingness to fight for us and support us in any way we need. Thank you all. 4 List of Figures Figure 1: Perceived Tendencies and Causes of Trauma...Page 23 Figure 2: Active and Passive Facilitation and Harm Questions ...Page 26 5 Introduction, Intellectual Context, Framework and Theory Description of Trauma Trauma is the physical and psychological response to an adverse experience (Center for Substance Abuse Treatment, 2014). An event may be especially traumatic to individuals if it is life-threatening or causes physical harm. Bonanno (2004) states that most of the population in the United States has experienced something traumatic at some point in their life. Recent research supports this estimation, stating that 90% of Americans have experienced a potentially traumatic event, such as natural disaster, assault, warzone exposure, serious illness to themselves or someone close to them, among many others (Kilpatrick et al., 2013). Both short-term and long-term outcomes of a traumatic event can vary greatly. Posttraumatic stress disorder (PTSD) is a common, well-known diagnosis for physical and psychological symptoms following trauma (Center for Substance Abuse Treatment, 2014). While many sources support the idea that a high number of Americans have been exposed to traumatic stressors (Hooyer et al., 2012; Kilpatrick et al., 2013), PTSD only arises in 5-10% of those individuals (Bonanno, 2004). Recent research has supported this claim, showing that PTSD is seen in approximately 7-8% of the general population (Pekez, 2018). Along with these negative long-term effects, many victims have reported posttraumatic growth (PTG) and resilience following trauma, which has helped them gain strength, hope, and positivity (Blackie et al., 2017; Carmil & Breznitz, 1991; Tedeschi & Calhoun, 1996;). These constructs will be discussed further later. Possible Diagnoses Trauma can impact peoples thoughts and what they believe about their lives (Beck et al., 2011). Common responses immediately following a traumatic event may include denial and 6 shock (Center for Substance Abuse Treatment, 2014). Along with psychological diagnoses and symptoms, trauma and PTSD can greatly impact physical and physiological outcomes as well. For example, long-term responses include chronic fatigue syndrome, widespread musculoskeletal pain that can manifest into various disorders (McBeth et al., 2007) such as fibromyalgia (Nilsen et al., 2007), irritable bowel syndrome (Talley & Spiller, 2002), and PTSD (Vieweg et al., 2007). The physiological outcomes may also include sensitization to triggers in ones environment (Heim & Nemeroff, 2001). Some effects that may not be seen as readily as muscle pain and emotional triggers, are hypertension (Kibler, Joshi, & Ma, 2009), gene expression (Duric & McCarson, 2006), cardiovascular disease (Kibler, Joshi, & Ma, 2009), obesity (Vieweg et al., 2007), and an overwhelming amount of additional physical, emotional, and psychological effects (McFarlane, 2010). For some, certain adverse experiences can be traumatic but may not develop into any diagnosis. Traumatic events that are most likely to cause PTSD are events that are intentional traumas caused by other humans, such as assault and torture (Schiraldi, 2000). Other factors that correlate with the prevalence of PTSD include positive affect and high levels of cognitive reactivity, or reacting negatively to a stressor (Giesbrecht et al., 2009). An individuals reaction to a traumatic event is formulated by the initial emotional response to the trauma. This response is then developed further by reevaluations, aiming to resolve any differences in beliefs between the traumatic experience and pre-existing beliefs. If a person does develop PTSD, its symptomatology tends to be maintained by feelings of mental defeat and negativity about the world and oneself (Beck et al., 2011). Short Term Effects of Trauma 7 The use of positive coping methods may enable an individual to persevere through their grief without developing severe effects of trauma. However, this is not always the case. Individuals may develop negative effects regardless of coping skills. Further, they may not have chosen positive coping skills initially, and therefore experience more negative effects. There are some negative effects of trauma that make an appearance right away and others that are not visible until later (Briere & Elliott, 1994). The initial period of time following trauma is critical, and most of the coping mechanisms develop within months following the traumatic event (Brewin et al., 1999; Shalev, 2002). Some examples of coping strategies that are used shortly after a traumatic event include the avoidance of thoughts, places, people, and conversations. In more serious cases, dissociation may occur. Additional changes observed after a traumatic event can include interpersonal difficulties, changed beliefs about the world, and emotional distress (Briere & Elliott, 1994). Coping is a comprehensive process in that is includes all aspects of a person, such as emotion, hormones, social life, and cognition (Pearlin & Schooler, 1978; Shalev, 1994; Tuval-Mashiach et al., 2004). Trauma affects an individual's narrative, which in turn, modifies their identity, which is why early adoption of positive coping methods is so essential (Tuval-Mashiach et al., 2004). Long-Term Effects of Trauma - Physical and Emotional In addition to the short-term effects of trauma, there are also significant, prolonged effects. Predictably, traumatic events can affect every aspect of an individual, whether it be physical, emotional, social, religious, or spiritual. However, in some cases, changes in physical health may be the most noticeable long-term change in a survivor (Briere & Elliot, 1994). Some of these difficulties may include increased blood pressure (Thurston et al., 2019), cardiovascular 8 disease, diabetes, gastrointestinal disorders, and even higher rates of cancer than seen in the general public (Kendall-Tackett, 2009). Beyond physical health, emotional health is also affected. Anxiety, depression, and sleep disturbances are found in higher rates among those who have endured trauma (Gordon, 2018; Greenberg, Brooks, & Dunn, 2015). Other symptomatology may include negative beliefs about the world, others, and oneself, memory failure, distorted cognitions, a negative emotional state that persists, low interest and inability to experience positive emotions, detachment, irritability and outbursts, recklessness, hypervigilance, and difficulties concentrating (Greenberg, Brooks, & Dunn, 2015). Further, childhood sexual abuse leads to higher rates of alcohol use (Galaif et al., 2001; Yuan, Koss, & Stone, 2006), suicide (Saunders et al., 1999; Yuan, Koss, & Stone, 2006), difficulties creating and maintaining relationships, poor coping strategies (Fritsch & Warrier, 2004; Yuan, Koss, & Stone, 2006), sexual dysfunction, and various personality disorders (Yuan, Koss, & Stone, 2006). Women who have been physically abused have lower perceived health, are more likely to obtain and be diagnosed with a sexually transmitted disease, experience various other gynecological problems, and are more likely to fail to seek necessary medical care (Campbell & Lewandowski, 1997). Regarding the general public, 64% of people who have chronic pain syndromes, such as chronic fatigue syndrome or fibromyalgia, have experienced at least one traumatic event in their life, and 39% of those people have had trauma in their childhood and adulthood, meaning that they have endured a lifelong history of adversity (Kendall-Tackett, 2009; Van Houdenhove, Luyten, & Egle, 2009). Long Term Effects of Trauma Social 9 The physical and emotional effects of trauma tend to be met with social repercussions. The misinformed and often antiquated beliefs of society lead to stigmatization specifically encompassing trauma surrounding domestic and sexual violence (Murray, Crowe, & Akers, 2016). These social repercussions are often termed stigma, referring to the pressure on individuals to conform to social standards (Goffman, 1959) especially those who are seen as deviant from the normal and are stigmatized in some way (Goffman, 1968). One area where the effects of stigma on trauma has been examined extensively is among veterans. The stigma associated with mental illness has been reported as the main barrier to receiving essential care after a traumatic event while serving in the military. Further, individuals with increased rates of mental illness reported worrying more about the stigma associated with their mental illness, which may contribute to trauma victims avoiding treatment in the effort to avoid receiving a diagnosis. The veterans reported feeling concerned that their peers may no longer be confident in them or that they would appear weak and jeopardize their careers (Hoge et al., 2004; Hooyer, 2012). There are many ways stigma can be distributed, such as commentary, rejection, exclusion, and other public approaches that signify the individual is different. Stigma can also be experienced internally or personally, such as when individuals feel that their illness does not fit with their identity or narrative (Hooyer, 2012). Victims of trauma also report feelings of avoidance, social anxiety, and isolation (Hofmann, Litz & Weathers, 2003). Long Term Effects of Trauma Spiritual Along with effects on an individual's social livelihood, adversity can demolish a persons world views and spirituality. Among veterans, intense levels of combat among combined with strong spirituality lead to the highest rates of PTSD symptoms in comparison to those who utilized less positive religious coping (Park et al., 2017). Along with religion, moderate or 10 loosely opinionated world views and spirituality contribute to increased struggle after a traumatic event. Carnelley and Janoff-Bulman (1992) explain the altered world views of those who have undergone trauma claiming that students who are victims of trauma view their world as more dangerous, have lower self-worth, and see the world as less meaningful. They argue that those who are not victims of trauma usually have higher self-worth, believe that everything happens for a reason, and believe that bad things wont happen to them. Experiencing adversity, such as trauma, can cause a dispute with these world views in such a way that the world views become shattered (Carnelley & Janoff-Bulman, 1992; Tuval-Mashiach et al., 2004). Adaptive Responses to Trauma-PTG, PPTG, and Resilience Although the negative effects of trauma are plentiful and can be severe, they are not the only changes seen in trauma survivors. For example, as mentioned previously, high levels of combat exposure combined with high levels of spirituality produced the highest PTSD symptomology. However, those individuals with higher symptomology also had more positivity following adversity and trauma, which is known as posttraumatic growth (Park et al., 2017). Interestingly, the positive effects of trauma are often observed and are now referred to as perceived posttraumatic growth (PPTG) or posttraumatic growth (PTG). Although it would be common for people who have not been through significant trauma to believe that going through traumatic experiences would create a shattered world-view and negative affect, some survivors exhibit higher percentages of positive world views, hopeful views of the future, and religious beliefs (Carmil & Breznitz, 1991). Many people who experience trauma report PTG, with as high as 83% of victims reporting some type of PTG following traumatic experiences (Blackie et al., 2017). Common factors of PTG include the perceived growth of self, relationships, and 11 philosophy due to the reevaluation of traumatic events and coping from trauma (Tedeschi and Calhoun, 1996). Emotional support is also associated with PTG (Kamen et al., 2016). Having a support system to listen to victims and assist in the reinterpretation of the meaning of the victims trauma helps trauma victims create a new personal narrative (Neimeyer, Wittkowski, & Moser, 2004; Kamen et al., 2016). The other end of this spectrum is also true: higher social stigma is associated with lower levels of PTG (Kamen et al., 2016). The perception of PPTG has also been studied with stroke survivors. Participants who had social support displayed higher levels of PPTG and considered their stroke to be a part of their identity, which improved their emotional distress from trauma. Individuals who claimed the stroke as part of their identity also had a higher appreciation of life and thoughtfully chose their relationships as a construct of positive change following their medical trauma (Kuenemund et al., 2016). PTG has additionally been studied in youth offenders. A 2010 study found that experiencing trauma between the ages of 18 and 24 is associated with a disruption to their belief in a just world and their perception of control. However, trauma also heightens the levels of spirituality and coping skills in these individuals (Maschi et al., 2010). Similar to PTG and PPTG, resilience is the personality factor attributed to the maintenance through adversity. For PTG to occur, the adverse experience must disrupt an individual's state of mind and beliefs to allow for growth. For example, these beliefs may include their general views, coping skills, and beliefs of purpose. (McMillen & Fisher, 1998; Tedeschi & Calhoun, 1996; Shakespeare-Finch et al., 2003). A study displaying these findings includes Siegal and Schrimshaw (2000) which showed that 83% of women who have HIV/AIDS were 12 positively affected because of their illness and the subsequent life changes (Siegal & Schrimshaw, 2000; Shakespeare-Finch et al., 2003). Outgroup Perceptions of an Individuals Trauma Gender and experiences of trauma can impact the attitudes and perceptions towards childhood abuse victims and other forms of childhood trauma (Cromer & Freyd, 2009). For example, identifying with a victim correlates with positive views and comments towards victims. Additionally correlated with the latter, being a parent (even if the victims are not their children) increased positivity towards victims (Barbis, 2018). Regarding gender, female participants and feminists are more likely to acknowledge childhood sexual abuse and realize their harm (Cromer & Freyd, 2009). Male victims are seen less favorably in comparison to female victims, and women are comprehensively more optimistic to all victims. Victims who are male are viewed more negatively than females, except for natural disaster victims (Mendelsohn & Sewell, 2004). Also concerning male victims, male participants who have a history of trauma view them more favorably than males with no reported history of trauma (Mendelsohn & Sewell, 2004). Stigma and How Victims Are Stigmatized Stigma is a term used to describe the actions and biases towards members of a specific group. Outsiders, and even the victims themselves, may be responsible for stigmatizing victims, and may include distancing themselves, discrimination, stereotyping, labeling, and acting in a prejudiced manner (Link & Phelan, 2001; Goffman, 1959; Goffman, 1968). Stigma can have negative, damaging effects on stigmatized individuals. These effects may include the failure to seek professional help (Clement et al., 2015), as well as reduced self-esteem, self-efficacy, and self-respect (Corrigan et al., 2016). When combined with a self-applied stereotype, this diminished self-respect can transcend into the "why try'' effect, which leads some to experience 13 reduced self-perceived purpose and lower motivation levels (Corrigan, Larson, & Ruesch, 2009). These effects can lead to depression and thus decreased recovery after traumatic events (Corrigan et al., 2016). These outcomes may be due to a victims decreased likelihood to seek help or fully participate in services to avoid labeling linked to stereotypes and stigma (Clement et al., 2015; Corrigan, 2004). Personal stigma can also include being uncomfortable with the fact that the public may stigmatize them, so they avoid care, treatment, and social support to diminish the likelihood of stigmatization (Hooyer, 2012). In addition to the publics perceptions of others that affect treatment, the public can sometimes place blame on victims of various traumas. The strongest found predictor of attitudes towards rape victims was their age, as younger people have more positive attitudes than older individuals. This predictor was not found in African American individuals, however, as their ages were not predictive of their attitudes. This indication is likely due to generational differences involving a heightened awareness of violence towards women in younger generations (Nagel et al., 2005). Additionally, lower socioeconomic status and reduced level of education are predictors of heightened negative attitudes and perceptions towards rape victims. Further, higher income is associated with heightened levels of sympathy towards rape victims (Nagel et al., 2005). In terms of respect for female victims, one study found that participants felt female victims of sexual assault were more at fault for the act when these women were also seen as less respectable and less impacted by the assault (Feldman-Summers & Lindner, 1976). Studies have found that men are more likely to experience victim-blaming, considering that they can be stereotypically assumed to be able to fight back and get away from the assault. Further, men are more likely to blame other victims (Davies & Rogers, 2006). Sadly, trauma victims often blame 14 themselves to some degree for what happened to them, which is often seen as a dysfunctional means of coping with the trauma (Davis et al., 1996). These findings contribute to the literature for the current study because the stigma given to victims of trauma will be studied. Specifically, findings of female participants being blamed more for causing their trauma is very similar to the current study, as questions about the cause of trauma are asked. Additionally, for any findings that indicate stigmatizing views, it can be inferred that victims would be less likely to seek treatment, which could potentially reduce PTG, feeding into a continuum of stigma surrounding victims. Why Stigma Matters As mentioned previously, trauma and its implications can have negative, and positive, effects on the victims. In specifics to stigma and its implications, one outcome is a delay in disclosure of assault (Alaggia, 2005). Ample research over the past two decades has consistently revealed that anywhere between 30-80% of victims do not plan on sharing their experiences of childhood sexual abuse before they reach adulthood, which in turn reduces access to the support they would more likely have had if they had disclosed their experiences (Alaggia, 2005; Arata, 1998; Lamb & Edgar-Smith, 1994; Paine & Hansen, 2002; Roesler & Wind, 1994; Smith et al., 2016). In cases of rape, victims are more likely to be blamed in interracial rape than intraracial, and questionable sexual acts were contemplated less if done within the same racial identities (Alaggia, 2005). In terms of revictimization, adult sexual assault is four times more likely in those who have experienced sexual abuse during childhood (Ullman, 2016). Further, most of these individuals blame themselves for the subsequent incident(s), even after years of aging and developing an understanding, compared to when the individual was a child. This self-blame is 15 believed to create a sense of control for the victims, which in turn helps them cope with the trauma they have experienced (Ullman, 2016; Janoff-Bulman, 1979). However, this behavioral coping mechanism is only found to be useful for traumatic events such as motor accidents, and not events like rape due to the lack of control of the situations (Janoff-Bulman, 1979). Interestingly, feelings of self-blame following trauma such as rape increase PTSD symptomology in victims (Frazier, 2003). Lead into Current Study As previously highlighted, trauma is surrounded by stigma from external (social) factors, and internal (personal) factors. A common example of this is with military veterans who encounter stigma from their societal and personal expectations to be strong, tough and display perseverance (Greene-Shortridge, Britt, & Castro, 2007). Additionally, personal stigma is a barrier to seeking treatment in the African American community (Brown et al., 2010; Dutton et al., 2013). External and internal stigma, as well as access to care, have been noted as the key reasonings to why some people suffering from traumatic events do not seek help (Murphy & Busutil, 2015). One way that stigma may be reduced is by educating the public about recovering after trauma through narratives. It is important to encourage the use of narratives in such a way that it positively highlights stigmatized individuals. Further, practice envisioning the perception of that victim so that the audience, and hopefully the general public, can learn to accept individuals who are experiencing stigma, rather than distancing themselves from the individual (Chung & Slater, 2013). A promising route to reducing the stigmatization of trauma is by educating different groups (patients, medical professions, and the general public) about the narratives of individuals who have been through trauma. One way in which this can be done is by exposing them to 16 stories of individuals who have experienced trauma so that they can have a fuller, more comprehensive understanding of trauma and its effects (Thornicroft et al., 2016; Yamaguchi, Mino, & Uddin, 2011; Knaak & Patten, 2016). A more comprehensive view of trauma and its effects would specifically include the positives that often come from adversity and trauma, once the initial coping and grieving subsides. These positives could include resilience, strength,a closer relationship with God, and more. As a result of reducing the stigma that these individuals experience, victims can live a fuller life and achieve their goals more readily (Corrigan, 2011). However, there is not enough evidence to suggest which method is best in reducing mental health stigma of each group of individuals who have undergone trauma and are affected by it long-term (Knaak & Patten, 2016; Rsch, Angermeyer, & Corrigan, 2005). The current study aims to examine whether stigma surrounding trauma may be altered by highlighting the positive or negative outcomes of trauma. This will be done by having participants read one of two versions of a vignette about a woman who experiences multiple traumatic incidents. Both versions address the negative outcomes of the trauma, while the experimental groups vignette additionally focuses on PTG of the individual. It is hypothesized that when individuals read the PTG vignette, they will have lower levels of stigma, which will be observable through more recognitions of trauma, fewer views of dangerousness, less desired social distance, and higher beliefs of the cause of trauma regarding the victim in an adapted version of the Link et al. (1999) scale. Method Participants Participants of the current study were United States citizens over the age of 18 recruited using Mechanical Turk (mTurk). The survey was completed by 119 participants via Qualtrics. 26 17 were omitted due to failure to successfully answer the attention checks, therefore, 93 of the participants were included in data analysis. The participants consisted of 57 male participants and 37 female participants from ages 18-74. 56 of the participants, the majority, were between the ages 25-34. Each participant received monetary compensation of $1 for completion of the 20minute study. Participants must have successfully completed 1,000 surveys via mTurk previously, also known as 1,000 HITS, which has been shown to help narrow in on quality responses (Peer, Vosgerau, & Acquisti, 2014). A minimum sample size of 102 participants was used to reach a power of 0.8, an effect size of 0.50, and an alpha level set at 0.05 which was determined using the G*Power calculator (see Appendix A). The survey remained open until the required responses were collected, and the 93 completed and reliable responses were kept for analysis. One of the attention checks was thrown out due to too many participants failing it. Upon consideration, the high fail rate was likely due to phrasing of the campus mall which was referenced in the vignettes. Participants may not know that a mall can be included as part of a school campus. If participants got all attention checks right except for this one, they remained in the study. If any attention checks were wrong besides this attention check, they were removed from the studys data due to a lack of evidence that they properly read the vignette. Materials After participants were recruited via mTurk, they were directed to a Qualtrics survey. They were asked basic demographic questions that adapted their survey based on the responses given (See Appendix B). The name of the victim in the vignette changed based on the ethnicity provided by each participant. For example, if a participant reported being Hispanic or Latino, the name in the vignette appeared as Ximena instead of Melissa to account for inclusion and to eliminate bias. Other examples include Leilani for Hawaiian or Pacific Islander, Melissa for 18 White or other, Imani for Black or African American, Aanisah for American Indian or Alaska Native, and Akemi for Asian. Having readers of various contexts read material that is inclusive of their race and ethnicity has shown to increase engagement and reflection (Harper, 2009; Nguyen Littleford & Nolan, 2013; Rouse, 2018). Qualtrics randomly assigned participants to one of two conditions to gather and temporarily store data. Participants were asked to read through one of two vignettes, which served as the manipulation in the current experiment. The vignettes describe a woman who has experienced traumatic events and it outlines the negativity she faced from this trauma in the first paragraph. Multiple traumatic experiences were detailed in the vignette to depict a story in which a victim would need increasing resilience to endure multiple traumatic events. This was done in an effort to allow positive views of the individual as she had experienced much adversity and has been very negatively impacted by it (PTS vignette) or has overcome it (in the PTG vignette). The experimental group were provided additional sentences outlining the vignette characters PTG to examine how knowledge of PTG may affect participants views of the individual in the vignette (see Appendix C). Along with the randomly placed attention checks, Link, Phelan, Bresnahan, Stueve, & Pescosolidos Publics Perceptions of Mental Illness scale (1999) was utilized to evaluate a comprehensive view of the participants' perceptions of the trauma victim described in the vignette (See Appendix D). Eight items were asked on a four-point Likert scale ranging from very likely, to very unlikely, regarding the participants opinions of what the victim in the vignette was like and what the cause of their trauma may be. Questions included In your opinion, how likely is it that the person described in the vignette has a mental illness?, In your opinion, how likely is it that the persons situation was caused by the persons own bad 19 character?, In your opinion, how likely is it that the persons situation was caused by a chemical imbalance in the brain?, In your opinion, how likely is it that the persons situation was caused by the way the person was raised?, In your opinion, how likely is it that the persons situation was caused by stressful circumstances in the persons life?, In your opinion, how likely is it that the persons situation was caused by a genetic or inherited problem?, In your opinion, how likely is it that the persons situation was caused by Gods Will?, and In your opinion, how likely is it that the person in the vignette would do something violent toward other people?. This scale additionally consists of 5 questions asked on a four-point Likert scale ranging from definitely to definitely not, which examined how likely the participant would be to do or allow certain things with the victim in the vignette. Participants answered questions regarding their willingness to Move next door to the person, Spend an evening socializing with the person, Make friends with the person, Start working closely with the person, and Have the person marry into the family. Following the study, regardless of which condition the participants were in, participants viewed an infographic summarizing findings of PTG (See Appendix E). This not only ensured that the study ended on an upbeat note but also ensured that every participant had access to the experimental condition PTG information. Procedure The current study gathered information about perceptions people have towards the survivors of trauma, and the stigma associated with trauma. After agreeing to partake in the study on mTurk, participants were taken to the Qualtrics survey and were first shown an informed consent page (Appendix F). Clicking to continue acknowledged their consent. Then they were asked basic demographic questions (Appendix B) and were randomly placed into one of the two conditions and asked to read one of the two versions of the vignette. Following the 20 reading, they were asked about their perceptions of the individual discussed in the vignette using questions and scaling from Link et al. (1999) in addition to being asked a few attention checks to ensure they read the vignette. To conclude the study, participants were shown an infographic that highlights the less widely known positive outcomes that tend to arise in individuals who have survived trauma (i.e. better social capital, perceived control of their identity and personal narrative, greater appreciation of life, etc.) and provide information and hope to the participants, which will also reduce the risk for the study (See Appendix E). Results A series of t-tests were run to examine the effects that each version of the vignette had on perceptions of the trauma victim in the vignette. Multiple, individual t-tests were run in conjunction with the Publics Perceptions of Mental Illness scale as seen in Link et al. (1999). Participants that answered that it was more likely for any of the items regarding cause of trauma or violent tendencies denoted an increase in stigma. Participants that answered that they would be less likely to do something with the victim in the vignette regarding facilitation and harm denoted an increase of trauma. It was anticipated that individuals who were exposed to the narrative that includes the positive long-term factors associated with trauma would have decreased stigma associated with trauma survivors and would rate the character in the vignette as more capable and more likable. See below for Figure 1 and Figure 2 graphs. Perceptions-Bad Character The effects of highlighting posttraumatic stress vs. posttraumatic growth on views of the victims bad character causing trauma are summarized in Figure 1. The posttraumatic stress 21 condition appeared to have lower beliefs of bad character (M=3.21, SD= 0.81) than the posttraumatic growth condition (M=3.37, SD= 1.08). Consistent with these observations, an independent-samples t-test revealed a significant effect for condition on overall perception score, t(91) = -.794, p = .048. Perceptions-Chemical Imbalance The effects of highlighting posttraumatic stress vs. posttraumatic growth on views of ones chemical imbalances causing trauma are summarized in Figure 1. The posttraumatic stress condition appeared to have lower beliefs of chemical imbalances as a cause for trauma (M= 3.06, SD= 0.92) than the posttraumatic growth condition (M=3.20, SD=0.96). Consistent with these observations, an independent-samples t-test revealed a non-significant effect for condition on overall perception score, t(91) = -.678, p = .460. Perceptions-Upbringing The effects of highlighting posttraumatic stress vs. posttraumatic growth on views of ones upbringing causing trauma are summarized in Figure 1. The posttraumatic stress condition appeared to have lower beliefs of upbringing as a cause for trauma (M=3.09, SD=0.97) than the posttraumatic growth condition (M=3.15, SD=0.97). Consistent with these observations, an independent-samples t-test revealed a non-significant effect for condition on overall perception score, t(91) = -.333, p = .965. Perceptions-Stress The effects of highlighting posttraumatic stress vs. posttraumatic growth on views of ones stress causing trauma are summarized in Figure 1. The posttraumatic stress condition 22 appeared to have lower beliefs of stress as a cause for trauma (M=1.91, SD=1.04) than posttraumatic growth condition (M=2.07, SD=1.02). Consistent with these observations, an independent-samples t-test revealed a non-significant effect for condition on overall perception score, t(91) = -.704, p = .423. Perceptions-Genetics The effects of highlighting posttraumatic stress vs. posttraumatic growth on views of ones genetics causing trauma are summarized in Figure 1. The posttraumatic stress condition appeared to have lower beliefs of genetics as a cause for trauma (M=3.15, SD=0.93) than the posttraumatic growth condition (M=3.39, SD=0.86). Consistent with these observations, an independent-samples t-test revealed a non-significant effect for condition on overall perception score, t(91) = .561, p = .561. Perceptions-Gods Will The effects of highlighting posttraumatic stress vs. posttraumatic growth on views of Gods will causing trauma are summarized in Figure 1. The posttraumatic stress condition appeared to have lower beliefs of Gods will as a cause for trauma (M=3.55, SD=0.86) than the posttraumatic growth condition (M=3.24, SD=1.14). Consistent with these observations, an independent-samples t-test revealed a significant effect for condition on overall perception score, t(91) = 0.05, p = .005. Perceptions-Violent Tendencies 23 The effects of highlighting posttraumatic stress vs. posttraumatic growth on views of ones likelihood of violent tendencies are summarized in Figure 1. The posttraumatic stress condition appeared to have lower beliefs of the likeliness of violent tendencies (M=3.00, SD=0.75) than the posttraumatic growth condition (M=3.35, SD=0.99). Consistent with these observations, an independent-samples t-test revealed a significant effect for condition on overall perception score, t(91) = -1.901, p = .007. Figure 1 Perceived Tendencies and Causes of Trauma PTSD Vignette PTG Vignette 4 3.5 * * * Mean Response 3 2.5 2 1.5 1 0.5 0 bad character chemical imbalance upbringing stress genetics god's will violent tendencies Questions Asked Note. This graph depicts the perceived tendencies and causes of trauma across both conditions. Facilitation and Harm-Neighboring The effects of highlighting posttraumatic stress vs posttraumatic growth on views of ones active and passive facilitation and ones activity and passive harm towards a victim of trauma are summarized in Figure 2. The posttraumatic stress condition appeared to have less willingness to move next door to the trauma victim (M=2.47, SD=0.88) than the posttraumatic 24 growth condition (M=1.98, SD=1.13). Consistent with these observations, an independentsamples t-test revealed a non-significant effect for condition on overall score, t(91) = 2.34, p = .424. Facilitation and Harm-Socialization The effects of highlighting posttraumatic stress vs posttraumatic growth on views of ones active and passive facilitation and ones activity and passive harm towards a victim of trauma are summarized in Figure 2. The posttraumatic stress condition appeared to have less willingness to spend an evening socializing with the trauma victim (M=2.40, SD=1.01) than the posttraumatic growth condition (M=1.83, SD=0.99). Consistent with these observations, an independent-samples t-test revealed a non-significant effect for condition on overall score, t(91) = 2.774, p = .407. Facilitation and Harm-Friendship The effects of highlighting posttraumatic stress vs posttraumatic growth on views of ones active and passive facilitation and ones activity and passive harm towards a victim of trauma are summarized in Figure 2. The posttraumatic stress condition appeared to have decreased willingness to make friends with the trauma victim (M=2.49, SD=1.04) than the posttraumatic growth condition (M=1.87, SD=1.07). Consistent with these observations, an independent-samples t-test revealed a non-significant effect for condition on overall score, t(91) = 2.837, p = .960. Facilitation and Harm-Coworking 25 The effects of highlighting posttraumatic stress vs posttraumatic growth on views of ones active and passive facilitation and ones activity and passive harm towards a victim of trauma are summarized in Figure 2. The posttraumatic stress condition appeared to have decreased willingness to move next door to the trauma victim (M=2.38, SD=0.77) than the posttraumatic growth condition (M=1.96, SD=1.15). Consistent with these observations, an independent-samples t-test revealed a non-significant effect for condition on overall score, t(91) = 2.103, p = .076. Facilitation and Harm-Become Family The effects of highlighting posttraumatic stress vs posttraumatic growth on views of ones active and passive facilitation and ones activity and passive harm towards a victim of trauma are summarized in Figure 2. The posttraumatic stress condition appeared to have decreased willingness for the trauma victim to marry into the family (M=2.53, SD=0.93) than the posttraumatic growth condition (M=2.07, SD=1.12). Consistent with these observations, an independent-samples t-test revealed a non-significant effect for condition on overall score, t(91) = 2.185, p = .511. 26 Figure 2 Active and Passive Facilitation and Harm Questions PTSD Vignette PTG Vignette 2.5 Mean Response 2 1.5 1 0.5 0 move in next door socialize for an evening make friends with Questions Asked start working closely with have the person marry into the family Note. This graph depicts the changes in facilitation and harm across both conditions. Discussion Trauma literature indicates that posttraumatic stress and posttraumatic growth are two of many common outcomes after facing adversity. However, PTG is seldom referenced, especially among the general public, as trauma is often highlighted solely as a negative experience. The goal of the current study was to determine whether participants who viewed the vignette highlighting long-term outcomes of posttraumatic stress versus long-term outcomes of PTG would show a change in stigma when responding to questions about the vignette. Interestingly, the significant results were contrary of what was predicted. Participants who viewed the vignette highlighting PTG exhibited heightened stigmatization towards the individual, specifically for three questions as discussed in the Results section. These intriguing findings reveal that highlighting PTG in the character impacted participants and their responses, but in such a way that was unexpected given the review of current literature and hypotheses 27 tested. These responses indicate that participants in the experimental condition believed that the trauma victim in the vignette was more likely to be violent towards others and that their trauma was seen as a response to Gods Will and the victims bad character. It was hypothesized that these stigmatizing views would be less common in participants who have read the PTG vignette. This was anticipated due to the idea that people would hold more favorable views of trauma and its victims after seeing the positivity that can arise after working through the negative aspects of adversity. Therefore, the finding that participants in the experimental, PTG group displayed stigmatizing views is noteworthy and peculiar. Active and Passive Facilitation and Active and Passive Harm were additionally measured with this scale. Specifically, this scale included measures of willingness to be a neighbor to the victim, socialize with the victim, become friends with the victim, work with the victim, and have the victim marry into the family. Interestingly, none of these measures were found to be significant. Overall, the results revealed that participants had stigmatizing views of the victim such that they believed them to be more violent, have bad character that caused their trauma, and that God planned trauma for them. However, the participants did not respond significantly to the willingness to be around the victims and socialize with them. Additionally, it is thoughtprovoking that individuals who experienced trauma but worked through it and came out stronger are seen to be in a more negative and stigmatizing light. One could imagine that participants would view these individuals as positive, strong, non-violent, and having good character, yet findings suggest otherwise. One way to interpret these unexpected results is to consider that the participants may have felt that the victim in the vignette ought to be experiencing negative outcomes, and thus reported increased stigma in certain areas. With participants viewing the vignette character in 28 this way, victims of trauma and adversity likely experience this stigma in society. Current literature is clear that once external stigma from society is internalized in an individual, it begins affecting that individuals health and wellbeing (Corrigan & Watson, 2002). This is a potential solution to the reason why posttraumatic growth is not as often recognized and discussed; the ever-present stigma allows any growth that could occur post-trauma to litigate. This may be why victims of trauma do not as often experience PTG instead of solely posttraumatic stress. In turn, it is promising that this internalization of stigma in victims increases the difficulty of resilience. The lack of PTG in conversation and common knowledge could be due to victims acceptance of such amounts of stigma to a point in which growth is challenging to achieve. This allows distress and posttraumatic stress to be more common, and therefore becomes noted as acceptable in society. Hence, people view victims negatively if they do not represent their idea of what trauma and its expected outcomes ought to look like. Literature aimed at better understanding stigma has identified certain characteristics of individuals that lead others to stigmatize them. Finkelhor & Asdigian (1996) define and describe three constructs that can be seen as victimization components. These characteristics include target vulnerability, target gratifiability, and target antagonism. Target vulnerability refers to characteristics that mark the victim as vulnerable to the outgroup (Finkelhor & Asdigian, 1996). For the current study, this is represented in the victim that has experienced trauma and adversity by gossip and rumors spread, being embarrassed and publicly shamed for a breakup based around rumors, and the death of a close family member. Secondly, target gratifiability refers to an individuals gender marking the victim as vulnerable to the outgroup (Finkelhor & Asdigian, 1996). The victim in the current studys vignette is female so this may have also led the outgroup (the participants) to heightened victimization. Lastly, target antagonism is the construct 29 describing undesirable characteristics that mark the victim in a negative light to the outgroup (Finkelhor & Asdigian, 1996). Some aspects of the current studys vignette may have created personality traits and other characteristics that the participants saw as undesirable, such as the slur regarding her sexual activities that she was given by her ex-boyfriend, which marked the individual to become stigmatized by participants. While the current study is an essential addition to the growing literature regarding trauma, posttraumatic growth, and stigma, it did have limitations that must be addressed. One such limitation is that the participants own traumatic history, or lack thereof, was not addressed. This inclusion would have aided the understanding of the impact of past trauma in an individuals life along with the severity and specific type of trauma and its impact on the views of the individual regarding trauma and its victims. Intriguing future studies may measure how participants view the victim in the vignette if the individual themselves have not been through something traumatic, and individuals who have been through trauma. Further, questions regarding the type of trauma and charting the severity of trauma would allow researchers to have additional insight on why someone may stigmatize, or not stigmatize, an individual who has been through traumatic events. A second limitation of the study includes the absence of certain traumatic experiences, such as rape or sexual assault, in the vignette. Rape and sexual assault have become more common in literature and news lately with the #MeToo movement. These words and descriptions may trigger some participants, but if done mindfully, they may have yielded meaningful data due to constructs such as victim-blaming, belief in a just world, and more. A third limitation is an absence of pretesting the vignette for its validity. This would have been a useful precaution; however, it was not included due to shortage of time and resources. 30 Lastly, there is the potential for the presence of Type 1 Error in the study due to the measures design that includes many individual tests to be run which could indicate a false rejection of the null hypothesis. This could account for the studys significant findings if there is in fact, no significance to the findings. Although the study does provide beneficial insight to the stigmatization of victims of trauma, there is still a need for further research. A future direction that should be taken would include a background knowledge of participants history of trauma. A future project could replicate the current study while including a measure to determine the level of trauma that each participant has or has not experienced to see how a history as a survivor influences stigma towards trauma and other victims. It may also be beneficial to determine what traumatic experiences that participants have had to better understand the causes of stigma. Further, a strong future study could include measuring the direct findings of the current study due to the unexpected results of the current study and the need for literature review after data collection from the new ideas that emerged. Conclusion There is extensive research regarding trauma, posttraumatic stress disorder, and less research surrounding stigma associated with trauma and posttraumatic growth. The current study combines these constructs in a single study and adds necessary information to the literature. The findings that PTG seem to increase stigma in our participants are surprising and add an interesting and new component to the literature that should be studied further. The findings of this study deem important to the understanding of not only PTG, resilience, and their commonplace in society, but also the stigmatization seen in trauma and its victims, as well as the 31 stigmatization of those who experience PTSD and PTG. Ideally, further research will be conducted surrounding these constructs and ideas so that there can be a better understanding of trauma, stigma, PTSD, and PTG, and to find ways to reduce stigma and increase growth seen in victims of trauma. Reflection & Future Directions This project has awarded me with tremendous amount of learning experiences. I have learned not only the ins and outs of creating, designing, proposing, executing, and writing up a true research study, but I have also experienced the tedious amounts of focused work required for such a project. This project has tested me in many ways, in many different circumstances, all of which have taught me one common theme: to be especially diligent at staying ahead of the game so that if any curveballs were to appear, I would be ready for them. For example, there was the prolonged stress of finding an advisor due to many advisors being out of commission for family reasons. Next, I was reminded of the required resilience needed for acceptance of my proposal, which endured three submissions to the Ron and Laura Strain Honors College. Once approved, there were many measurement changes of scales to use for the project which required perseverance to read vast amounts of literature and comprehend method and procedure sections of studies that were completely foreign to me. Due to some of these overwhelming changes and stress, there was some confusion of measures in which I failed to include the questions from the Cuddy, Fiske, & Glick (2007) BIAS map scale that was in the proposal. However, these questions were asked in the Qualtrics study, so this will hopefully be able to be used in a future article submission in hopes of being published in a psychological research journal. Another challenge of this study that reminded me of the need for diligence and proactiveness was the 32 COVID-19 pandemic which affected meetings with my advisor on campus, as well as many extreme changes to everyones life which filled us all with uncertainty and anxiety. Despite the occasional stress from setbacks regarding the study, I am immensely grateful for the opportunity to participate in such academically relevant research with a well-loved psychology professor as my advisor, and another as the reader of my proposal submissions and now my final manuscript. It is my goal to complete more research in the future as I continue onto graduate school for a masters degree in School Social Work. Additionally, my advisor and I plan to complete an article including everything from this Honors Manuscript, as well as extra data to be included from the survey. Ideally, this article will be published in an undergraduate research journal, and maybe even an academic journal, that my advisor and I will be working on over the coming summer, due to the surprising results of the current study. In future research, I will have this informed background which will help me to acknowledge the time and efforts that a project like this demands and I will have the experience to know how to be even more thorough and organized, especially in understanding scales and utilizing them in a more effective manner. 33 References Alaggia, R. (2005). Disclosing the trauma of child sexual abuse: A gender analysis. Journal of loss and trauma, 10(5), 453-470. Arata, C. M. 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Association of sexual harassment and sexual assault with midlife womens mental and physical health. JAMA internal medicine, 179(1), 48-53. Tuval-Mashiach, R., Freedman, S., Bargai, N., Boker, R., Hadar, H., & Shalev, A. Y. (2004). Coping with trauma: Narrative and cognitive perspectives. Psychiatry: Interpersonal and Biological Processes, 67(3), 280-293. Van Houdenhove, B., Luyten, P., & Tiber Egle, U. (2009). Stress as a key concept in chronic widespread pain and fatigue disorders. Journal of Musculoskeletal Pain, 17(4), 390-399. Vieweg, W. V. R., Julius, D. A., Bates, J., Quinn III, J. F., Fernandez, A., Hasnain, M., & Pandurangi, A. K. (2007). Posttraumatic stress disorder as a risk factor for obesity among male military veterans. Acta Psychiatrica Scandinavica, 116(6), 483-487. Yamaguchi, S., Mino, Y., & Uddin, S. (2011). Strategies and future attempts to reduce stigmatization and increase awareness of mental health problems among young people: a narrative review of educational interventions. Psychiatry and clinical neurosciences, 42 65(5), 405-415. Yuan, N. P., Koss, M. P., & Stone, M. (2006). The psychological consequences of sexual trauma. Retrieved September, 27, 2007. 43 Appendix A 44 Appendix B 1. What is your age? 1. Under 18 2. 18-24 years old 3. 25-34 years old 4. 35-44 years old 5. 45-54 years old 6. 55-64 years old 7. 65-74 years old 8. 75 years or older 2. Please specify your ethnicity: 1. White 2. African American or Black 3. Hispanic or Latino 4. American Indian or Alaska Native 5. Asian 6. Native Hawaiian or Pacific Islander 7. Other 3. Please specify your gender: 1. Male 2. Female 3. Transgender male 4. Transgender female 45 5. Other 6. Prefer not to answer 4. What is the highest degree or level of school you have completed? If currently enrolled, highest degree received. 1. No school completed 2. Nursey school to 8th grade 3. Some high school, no diploma 4. High school graduate or GED equivalent 5. Some college credit, no degree 6. Trade/technical/vocational training 7. Associates degree 8. Bachelors degree 9. Masters degree 10. Professional degree 11. Doctorate degree 5. What is your martial status? 1. Single, never married 2. Married or domestic partnership 3. Widowed 4. Divorced 5. Separated 6. Employment status 1. Employed for wages 46 2. Self employed 3. Out of work and looking for work 4. Out of work but not currently looking for work 5. A homemaker 6. A student 7. Military 8. Retired 9. Unable to work 7. Household income 1. Below $10k 2. $10k-$50k 3. $50k-$100k 4. $100k-$150k 5. Over $150k 47 Appendix C Beginning of vignette (negatives only). Melissa is a 25-year-old woman who lives in the U.S. and has unfortunately already experienced multiple traumatic events. Melissas mother was diagnosed with breast cancer and passed away after a long, hard battle when Melissa was 19 years old. A few years later in college, she experienced a humiliating and debilitating break up with her long-term boyfriend, in which he accused her of cheating and called her a sexually-shaming, and untrue, slur. To make matters worse, Melissa was publicly embarrassed during this traumatic experience because it took place on the campus mall in front of many people, and spread around campus as a rumor of her sleeping around and being easy. Following these events, Melissa felt lonely, frequently woke up having nightmares and flashbacks, felt easily triggered, and would avoid people, places, and conversations that reminded her of the events. Melissa also found herself struggling with finding meaning in life, so she consequently participates in risky behavior such as self-harm, unprotected sex, and drug use. Vignette (positives): Melissa is a 25-year-old woman who lives in the U.S. and has unfortunately already experienced multiple traumatic events. Melissas mother was diagnosed with breast cancer and passed away after a long, hard battle when Melissa was 19 years old. A few years later in college, she experienced a humiliating and debilitating break up with her long-term boyfriend, in which he accused her of cheating and called her a sexually-shaming, and untrue, slur. To make matters worse, Melissa was publicly embarrassed during this traumatic experience because it took place on the campus mall in front of many people, and spread around campus as a rumor of her 48 sleeping around and being easy. While Melissa did notice some long-term negative changes in her life, the majority of the effects she noticed after working through these traumatic incidents were actually very positive. She now feels like her relationships have improved, and she feels empowered and strong due to her resilience through adversity. She finds herself to be more confident in any situation and has identified new possibilities in life that she previously didn't see for herself. She also has grown to be more spiritual and feels that she has an all-around greater appreciation of life. 49 Appendix D Please rate each question below as either 1-4. 4 = very likely 3 = somewhat likely 2 = somewhat unlikely 1 = very unlikely 1. How likely is it that the person described in the vignette has a mental illness? 2. In your opinion, how likely is it that the persons situation was caused by the persons own bad character? 3. In your opinion, how likely is it that the persons situation was caused by a chemical imbalance in the brain? 4. In your opinion, how likely is it that the persons situation was caused by the way the person was raised? 5. In your opinion, how likely is it that the persons situation was caused by stressful circumstances in the persons life? 6. In your opinion, how likely is it that the persons situation was caused by a genetic or inherited problem? 7. In your opinion, how likely is it that the persons situation was caused by Gods Will? 8. In your opinion, how likely is it that the person in the vignette would do something violent toward other people? 50 Please rate each question below as either 1-4. 4 = definitely 3 = likely 2 = unlikely 1 = definitely not How willing would you be to. 2. Move next door to the person? 3. Spend an evening socializing with the person? 4. Make friends with the person? 5. Start working closely with the person? 6. Have the person marry into the family? 51 Appendix E 52 Appendix F Psychological Sciences KEY INFORMATION FOR POTENTIAL RESEARCH PARTICIPANTS Consent is being sought for research that is designed to measure the perceptions people have towards others. Participation in this study is voluntary and should last around 15 minutes. The procedure includes reading a vignette and answering questions in multiple choice formats regarding the passage. There are no risks or benefits to the participant associated with this study. There are no alternative procedures to this study. CONSENT TO PARTICIPATE IN RESEARCH STUDY Perceptions of Trauma Study Principal Investigator (PI): Melissa Loria UIndy Email: loriam@uindy.edu UIndy Telephone: (317) 788-3222 Melissa J. Loria, Ph.D. in the Department of Psychological Sciences at the University of Indianapolis (UIndy) is conducting a research study. Why is this study being done? This study is designed to assess and better understand the perceptions individuals have towards trauma and what it encompasses. What will happen if I take part in this research study? If you volunteer to participate in this study, the researcher will ask you to do the following: Read a short passage a paragraph in length that describes an individual and his or her experiences. This section of the study is experimental as the passages will change slightly for each participant. Answer questions about the individual based on opinion and fact that reference the person in the vignette. o These questions include multiple choice and scaled questions. The entirety of the study will take place online via MechanicalTurk. How long will I be in the research study? Participation will take a total of about 15 minutes. Are there any potential risks or discomforts that I can expect from this study? 53 There are no anticipated risks or discomforts.] Are there any potential benefits if I participate? You will not directly benefit from your participation in the research] The results of the research may bring knowledge to the perceptions of trauma and its victims which can, in turn, assist in further research, reduce stigma towards victims, further psychological treatment, create or modify prevention methods, and more. Will I be paid for participating? You will receive monetary compensation of $1 for completing the study. Will information about me and my participation be kept confidential? The results of this study may be published in a scholarly book or journal, presented at professional conferences or used for teaching purposes. However, only aggregate data will be used. Personal identifiers will not be used in any publication, presentation or teaching materials. Will the data from my study be used in the future for other studies? It is possible that de-identified data from this study could be used for future research or shared with other researchers for use in studies, without additional informed consent. De-identified means that any codes and personal information that could identify you will be removed before the data is shared. What are my rights if I take part in this study? You can choose whether or not you want to be in this study, and you may withdraw your consent and discontinue participation at any time. Whatever decision you make, there will be no penalty to you, and no loss of benefits to which you were otherwise entitled. Who can I contact if I have questions about this study? The Research Team: If you have any questions, comments or concerns about the research, you can talk to the one of the researchers. Please contact: Melissa Loria at 317-788-3222 or loriam@uindy.edu The Director of the Human Research Protections Program (HRPP): If you have questions about your rights as a research participant, or you have concerns or suggestions and you want to talk to someone other than the researchers, you may contact the Director of the Human Research Protections Program, by either emailing hrpp@uindy.edu or calling 1 (317) 781-5774 or 1 (800) 232-8634 ext. 5774. How do I indicate my informed consent to participate in this study? 54 If you consent to participate in this study, then you affirm that you satisfy inclusion criteria and your consent is voluntary. To indicate your voluntary consent and proceed with the questionnaire, click begin to continue to the next page and begin the study.  ...

... Quantification of Ampicillin and Cloxacillin in the Antibiotic Ampiclox using Reversed-Phase HighPerformance Liquid Chromatography By Rachel Caitlin Hurrell Honors Project Thesis Submitted in Partial Fulfillment Of the Requirements for a Baccalaureate Degree With Distinction In the Ron and Laura Strain Honors College of THE UNIVERSITY OF INDIANAPOLIS May 2020 Faculty Advisor: Levi H. Mielke, Ph.D. Executive Director of Honors: James B. Williams, Ph.D. Rachel Hurrell 2020. Copyright in this work rests solely with the author. Please ensure that any reproduction or re-use is done in accordance with relevant national copyright protection. i Abstract Substandard and falsified medical products are increasingly prevalent in developing nations. These products pose a significant threat to human health and place a strain on many resources in the medical industry. Included are pharmaceuticals that contain a different amount or identity of the active ingredient. Such a discrepancy may result in ineffective treatment of illness and inflict harm on the patient. Antibiotics are among the most widely reported classes of substandard and falsified drugs. It is important to the well-being of society to address this growing issue. One effort currently employed to identify these pharmaceuticals is reversed-phase high-performance liquid chromatography (RP-HPLC). Initially, this investigation focused on the development of a method using RP-HPLC to quantify the ampicillin and cloxacillin contained in the antibiotic Ampiclox. Strict guidelines, regulations, and quality assurance procedures specified by the United States Pharmacopeia (USP) must be followed during pharmaceutical analyses. The method was tested to ensure that the separation produces parameters that satisfy USP standards. Following validation, the method was used for the analysis of 21 Ampiclox tablets collected in Liberia. These analyses indicate promising application of the method to future analysis of Ampiclox samples. ii Table of Contents Acknowledgements List of Tables List of Figures Statement of Purpose Introduction Methods/Procedure Results Analysis/Conclusion Reflection References Appendix 1 Appendix 2 iii iv v 1 2 16 28 35 39 42 46 48 iii Acknowledgements Without the generous support of the Shaheen College of Arts and Sciences through their Undergraduate/Scholarly Creative Activity research grant, this investigation would not have been possible. These funds were used to purchase supplies crucial to the investigation. Additionally, the funds were used to support an oral presentation accepted to the National Conference on Undergraduate Research March 2020. Additionally, the support and guidance from the Distributed Pharmaceutical Analysis Laboratory (DPAL) and the University of Notre Dame played a pivotal role throughout the course of the investigation. Specifically, Dr. Marya Lieberman who heads DPAL, as well as Sarah Bliese, offered individual support throughout the method development and pill analysis procedure. Furthermore, Dr. Robyn Goacher from Niagara University also provided support and advice throughout the method development process. Dr. Levi Mielke is an outstanding advisor who offers an expertise in analytical chemistry. Without his guidance this investigation would not have been possible. His extensive knowledge of chromatography techniques fostered my growth and success throughout the process. Finally, the Department of Chemistry at the University of Indianapolis offered extensive support including research space, equipment, reagents, and advice throughout this investigation. Specifically, Dr. Joe Burnell provided guidance throughout the method development process with his experience in HPLC. Dr. Styers-Barnett also provided important input and helped with the writing process. iv List of Tables Table 1: Summary of Preparation of Calibration Dilutions Table 2: Summary of Results for Ampicillin Table 3: Summary of Results for Cloxacillin 20 31 33 v List of Figures Figure 1: Structures of the b-lactam antibiotics ampicillin and cloxacillin Figure 2: Representative chromatogram Figure 3: Example images used in pill documentation Figure 4: Representative calibration curve for ampicillin Figure 5: Representative calibration curve for cloxacillin Figure 6: Intensity versus time plot for ampicillin Figure 7: Intensity versus time plot for cloxacillin 3 21 25 29 30 36 36 1 Statement of Purpose The purpose of this investigation was two-fold. The primary focus of this project was the investigation of drug purity for pharmaceuticals, specifically Ampiclox, sold in developing nations. Pharmaceuticals that contain less than the specified quantity of active ingredient are considered substandard and are increasingly common in developing nations. Such products pose significant risk to the health and well-being of these communities. A secondary focus of the project was to develop a reverse-phase highperformance liquid chromatography (RP-HPLC) method to simultaneously quantify ampicillin and cloxacillin contained within the antibiotic Ampiclox. First, the method was approved by the Distributed Pharmaceutical Analysis Laboratory (DPAL) to confirm that it yields accurate and reproduceable results. Subsequently, the method was used for analysis of a subset of 21 Ampiclox samples obtained from a developing nation. Such an analysis provides insight into the quality of the samples and facilitates a conclusion regarding the purity of the pharmaceuticals. 2 Introduction Antibiotics have played a critical role in society especially since their rising availability between 1940 and 1970.1 This class of drugs alone has doubled the human life expectancy in the 20th century.1 Antibiotics are diverse medications with a widely accepted definition being any chemical substance produced by micro-organisms, which has the capacity to inhibit growth of and even to destroy bacteria and other microorganisms. The action of an antibiotic against micro-organisms is selective in nature, some organisms being affected and others not at all or only to a limited degree; each antibiotic is thus characterized by a specific antimicrobial spectrum.1 The specific antimicrobial spectrum of each antibiotic is an important feature of this drug class and allows for targeted and effective therapy development. One very important and widely used class of antibiotics is the b-lactam class, which will be the focus of this investigation. The b-lactam class is defined by the presence of a highly strained and reactive 4-membered, cyclic amide b-lactam ring. This ring plays an important role in the mechanism by which antibiotics inhibit the growth of bacteria and other microorganisms.2 Specifically, this ring structure binds to the active site of the penicillin binding protein to inhibit the synthesis of the cell wall leading to cell-lysis and death of the bacterium.3 3 Cl NH2 H N H N S O H N H S O O O O O HO O HO Figure 1: Structures of the -lactam antibiotics ampicillin (left) and cloxacillin (right) Antibiotics, especially those in the b-lactam family, have proven to be incredibly effective in their treatment of bacterial infections and ultimately in the improvement in the quality of human life. b-lactam antibiotics are also among the most commonly reported substandard antibiotics.4 A substandard medical product, according to the World Health Organization (WHO), is one that fail[s] to meet either [its] quality standards or specifications or both.5 In addition, medical products can also be falsified meaning that they deliberately/fraudulently mispresent their identity, composition, or source.5 The distinction between substandard and falsified is important in multiple regards. Primarily, while both substandard and falsified medical products pose a significant risk to society, a substandard drug contains some quantity of active drug, but less than the recommended therapeutic dosage. In contrast, a falsified drug may contain no active ingredient, and may even have an alternative substance replacing the active drug which, in some cases, has been a known human toxin making these falsified products particularly dangerous.6 b-lactam antibiotics are not the only medication with reports of substandard samples being distributed to the public. Anti-malarial drugs are most commonly reported 4 as substandard, however, many medicines, vaccines, and other therapeutic categories5 have failed to meet quality standards. Low-quality medical products have become increasing prevalent in society, particularly in those nations faced with widespread poverty. The WHO states that approximately 10% of medical products produced in these nations are either substandard or falisified.5 These products are not restricted to these regions, however. Falsified and substandard medical products impact every region of the world.5 Both substandard and falsified pharmaceutical and therapeutic products pose a serious threat to society and have wide-reaching impacts. Most generally, they increase morbidity (the state of being diseased) and mortality.6 Several specific factors contribute to such drastic and impactful consequences. One such contributing factor is the spread of anti-bacterial resistance.7 When a patient is prescribed a substandard or falsified antibiotic to treat a bacterial infection, not all of the bacteria will be killed due to the lower concentrations of active ingredient. Those bacteria not destroyed by the low dosage of the antibiotic contain mutations that make the bacteria resistant to the drug.7 These remaining bacteria will reproduce and rapidly multiply increasing the population of resistant bacteria.7 The impacts of resistant bacteria do not end with this patient, however. As the patient with resistant bacteria travels and exposes other members of society to the bacteria, the resistance will spread on a large scale.7 In addition, the medication will fail to cure the patients ailment, reducing quality of life and leaving the patient susceptible to complication. Subsequently, substandard drugs also have an economic impact. As 5 doctors search to determine why treatments are ineffective, there is a great deal of money and time lost to superfluous research for alternative treatments.7 It is also important to understand how and why these falsified and substandard products are made available for human consumption. According to the WHO, these medical products make it into the market most commonly when they fill a vacuum.7 In impoverished nations, there is high demand for medicines and diagnostics tests with an extreme lack of resources available to meet those needs.7 As a result, falsified medical products are used in place of their safe and effective counterparts. Manufacturers of these falsified products are driven by an unsavory combination of the ill-informed, the careless, the unprincipled and the criminal allowing them to thrive in places where the technical capacity is poor and the risk of detection is low.7 Healthcare institutions will do whatever it takes to obtain medicines in the face of a rampant outbreak, including sourcing their products from unreliable producers.7 It is clear that the repercussions of substandard and falsified drugs are immense. Unfortunately, this problem is not being adequately addressed. Only 5-15% of the 191 member states of the WHO report cases of counterfeit drugs.6 The WHO suggests that there are various reasons for the staggeringly low numbers of reported cases. One is that reporting processes in affected nations are incredibly complex or, in some cases, nonexistent.7 Additionally, there is a general fear among officials that reporting cases of substandard or falsified products will have grave repercussions.7 For example, prescribers of falsified medicines fear that public exposure of the use of a low-quality product will leave them susceptible to retribution by supervisors, leading to loss of employment, or, in 6 severe cases, prosecution or civil action.7 The WHO is aware of these limitations and is implementing several systems by which such substandard and falsified drugs can be more easily reported. The WHO, for instance, has developed a Global Surveillance and Monitoring System (GSMS). This system is an organized attempt amongst WHO member states to reduce and eliminate the prevalence of falsified and substandard medical products circulating on the market.7 There are several main objectives of GSMS to mitigate the impacts of falsified products on society including, but not limited to, measures to make reporting easier and more accurate, designating focal points throughout the world to allow communication between national and global authorities, and the creation of a global database to coordinate and track reports of falsified products.7 Another simple, yet critical component of these mitigation efforts, is to spread awareness, especially to those directly impacted.7 While there are many areas in which improvements can be made to reduce the harm that these medical products are causing to the public, one area of particular interest is the use of various detection methods both in the field and in the lab. There are several methods currently being employed to take such measurements. Most commonly, falsified and substandard drugs are detected through the use of inspection, colorimetric methods, chromatography, and spectrometry.4 This list is not exhaustive, and each of the aforementioned techniques has several variations that can be used to detect quantities of active drug. Both qualitative and quantitative measurements are paramount to understanding the repercussions of these medical products throughout society. It is important to distinguish between a qualitative and quantitative analysis of a medical 7 product. A qualitative measurement can be used to indicate the presence of an active ingredient.13 As a result, such an analysis is used to quickly distinguish between those drugs with and without the active ingredient.13 A quantitative analysis is used to determine the exact quantity of the active ingredient (or other substances contained in the product) and conclusions using such data can be extended to understand how the drug may impact a patient.13 Both types of analyses are required to complete a comprehensive conclusion regarding drug quality.13 Of current interest to many groups investigating the prevalence of substandard medical products is the availability of rapid testing that can be used in the field to yield immediate conclusions regarding the active drug content of the pharmaceutical. One very important initial screening that can be conducted in any location is a visual inspection. The WHO created a checklist to aid in the identification of potential fraudulent medications using only a preliminary visual inspection. The checklist suggests special attention should be given to the packaging and labeling, as well as the physical characteristics of the drug tablet or capsule itself.8 Such an inspection requires little to no training and does not require access to expensive instrumentation, making it a valuable technique. Although visual inspection can provide a good first indication of the presence of counterfeit products, a more accurate analysis that can offer quantitative and qualitative data is necessary to confirm fraudulent practices. Some other rapid testing methods include portable technologies such as Raman and Near-infrared Spectroscopy (NIR).9 Both NIR and Raman spectroscopy are used to determine a molecular fingerprint based on the vibration of the chemical bonds of the molecule being 8 analyzed.9 In order to yield useful conclusions from the spectra produced by these instruments, a comparison to spectra for known compounds must be completed.10 This is a limiting factor as research must be conducted before a conclusion can be made. Furthermore, these handheld instruments can cost up to $50,000 and thus place an economic restraint on their use.10 On the leading edge of portable technologies for the analysis of pharmaceutical samples is the development of various paper assays that use chromatographic (separation of constituents in the sample) and colorimetric (color change to indicate presence of the component of interest) techniques to indicate the presence of active drugs.10 Such an assay provides a qualitative assessment of the product. When a liquid sample of the medication is placed on the card, capillary action carries the drug up several separate channels, each containing a different reagent used to indicate the presence of the drug.11,12. As the drug comes into contact with the reagent, a reaction, often one that results in a color change, occurs to indicate the presence of the active drug.11,12 Several studies regarding these paper based analyses indicated that overall, these tests show high sensitivity and specificity for analytes and provide a useful chemical profile of active pharmaceutical ingredients (APIs) and excipients within a few minutes.12 One limitation of these paper assays is their inability to distinguish between samples with little active ingredient and no active ingredient.11 It is clear that while portable methods for detection of substandard medical products is important, there are also several limitations on the quantitative data these techniques provide. Often, only techniques carried out in a laboratory by a trained 9 technician allow for quantification of the active ingredient contained in a pharmaceutical sample. One commonly used technique for a quantitative analysis of the sample is highperformance liquid chromatography (HPLC).10 HPLC is generally regarded as the definitive technique for drug content analysis.13 HPLC is the focus of this investigation. While there are many different types of chromatography, they all operate on the same principle: using the different physical properties of each component of a mixture (in this case, the drug) to separate the mixture into its individual components.14 More specifically, chromatographic techniques involve two main components, a stationary phase and a mobile phase, both usually contained in a column.14 The stationary phase (the one that stays in place inside the column) is often a viscous liquid chemically bonded onto the surface of solid particles packed into the column.14 The mobile phase, however, moves through the column freely and is either a liquid or a gas.14 One of the two phases will be polar and the other is nonpolar.14 As a result, the components of the mixture that are polar will be attracted to the polar phase while those that are nonpolar will remain attracted to the nonpolar phase.14 A chromatogram that relates detector response to elution time (the time required for the compound of interest to pass through the column) allows for identification of each component of the mixture.14 Chromatograms produced from chromatography instruments resemble a graph with a number of peaks corresponding to the number of separated analytes. Each peak corresponds to an analyte. Peaks can be identified by their retention time, or the time required for the solute to reach the detector following injection.14 Two important characteristics of chromatograms include the peak height and peak area of each eluted 10 peak. The peak area is found by integrating the peak of interest (to determine the area beneath the curve) while the peak height is the distance from the base of the peak to the top of the peak.14 Each of these quantities is proportional to the concentration or amount of the analyte which reached the detector. Thus, the peak height and area allow for the construction of a calibration curve. A calibration curve relates the instrument response (peak height or area) to the concentration of the analyte. Thus, this plot may be used to determine the concentration of an analyte in an unknown solution. Chromatography is particularly applicable to the identification of pharmaceutical samples as it uses principles which match perfectly with the physicochemical properties of drugs.15 This investigation will specifically use reverse-phase high-performance liquid chromatography (RP-HPLC). Reverse-phase liquid chromatography indicates that a polar mobile phase and a nonpolar stationary phase are used.15 Liquid chromatography must be used in the analysis of pharmaceutical compounds because the sample cannot be volatilized for analysis by gas chromatography.14 In HPLC, high pressure [is used] to force solvent through closed columns containing fine particles that give high-resolution separations.14 The solvent consists of two components: an organic solvent, and a buffered solution. The buffer is particularly important when the analyte is ionizable (can gain or lose a proton due to changes in pH). A buffered solution resists changes in pH so that the analyte remains in only one form. A variety of detectors may be used to identify compounds separated using HPLC. The HPLC used in this investigation utilizes a spectrophotometer. A spectrophotometer is an instrument that determines the absorption of a selected wavelength of light (specific to the compound of interest).14 11 In this investigation, the technique of reverse-phase high-performance liquid chromatography was used to analyze the quantity of active drug contained in Ampiclox. Ampiclox is a combination of the two b-lactam antibiotics ampicillin and cloxacillin (Figure 1).16 Individually, both antibiotics are effective in their treatment of bacterial infections. Both drugs, due to their classification as members of the b-lactam family, inhibit bacterial growth using the mechanism described earlier. More specifically, ampicillin is a broad-spectrum antibiotic that is not resistant to beta-lactamases, the enzymes responsible for resistance to b-lactam antibiotics.17 Cloxacillin is a more narrow-spectrum antibiotic that is resistant to beta-lactamases.17,18 Cloxacillin, however, is limited in its use as it is incompletely absorbed from the gastrointestinal tract.18 The lack of beta-lactamase resistance of ampicillin is satisfied by the resistance of cloxacillin to these enzymes. Furthermore, the malabsorption of cloxacillin by the body is fulfilled by the high bioavailability of ampicillin making the combination of the two drugs a very powerful antibiotic treatment for many bacterial infections.16,17 The focus of this investigation was to develop a RP-HPLC method to simultaneously analyze the presence of both ampicillin and cloxacillin in Ampiclox tablets obtained from third world nations. Specifically, the method development process determined parameters such as solvent, mobile phase, detector wavelength, injection volume, and run time required for the successful separation of the active ingredients contained in pill samples. Samples of this antibiotic are obtained from the Distributed Pharmaceutical Analysis Laboratory (DPAL) led by the University of Notre Dame. DPAL consists of a collection of several academic institutions who are determin[ing] 12 analytically the quality of medicines collected from low- and middle-income countries (LMICs).19 Beginning in the Fall of 2018, the University of Indianapolis became a member of DPAL, granting the university access to pharmaceutical samples for HPLC analysis. Participation in DPAL requires strict system suitability requirements and thus ensures that high quality accurate data are produced.19 These measures of system suitability are concerned with monitoring the accuracy and reproducibility of methods used to analyze pharmaceutical samples. There is strict regulation throughout the method development process, sample analysis, and data processing. The specific metrics that are indicative of the precision of the method are detailed in the methods section. Despite the high level of integrity through which laboratory and data analysis are conducted, DPAL is not a certified pharmaceutical analysis laboratory and any suspicious samples are reported to the WHO and sent to the regulatory agencies that are equipped to perform the compendial testing.19 Not only does working in conjunction with DPAL ensure highly regulated and accurate results, it provides access to pharmaceutical samples for analysis. Samples of pharmaceuticals are collected from small medicine shops across Africa.19 The samples are collected at a central location, the Moi Teaching and Referral Hospital (MTRH), where defining characteristics that can be used to track the drug are recorded before distribution to DPAL.19 It is important to note that antibiotics are not highly regulated in many developing nations. In fact, antibiotics are widely available over the counter, without prescription and through unregulated supply chains making access for analysis by groups such as DPAL easily attainable.20 13 Several successful analyses of multidrug antibiotics have been conducted previously. One such example is the successful separation of Novaclox into its amoxicillin and dicloxacillin constituents by Patil et al. (2014) in their Development and Validation of RP-HPLC Method for Simultaneous Estimation of Amoxicillin and Dicloxacillin in Bulk Drug and Capsules.21 Another successful separation of a multidrug antibiotic was completed by Battula and Shivaraj in their RP-HPLC Method for Simultaneous Estimation of Ampicillin and Cloxacillin in Capsule Dosage Form.22 This paper was also focused on the analysis of Ampiclox. The completion of the simultaneous analysis of the active drugs in Ampiclox previously indicates that method development to detect the active drugs present is attainable. The parameters used in this investigation will be different than those used by Battula and Shivaraj as the method is sensitive and specific to the instrument used. Several adjustments were made to optimize the method for the instruments used in this investigation. Upon initiation of the method development process, it was determined that the method developed in the paper by Battula and Shivaraj was not entirely compatible with the HPLC belonging to the University of Indianapolis (UIndy) Department of Chemistry. Thus, to successfully develop a reliable and accurate method for the analysis and quantification of ampicillin and cloxacillin in Ampiclox, several additional published methods were referenced. Specifically, a method used previously to successfully analyze another drug, Metformin, with the HPLC at UIndy was referenced to estimate a more optimal buffer concentration than that suggested in the method developed by Battula and Shivaraj. This method for analysis of Metformin was published in the HPLC 14 Methodology Manual which is distributed by DPAL. Additionally, a procedure developed by Ashinagar and Naseri to analyze three penicillin antibiotics using HPLC indicated that the wavelength of the detector should be set to 225 nm as opposed to the 254 nm as suggested by Battula and Shivaraj.27 To solidify and further optimize the method, United States Pharmacopeia guidelines for analysis of ampicillin and cloxacillin were referenced. The methods used by the USP suggested that an isocratic analysis, one that maintains a composition of mobile phase, is preferable as compared to a gradient analysis in which the composition of the mobile phase changes over time.24,28 The USP guides also confirmed that a potassium phosphate buffer should be used and that the detector should be set to a wavelength of 225 nm.24,28 The guides were also crucial in the method validation process as they specified the USP requirements for analytical metrics including tailing factors (an indication of the symmetry of the peak), resolution (the degree of separation between peaks), and theoretical plates (the efficiency of the separation). USP guidelines detail stringent requirements for the shape of each eluted peak on the chromatogram. As a result, several resources were used to build a comprehensive understanding of factors which impact peak shape. Primarily, information from Agilent Technologies regarding peak shape in HPLC was used. Most notably, poor peak shape in HPLC is often due to unwanted interactions between the stationary phase and analyte.29 Thus, it is crucial for the production high resolution peaks to minimize these interactions. An HPLC column is most commonly packed with silica which contains an ionizable silanol side chain; this meaning that under certain levels of pH, the groups may become 15 negatively charged.30 Most often, these interactions are the result of working with a buffer that is basic, promoting the transfer of a hydrogen ion from the silanol group on the column to the buffer. This negatively charged group within the column can then interact with the analyte moving through the column, especially if the analyte is positively charged (a common characteristic of many b-lactam antibiotics). Such an interaction results in poor peak shape due to tailing and altered retention times.30 One solution to this problem involves the addition of an acidic mobile phase modifier such as trifluoroacetic acid.29 This mobile phase modifier minimized interactions between the ionized stationary phase and the analyte by protonating the deprotonated silanol groups.29 A successful analysis of Ampiclox has important and wide-reaching implications. If the amount of active drug contained in the pharmaceutical sample obtained from DPAL and MTRH is unusual, the data will be reported to the WHO for further investigation. Reporting suspicious drug samples to the WHO is important in the mitigation of the negative impacts that substandard and falsified medical products present in developing nations. An increased awareness and detection of these products can ultimately be used to reduce the number of the products available for consumption, thus reducing their negative impacts on society. 16 Methods/Procedure Sampling: In this investigation, a method to separate ampicillin and cloxacillin contained in the antibiotic Ampiclox using reversed-phase high-performance liquid chromatography (RP-HPLC) was developed. Additionally, this method was used to quantify the APIs present in Ampiclox tablets obtained from a developing nation. The Ampiclox tablets supplied by the Distributed Pharmaceutical Analysis Laboratory (DPAL) were obtained from drug stores in Liberia.19 Pharmaceuticals in developing nations such as Liberia are easy to obtain as the drugs are commonly available over the counter, without prescription and through unregulated supply chains.20 Upon receipt, DPAL processed the pharmaceuticals, and assigned them identification numbers and identified defining characteristics such as packaging details.19 These identification numbers were used throughout the analysis to ensure the identity of each pill was preserved. The specific brand and dosage (amount of active pharmaceutical ingredient [API]) of the pharmaceutical are unknown. Twenty-one pills were obtained for quantification of API. Sample Preparation: Safe Handling: Throughout this investigation, chemicals that have the potential to be toxic or dangerous were used. Appropriate precautionary measures were taken to minimize the risk for any harm. The hazards posed by each chemical were thoroughly studied before handling the chemical (links to relevant Safety Data Sheets (SDS) included in Appendix 17 1). Proper laboratory attire (long pants, closed toed shoes) and protective equipment (goggles and nitrile gloves) were worn at all times. Special care was given when handling the pharmaceutical products since they contain active pharmaceutical ingredients with known biological impacts. System Suitability: DPAL requires that all of its collaborators observe strict system suitability requirements to ensure accurate and precise data are collected. Before proceeding with the quantification of the API present in Ampiclox pills, several metrics demonstrating the reliability of the method were met. First, several standard solutions of ampicillin and cloxacillin were prepared. Standard solutions were used to create a calibration curve which was used to quantify the API present in the Ampiclox pills obtained from DPAL. A calibration curve relates the area beneath the peak on the chromatogram to the concentration of that analyte. DPAL requires a calibration curve with a correlation coefficient of 0.98 or greater.23 The high correlation coefficient indicates that the standards used were properly prepared and lie within the linear dynamic range of the instrument; the range of concentrations over which the instrument response and concentration vary according to a linear relationship.14 This is a metric that ensures accurate quantification of API. All standard solutions were prepared in calibrated glassware. Glassware is designed to give accurate measurements at 25 C and assumes no imperfections. Laboratory conditions do not always match these ideal conditions. This correction 18 eliminated any error resulting from the expansion and contraction of borosilicate glass at temperatures other than 25C or any other imperfections in the glassware. The procedure for the calibration of all glassware used in the experiment is as follows. First, the ambient room temperature was measured in order to determine the corresponding density of water. Next, the selected piece of glassware was carefully filled to its appropriate capacity with de-ionized (DI) water. The mass of this water was then measured with a balance and recorded. The glassware was dried, and the procedure was repeated twice more to yield a total of three masses of water. Each of these measurements was corrected for buoyancy and the expansion of borosilicate glass.14 Following this correction, using the density of water, the true volume of water held by each piece of glassware could be accurately determined. An average of the corrected volume for each of the three trials was used in any calculation in which the volume of the solution was relevant. To prepare the standard solutions that were used to produce a calibration curve, five samples with concentrations ranging from approximately 5%-200% of the expected quantity of active ingredient was prepared. In this investigation, the Ampiclox pills are expected to contain 250 mg ampicillin and 250 mg cloxacillin. The specific range of calibration standards prepared were as follows: 500, 350, 250, 100, 50, and 25 parts per million(ppm) or mg/L. The standard solutions were made from secondary pharmaceutical standards of ampicillin and cloxacillin obtained from the chemical sales company Sigma Aldrich. A stock solution of ampicillin and cloxacillin of concentration 500 ppm was prepared in a calibrated 250 mL volumetric flask. To prepare this stock solution, a mass of 0.1250 g each of ampicillin and cloxacillin was obtained and transferred to the 19 volumetric flask. Approximately 125 mL of DI-water was added to the flask to allow for the APIs to be dissolved and thoroughly mixed. Once the solution was homogenous, the remaining water was added to bring the solution to the 250 mL mark on the glassware. One must account for the purity of each standard to determine the exact concentration of the stock solution. Sigma Aldrich provides a certificate of analysis that lists the exact purity of each lot of standard (appendix 2). The purity (91.6% for ampicillin and 94.4% for cloxacillin), in combination with the corrected volume of the calibrated volumetric flask, was used to determine an exact concentration for prepared stock solutions. To prepare the remaining calibration standards, the 500 ppm ampicillin/cloxacillin stock solution was diluted using appropriate volumetric pipets and 50 mL volumetric flasks. To prepare the 350 ppm standard, 35 mL of the 500 ppm stock solution was measured and transferred to the 50 mL volumetric flask. This solution was brought to 50 mL with DI-water and inverted to mix thoroughly. As above, the exact concentration of each dilution was determined by considering the exact volume of the glassware and the purity of the standards. This general procedure was repeated for all remaining concentrations of standard solutions; the calculated volumes necessary to prepare each dilution are summarized in Table 1. 20 Table 1: Summary of Preparation of Calibration Dilutions Desired Concentration (ppm) Volume of Stock Solution (mL) 350 35 250 25 100 10 50 5 25 2.5 The 0.01M potassium dihydrogen phosphate (KH2PO4) buffer at a pH of 3 with 0.1% reagent grade tri-fluoroacetic acid (TFA) was used for all analyses on the instrument. 1.0 L of the buffer was prepared at a time to minimize risk for algal growth. The buffer was prepared by dissolving 1.3619 g KH2PO4 in less than 1.0 L of DI water. Once all KH2PO4 was thoroughly dissolved, 1 mL of TFA was added and mixed. The pH was adjusted to 3 with the addition of H3PO4 and KOH. These components were added dropwise while monitoring the pH with a pH probe. Once the desired pH was achieved, the buffer was transferred to a 1.0 L volumetric flask and was brought to a volume of exactly 1.0 L. All prepared solutions must be filtered using specialized HPLC filtration systems to prevent clogging and contamination of the HPLC column. The buffer was filtered using a vacuum filter and the standards were filtered using Whatman 0.45 micron PTFE disposable syringe filters. This allows for filtration of only small volumes as necessary. 21 Additionally, Sigma-Aldrich HPLC grade acetonitrile was purchased so that possible chemical interferences were not introduced into the instrument. The HPLC used throughout this investigation is an Agilent InfinityLab LC Series 1220 Infinity II LC System fitted with an Agilent Eclipse C18 column. A gradient elution proceeding from 90% buffer to 36% buffer (the remaining percentage is acetonitrile) over 12 minutes maximized the separation and resolution of the APIs (Figure 2). Between injections, a four-minute re-equilibration time was incorporated to prepare the instrument for the next separation. During this re-equilibration period, the instrument was returned to the initial 90% buffer 10% acetonitrile ratio. The 16-minute run begins at time 0.0 when the sample is injected. The flow rate of the instrument was 1.0 mL/min with an injection volume of 20 L.22 The detection wavelength was set to 225 nm.22 Normal Ampiclox Chromatogram 1600 Intensity (mAu) 1400 1200 1000 800 600 400 200 0 -200 0 5 10 Time (min) 15 20 Figure 2: Representative Chromatogram (ampicillin left peak, cloxacillin right peak) 22 These parameters were used to produce the linear calibration curve. The calibration was constructing according to the following steps. A small aliquot of each calibration standard was filtered into an HPLC vial. These vials were labeled and inserted into the HPLC for analysis. Further system suitability requirements included demonstrating the precision of the instrument through the calculation of the relative standard deviation (RSD) of 6 consecutive injections of the known normal standard.23 The RSD value for these replicates should be less than 2%.23 The 250 ppm external standard (prepared as described above) in the middle of the linear dynamic range of the instrument was used to satisfy this requirement. This test ensured that the instrument yielded a consistent response over the course of several injections. Consistency is imperative because several consecutive injections into the instrument were made during the analysis of Ampiclox pills. Consistency throughout the injections is critical to the accuracy of API quantification. In addition, quantification of API in an overdosed, normal, and deficient sample (a standard containing ~150%, ~100, and ~35% of the normal concentration of API respectively), was determined.23 Three replicate injections of each sample were run with an external standard run every fourth measurement to ensure the instrument was yielding reproducible data.4 To meet system suitability requirements, the RSD needed to be less than 2% for the external standard and each of the overdosed, normal, and deficient standards should be within 2% of the true concentration.23 The successful completion of this requirement demonstrated that if an Ampiclox pill contained an amount of API different than expected, the method accurately quantified the amount of API present. 23 Accuracy of the method using a spike recovery was also demonstrated. An Ampiclox tablet from a known source (one that contains a known concentration of API) obtained from DPAL was prepared by dissolving the pill in DI water and diluting it to a concentration within the linear range of the instrument. An aliquot of this pill solution was spiked with ~30% API from the external standards.23 The concentration of API in this spiked pill solution was determined and had to fall within 90-110% of the true value.23 This metric indicated that the instrument response was proportional to changes in concentration. The specificity of the instrument was confirmed by the successful completion of a spike recovery from a degraded dosage form matrix.23 This solution was created by baking the tablet for an hour at 60 C, then spiking the pill with an extra ~30% of the pure API and measure the spike recovery.23 This metric measured the ability of the instrument to detect the API in the presence of a complex matrix, or one containing unexpected and unpredictable compounds. Such a matrix could potentially be present in pills that are substandard or falsified. It is important that even in the presence of such an obscure matrix, the API could still be accurately quantified and detected by the instrument. Finally, the limit of detection (LOD) and lower limit of quantification of the instrument must be reported (LLOQ). This was determined by calculating the standard deviation of the peak area of a standard solution near the estimated LOD (from the calibration curve) and the slope of the calibration curve. The LLOQ is a measure of the smallest possible quantity of drug that can be accurately quantified by the HPLC 24 instrument. The LOD is the smallest amount of drug that is statistically different from the blank. These limitations are important to determine as pill solutions should not be prepared below these concentrations to ensure accurate quantification of API. Throughout the sample analysis, an external standard was injected after every fifth run of the instrument. The peak height for this standard was compared to the six replicate injections. If the RSD was within 2%, the instrument was yielding precise, consistent data, and analysis could proceed. If the peak heights differed by more than 2%, the linearity of the instrument had to be reestablished with five external standard solutions as described above. Sample Analysis: System suitability requirements were met in May 2019, resulting in the authorization of UIndy to receive Ampiclox sample pills from DPAL. Quantification of ampicillin and cloxacillin contained in each pharmaceutical tablet were determined using the same RP-HPLC and method parameters used in the system suitability process as described above. Similar to the system suitability and method development process, there are strict requirements that must be followed throughout the analysis of Ampiclox pills. Most notably, tracking the identity of all pills is crucial to the integrity of the project. Each pill has a unique identification code, the Notre Dame Identification Number (NDID). This coding system is used to track each sample pill so that the results may be reported back to DPAL. Each NDID has three individual pills associated with it. All three pills have been obtained from the same manufacturing facility. Each pill in a set is 25 labeled pill A, B, and C upon receipt at UIndy. This designation is arbitrarily made by the analyst. Such a classification allows for additional tracking, as well as an additional level of quality control. Additionally, a photograph of the pills was captured and catalogued alongside the NDID and any other identifying information obtained upon visual inspection (Figure 3). The left and center images show the packaging and Ampiclox capsules. The rightmost image shows the contents of an Ampiclox capsule. Figure 3: Example images used in pill documentation Before analysis of any pills can be done, five injections of a 250 ppm external standard must yield a peak area within 2% relative standard deviation and the retention times of these peaks must be within 30 seconds.23 Throughout the analysis, after every fifth injection of the pill, a 250 ppm external standard was run, and its intensity had to be within 2% of the five initial injections preceding initiation of analyses. Each injected pill was considered to pass analysis if the measured quantity of ampicillin and cloxacillin was within 90-110% of the advertised amount.23 Pill A is the first pill to be analyzed. If this sample fails analysis, a second portion of pill A would be analyzed. If this pill fails a second time, a portion of pill B would analyzed. If the pill is still outside of the range of 90-110%, the sample is returned to DPAL for further analysis. If the sample fails at this level, a letter is sent to the WHO for possible action. 26 All Ampiclox pills received in this investigation were gel-capsules. After all procedures for documenting the pill were completed, the capsule was removed from the packaging and the mass of the whole pill was recorded. Subsequently, the gel-capsule was gently separated, and the contents were removed. The mass of the empty capsule was recorded, and the mass of the contents was determined by difference. The theoretical dosage of each pill was 250 mg ampicillin and 250 mg cloxacillin. Due to excipients found in pharmaceutical products (non-API additives to aid in the production and increase bioavailability of the drug to the patient31), the mass of the contents of the pill was greater than 500 mg. As a result, the mass to be weighed was determined using ratios. If the entire pill was to be dissolved in a 1.0 L flask, the concentration of the resulting solution would be 250 ppm of each API. Instead, only a tenth of the pill was prepared at a time. Thus one-tenth of the mass of the whole pill was dissolved in a 100 mL calibrated volumetric flask. The exact mass of the portion of pill used in the analysis was recorded for each sample. As the external standards were prepared, the contents of the pill were dissolved in DI-water. To ensure that the entire pill was thoroughly dissolved, sonication was used. The final step in the preparation for analysis was filtration of the solutions using syringe filters as described in the system suitability procedures above. Sample Storage: Once prepared, any solutions containing the API were stored in a chemical only refrigerator (2C) available in the Department of Chemistry to minimize the degradation 27 of the active ingredients over time. The prepared solutions were stored in the refrigerator for up to a month before fresh solutions were prepared to guarantee the integrity of the external standards and pill solutions were maintained. It was found that the ampicillin and cloxacillin standards degraded if stored for periods longer than one week. Thus, new external standard solutions were prepared before all pill analyses. Filtered buffers and mobile phases that did not degrade at room temperature were stored in covered 1.0 L bottles to prevent contamination. 28 Results Calculations: Before a determination regarding the quality of the pill can be made, the theoretical concentration of each capsule solution must be determined. This calculation considers the mass of the excipients in the pill as well as the calibrated volume of the glassware used to prepare the solution. () = () () () () Despite the fact that there are two active ingredients in each capsule, the calculation for theoretical concentration is the same for both active pharmaceutical ingredients (APIs). This is attributed to the fact that the pills contain both ampicillin and cloxacillin as a part of a complex matrix of excipients. The theoretical mass of Ampiclox for all samples is 250 mg as indicated by the manufacturer. The volume of solution is known as a result of the calibration of all glassware, and the remaining masses were recorded during the preparation of the sample solutions. Additionally, it was discovered that the dosage form of the standard ampicillin and cloxacillin needed to be taken into consideration. That is, both ampicillin and cloxacillin are manufactured and distributed in their salt form (ampicillin sodium and cloxacillin sodium). Thus, not only must the concentration of the standards be corrected 29 for their purities, but also for the difference in molar mass due to the presence of sodium. This correction is applied as shown below. = () = () Once the theoretical concentration has been determined, the actual concentration determined via analysis with high-performance liquid chromatography (HPLC) can be calculated. Figures 4 and 5 show a characteristic calibration curve for ampicillin and cloxacillin, respectively. These plots were constructed from six concentrations of standard ampicillin and cloxacillin within the linear dynamic range of the instrument. Ampicillin Calibration Curve 7000 6000 Peak Area 5000 4000 3000 y = 13.338x + 33.033 R = 0.9997 2000 1000 0 0.00 50.00 100.00 150.00 200.00 250.00 300.00 350.00 400.00 450.00 500.00 Concentration (ppm) Figure 4: Representative calibration curve for ampicillin 30 Cloxacillin Calibration Curve 20000 18000 16000 Peak Area 14000 12000 10000 8000 y = 37.301x + 145.97 R = 0.9992 6000 4000 2000 0 0.00 50.00 100.00 150.00 200.00 250.00 300.00 350.00 400.00 450.00 500.00 Concentration (ppm) Figure 5: Representative calibration curve for cloxacillin The linear regression lines for these plots yield an equation from which the concentration of the pill sample may be determined. The generic equation used to determine the concentration of ampicillin is shown below. () = (-) In this equation, the measured peak area was the y-value, and the equation was solved algebraically for x (the concentration in ppm). This calculation was repeated for all ampicillin and cloxacillin samples. Once the theoretical and experimentally measured concentrations had been determined for all pill samples, the percent difference was calculated as shown below. 31 = 100% Using this equation, a positive percent difference indicates an overdosed pill while a negative percent difference indicates a deficient pill. Following Distributed Pharmaceutical Analysis Guidelines (DPAL) guidelines, a pill fails the analysis when the percent difference is greater than 10%. Tables 2 and 3 provide a summary of the theoretical, experimentally determined, and percent difference in concentration for all 21 analyzed pill samples. Data: Table 2: Summary of Results for Ampicillin Pill ID NDID-19L0001 NDID-19L0002 NDID-19L0003 NDID-19L0004 NDID-19L0005 NDID-19L0006 NDID-19L0007 NDID-19L0008 Theoretical Concentration Ampicillin (ppm) Measured Concentration Ampicillin (ppm) Percent Difference (%) Preliminary Assessment of Pill Quality 252.43 265.06 5.00 N/A 250.81 282.48 12.63 Overdosed 253.75 201.81 -20.47 Deficient 249.52 246.27 -1.30 Pass 250.99 267.71 6.66 Pass 250.54 264.39 5.53 N/A 248.07 256.00 3.20 Pass 251.78 342.09 35.87 Overdosed 32 Table 2, Continued Pill ID NDID-19L0009 NDID-19L0010 NDID-19L0011 NDID-19L0012 NDID-19L0013 NDID-19L0014 NDID-19L0015 NDID-19L0016 NDID-19L0017 NDID-19L0018 NDID-19L0019 NDID-19L0020 NDID-19L0021 Theoretical Concentration Ampicillin (ppm) Measured Concentration Ampicillin (ppm) Percent Difference (%) Preliminary Assessment of Pill Quality 214.37 254.49 18.71 Overdosed 246.39 201.30 -18.30 Deficient 252.05 256.81 1.89 Pass 251.20 263.19 4.77 N/A 215.98 260.02 20.39 Overdosed 213.33 266.30 24.83 Overdosed 249.95 270.78 8.34 N/A 249.58 259.77 4.08 Pass 249.24 266.91 7.09 Pass 253.99 247.18 -2.68 N/A 249.85 216.78 -13.24 N/A 248.55 265.76 6.93 Pass 216.24 260.68 20.55 Overdosed 33 Table 3: Summary of Results for Cloxacillin Pill ID NDID-19L0001 NDID-19L0002 NDID-19L0003 NDID-19L0004 NDID-19L0005 NDID-19L0006 NDID-19L0007 NDID-19L0008 NDID-19L0009 NDID-19L0010 NDID-19L0011 NDID-19L0012 NDID-19L0013 NDID-19L0014 NDID-19L0015 NDID-19L0016 NDID-19L0017 NDID-19L0018 Theoretical Concentration Ampicillin (ppm) Measured Concentration Ampicillin (ppm) Percent Difference (%) Preliminary Assessment of Pill Quality 252.43 152.15 -39.73 N/A 250.81 266.25 6.16 Pass 253.75 26.66 -89.49 Deficient 249.52 253.23 1.49 Pass 250.99 233.10 -7.13 Pass 250.54 161.65 -35.48 N/A 248.07 201.41 -18.81 Deficient 251.78 289.73 15.07 Overdosed 214.37 138.36 -35.46 Deficient 246.39 26.79 -89.13 Deficient 252.05 223.44 -11.35 Deficient 251.20 240.86 -4.12 N/A 215.98 215.13 -0.40 Pass 213.33 229.75 7.70 Pass 249.95 205.27 -17.87 N/A 249.58 245.45 -1.66 Pass 249.24 274.34 10.07 Overdosed 253.99 265.47 4.52 N/A 34 Table 3, Continued Pill ID NDID-19L0019 NDID-19L0020 NDID-19L0021 Theoretical Concentration Ampicillin (ppm) Measured Concentration Ampicillin (ppm) Percent Difference (%) Preliminary Assessment of Pill Quality 249.85 239.16 -4.28 N/A 248.55 243.76 -1.93 Pass 216.24 230.44 6.57 Pass When making the preliminary assessment of pill quality, any pill with a percent difference within the 10% range passed analysis. Any pill with a percent difference less than -10% or greater that +10% was deemed deficient or overdosed, respectively. It was not possible to make any conclusions regarding pills that were analyzed when the relative standard deviation of the intensity of 250 ppm external standard was outside of the accepted 2% range (indicated by N/A in Tables 2 and 3). 35 Analysis/Conclusion The results obtained in this analysis are a reflection of a preliminary analysis of 21 Ampiclox pills obtained from the Distributed Pharmaceutical Analysis Laboratory (DPAL). Overall, this was a successful analysis yielding meaningful results. Not only can the results indicate the quality of the Ampiclox sample pills, but they are also a reflection of the successful development of a method to analyze these samples. Despite these significant successes, it was observed that a remarkably high number of samples were discovered to be outside of the accepted range of 10% difference. Such a result suggests that there may be some weakness in the methods used and that revisions are necessary. These surprising results prompted conversations with DPAL and a collaborating university (Niagara University) also developing a method for analysis of Ampiclox. After conversations with Dr. Robyn Goacher at Niagara University, and upon thorough analysis of data and procedures, a major concern developed regarding the stability of the prepared external standard solutions. A plot of the intensity of the peak area of the 250 ppm external Ampiclox standard over time revealed that there is a significant decrease in intensity corresponding to the degradation of the standard (Figures 6 and 7). The upper and lower limits for the intensity are denoted by the dashed lines. 36 Ampicillin External Standard PEak Area (mAb) 3300 3250 3200 3150 3100 3050 3000 0 5 10 15 20 25 Days Since Calibration Figure 6: Intensity versus time plot for ampicillin Cloxacillin External Standard 9900 Intensity (mAb) 9800 9700 9600 9500 9400 9300 0 5 10 15 20 25 Days Since Calibration Figure 7: Intensity versus Time Plot for cloxacillin The most significant concern regarding the analysis of pill solutions was the stability of the standard solutions over the period required for preparation and analysis of all pills. To most effectively remove the errors in the results associated with degradation of the standard over time, additional pill analyses will be completed within one week of 37 calibration and preparation of the standard solutions. New standard solutions will be made before the analysis of any new group of pills. Although the majority of the data was deemed inconclusive and requires reevaluation, there are several pills with a percent difference of greater than 35%. Most notably, NDID-19L-003 and NDID-19L-0010 had 89.49% and 89.13% deficiency in cloxacillin, respectively. Without further analysis, the significance of these results may not be directly concluded. Such a large deficit, however, is concerning and confirms that high-performance liquid chromatography (HPLC) analysis, with refinements, may be a valuable tool in quality control testing of pharmaceutical products. Future directions of this project will focus largely on re-analyzing pill samples with consideration for the lack of stability of the standards. Once the re-evaluation of this initial subset of 21 Ampiclox capsules is completed, findings will be reported to DPAL. Subsequently, the method may be used to analyze additional pills obtained from DPAL. The purpose of this investigation is multi-faceted. Not only was it intended to achieve the broad goal of yielding results that are meaningful in detecting substandard and falsified medications, but also to develop a novel method for the analysis of Ampiclox using RP-HPLC. It is important to highlight that although the results acquired in the first round of analyses are not believed to be a true and accurate representation of the quality of the pills, the data did provide important insights into the success of the method development, as well as the validity of the procedure of analysis. By assessing the success of each of these purposes, important changes to future procedures were determined. Furthermore, throughout the analysis process, the chromatograms were 38 consistent and reproducible. The tailing factors, resolution, and retentions times remained well within DPAL specifications indicating the method is an effective separation of the active pharmaceutical ingredients (APIs). 39 Reflection This honors investigation was successful and facilitated growth and learning in a multitude of disciplines. The first and major goal of this project was the development of a reverse-phase high-performance liquid chromatography (RP-HPLC) method that could be used to simultaneously separate and quantify ampicillin and cloxacillin in Ampiclox. This method development process, although ultimately successful, was long, extensive, and frustrating. In the earliest stages, previously published literature was used as a guideline for development. It was rapidly realized that the methods published in the literature were not compatible with the instrumentation used in this project. As a result, I began to build a knowledge-base and skillset from which I could develop a method based on experimentation alone. Throughout this process, I faced many setbacks and challenges. Within the first 24 hours of beginning my research, I used a very concentrated buffer which precipitated with the organic acetonitrile mobile phase to clog the HPLC. This occurrence impeded progress in my work early on and for an extended period. I spent time on the phone with Agilent customer support to replace parts and remove precipitate from the tubing in the instrument. A few days later, I not only emerged victorious, but also well versed in the inner workings of the HPLC. Conversations with instrument manufacturers (such as my conversation with Agilent Technologies described above) were important in developing the skill of troubleshooting. It is important to be able to convey the problem concisely and accurately when talking in real-time to experts in the field. Throughout the investigation, I was forced to identify and locate resources to help me solve encountered problems. Broadly, 40 this trouble-shooting process facilitated the development of a valuable skillset that is widely applicable in many situations. Shortly after overcoming this first set-back, I encountered another challenge. After many manipulations of buffer composition and gradients of elution, I had achieved adequate separation of ampicillin from cloxacillin. The peak shape as indicated by the tailing factor, however, was far outside of United States Pharmacopeia (USP) guidelines. After extensive research and discussion, in addition to much trial and error, it was theorized that the poor peak shape was due to interaction of the charged groups on the active pharmaceutical ingredients (APIs) with ionized silanol groups inside of the column. To solve this problem, a competitor acid was added to the buffer. This acid protonates the ionized silanol groups in the column and prevents unnecessary interactions in the column from producing warped peaks. With this correction, the method passed all system suitability requirements as specified by the Distributed Pharmaceutical Analysis Laboratory (DPAL), and the method was accepted for analysis of pharmaceutical samples. Analysis of Ampiclox pills facilitated the larger goal of the project to be achieved; the quality of an antibiotic obtained from a developing nation could be successfully and adequately assessed. Throughout the analysis of pill samples, there are strict guidelines and requirements that must be met to ensure the results are meaningful and significant. Thus, skills of communication, diligence, and organization are required to successfully complete the analysis. Upon collection of preliminary data, an evaluation of the validity of the analysis procedure was required. A critical evaluation of results revealed that there 41 was some error in the treatment of data to be addressed before proceeding in sample analysis. Specifically, it was important to realize that the external standards degrade over time. This finding has inspired me to pursue further analysis of this initial set of 21 Ampiclox pills. Completion of this task will ultimately fulfill my desire to make an impact on the lives of others by reducing the distribution of substandard and falsified pharmaceuticals. All aspects of this project required critical thinking and problem solving; skills that are invaluable both personally and professionally. I use the skills I developed while completing this project every day. They are particularly applicable to me as I will be attending graduate school in chemistry in the fall and will be continuing with research in this field. Not only did I learn important scientific procedures and techniques, but I have developed skills of writing and communication that are important in sharing results with a larger group. Importantly, I found great joy and satisfaction in working on a project with such relevance and importance. I am so grateful to have been given the opportunity to learn and execute these skills and feel that they will facilitate much success in my future endeavors. 42 References 1. Demain, D.L., Sanchez, S., Eds. Antibiotics: Current Innovations and Future Trends; Caister Academic Press: Norfolk, UK, 2015; pp xi-15. 2. Amador, P; Fernandes, R; Prudencio, C. b-Lactams: chemical structure, mode of action and mechanisms of resistance. Rev. in Med. Microbiol. 2013, 24, 78. 3. Lin, J; Ximin, Z. Beta-Lactamase induction and cell wall metabolism in Gramnegative bacteria. Front. Microbiol. 2013, 4, 1-2. 4. Falagas, M.E.; Kelesidis, T. Substandard/counterfeit antimicrobial drugs. Clin. Microbiol. 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Battula, S.; Shivaraj, A. RP-HPLC Method for the Simultaneous Estimation of Ampicillin and Cloxacillin in Capsule Dosage Form. Int. J. App. Pharm. Sci. Research 2017, 2, 93-98. 23. Lieberman, M. HPLC Methodology Manual Distributed Pharmaceutical Analysis Laboratory (DPAL). unpublished work. 24. United States Pharmacopeia and National Formulary [online]; USP 42-NF 371107; United States Pharmacopeial Convention: Rockville, MD, 2019. https://online.uspnf.com /uspnf/docu ment/GUID-7 ED2261C-2EA5457A-B076-7AB6D07017CF_1_en-US 25. Compound Summary for CID 6249: Ampicillin. https://pubchem.ncbi.nlm. nih. gov/comp ound/ampicillin#section=2D-Structure (accessed March 7, 2019), Pub Chem Open Chemistry Data Base. 26. Compound Summary for CID 6098: Cloxacillin. https://pubchem.ncbi.nlm.nih. gov/comp ound/cloxacillin#section=2D-Structure (accessed March 7, 2019), Pub Chem Open Chemistry Data Base. 27. Ashnagar, A., Naseri, N.G. Analysis of Three Penicillin Antibiotics (Ampicillin, Amoxicillin, and Cloxacillin) of Several Iranian Pharmaceutical Companies by HPLC. E-J. Chem. 2007, 4, 536-545. 28. United States Pharmacopeia and National Formulary [online]; USP 42-NF 37322; United States Pharmacopeial Convention: Rockville, MD, 2019. https://online.uspnf.com /uspnf/docu ment/GUID-57A2342F-D116-4DC3920C-5AFD817C099_3_en-US 29. The Secrets of Good Peak Shape in HPLC. https://www.agilent.com/cs/ library/eseminars/ Public/secrets %20of %20good%20peak%20shape %20in%20hplc.pdf 30. Neue, U.D.; Niederlanender, C.L.;Peterson, J.S. Liquid Chromatography Stationary Phases with Reduced Silanol Interactions. 5,374,755. Dec. 20, 1994. 31. Rowe, R.C.; Sheskey, P.; Quinn, M. Preface. Handbook of Pharmaceutical Excipients,6; Pharmaceutical Press: London, UK: 2009; x. 32. Certificate of Analysis: Ampicillin Sodium. https://www.sigmaaldrich.com/ catalog/CertOfAnalysisPage.do?symbol=PHR1424&LotNo=LRAA9206& brandTest=SIAL&returnUrl=%2Fproduct%2FSIAL%2FPHR1424 (accessed Mar 22, 2020). Sigma-Aldrich. 45 33. Certificate of Analysis: Cloxacillin Sodium. https://www.sigmaaldrich.com/ catalog/CertOfAnalysisPage.do?symbol=PHR1922&LotNo=LRAB1227& brandTest=SIAL&returnUrl=%2Fproduct%2FSIAL%2FPHR1922 (accessed Mar 22, 2020). Supelco. 46 Appendices Appendix 1: SDS Sheets Ampicillin Sodium: https://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=US&langu age=en&productNumber=PHR1424&brand=SIAL&PageToGoToURL=https%3A%2F% 2Fwww.sigmaaldrich.com%2Fcatalog%2Fproduct%2Fsial%2Fphr1424%3Flang%3Den Cloxacillin Sodium: https://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=US&langu age=en&productNumber=PHR1922&brand=SIAL&PageToGoToURL=https%3A%2F% 2Fwww.sigmaaldrich.com%2Fcatalog%2Fproduct%2Fsial%2Fphr1922%3Flang%3Den HPLC Grade Acetonitrile: https://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=US&langu age=en&productNumber=437557&brand=SIAL&PageToGoToURL=https%3A%2F%2F www.sigmaaldrich.com%2Fcatalog%2Fproduct%2Fsial%2F437557%3Flang%3Den Tri-fluoroacetic Acid https://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=US&langu age=en&productNumber=T6508&brand=SIGALD&PageToGoToURL=https%3A%2F% 2Fwww.sigmaaldrich.com%2Fcatalog%2Fsearch%3Fterm%3Dtrifluoroacetic%2Bacid% 26interface%3DAll%26N%3D0%26mode%3Dmatch%2520partialmax%26lang%3Den% 26region%3DUS%26focus%3Dproduct 47 Potassium Dihydrogen Phosphate: https://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=US&langu age=en&productNumber=1.04873&brand=MM&PageToGoToURL=https%3A%2F%2F www.sigmaaldrich.com%2Fcatalog%2Fsearch%3Fterm%3Dpotassium%2Bdihydrogen% 2Bphosphate%26interface%3DAll%26N%3D0%26mode%3Dmatch%2520partialmax%2 6lang%3Den%26region%3DUS%26focus%3Dproduct 48 Appendix 2: Certificates of Analysis for Ampicillin and Cloxacillin32,33 49  ...

... Prosocial Music and Mood By Skylar Haywood Honors Project Thesis Submitted in Partial Fulfillment Of the Requirements for a Baccalaureate Degree With Distinction In the Ron and Laura Strain Honors College of THE UNIVERSITY OF INDIANAPOLIS May 2020 Faculty Advisor: Kathryn L. Boucher, Ph.D. Executive Director of Honors: James B. Williams, Ph.D. Skylar Haywood 2020. Copyright in this work rests solely with the author. Please ensure that any reproduction or re-use is done in accordance with relevant national copyright protection. i Abstract Past research has identified increases in prosocial behavior in people who listen to a prosocial song, relative to those who listened to a neutral song. However, research explaining how prosocial music can lead to prosocial behavior is understudied. In the current study, I aimed to investigate the role of mood as it related to a prosocial songs influence on prosocial intentions. It was hypothesized that those who listen to the prosocial song would report greater positive affect than those who listen to the neutral song. Additionally, it was hypothesized that those who listen to the prosocial song would report greater prosocial intentions than those who listen to the neutral song. This study used a between-subjects research design where participants were randomly assigned to listen to a prosocial (experimental condition) or a neutral (control condition) song. Participants listened to their assigned song, then reported their affect, performed a manipulation check, and, finally, were assessed on their prosocial intentions. Neither hypothesis was supported, but an interaction revealed that the neutral condition had higher positive affect ratings than the prosocial condition. Also, the mean scores of prosocial intentions revealed that those in the neutral condition had greater prosocial intentions than those in the prosocial condition. Keywords: prosocial music, mood, prosocial intentions ii Table of Contents Abstract...i List of Tables.iv List of Figures.v Introduction1 Effects of Prosocial Music...1 Positive Mood and Helping Behavior..2 Music and Mood..4 Musical Influence on Mood and Prosocial Behavior...5 The Present Study6 Method7 Participants...7 Ethics and Confidentiality8 Procedure.8 Experimental Manipulation of Music..9 Measures10 Data Storage and Handling11 Data Ownership.12 Results...12 Discussion.15 Limitations.18 Future Directions...20 Conclusion21 iii Reflection..22 References.25 Appendix A...30 Appendix B...31 Appendix C32 Positive and Negative Affect Scale (PANAS)...32 Content Semantic Differential...33 Hypothetical Non-Profit Organization: Prosocial Intent Measure.34 iv List of Tables Table 1 Descriptive Statistics for Scale Items Table 2 Pearson Correlation for Correlation of Outcome Variables v List of Figures Figure 1 Mean Affect Score by Affect Type and Condition 1 Prosocial Music and Mood Have you ever thought about the influence of the music you listen to on your behavior? For instance, the song "Imagine" by John Lennon contains lyrics such as "Imagine all the people living life in peace" (Lennon, 1971). Have you ever considered how influential those lyrics and that song could be on behavior? Music is pervasive in everyday life. It is so pervasive that the act of listening to music occurs several times a day in a person's day (Juslin & Laukka, 2004). Considering music's pervasiveness, it is important to understand how music can alter not only how we think, but also how we act. Not only how it might bring us to negative actions such as violence or aggression, but also positive actions such as helping. How music can influence engaging in prosocial behavior is important to understand in the framework of the media's influence on behavior. The issue lies in how research on the influence of prosocial music is lacking. Only a small amount of research exists focusing on prosocial music and its connection to prosocial behavior. Limited research means there are gaps in the understanding of the relationship between these two factors. That is why the focus of this study is the prosocial influence of prosocial music. Effects of Prosocial Music Previous research has investigated the effect of music with prosocial content on a multitude of outcomes. For instance, after listening to a song with the prosocial lyrics there are measurable increases in prosocial thought and empathy (Greitemeyer, 2009). Furthermore, after listening to music with prosocial lyrical content participants had less accessibility to antisocial thoughts, affect attitudes toward subjects such as war and violence and had lowered hostile feelings (Grietemeyer, 2011). Prosocial thoughts are believed to lead to prosocial behavior, but the effect of prosocial music on actual prosocial behavior is understudied. 2 Grietemeyer (2009) had participants listen to music with prosocial lyrics or music with neutral lyrics. Prosocial music led participants to donate more to a non-profit charity relative to participants who listened to neutral music. Additionally, Kennedy (2013) found that participants who had listened to prosocial music helped researchers pick up more pens than participants who listened to neutral music. Field research has also addressed the effects of prosocial music on prosocial behaviors. A study testing prosocial lyrical content in a French restaurant found that those who heard the prosocial music gave bigger tips than those who did not hear prosocial music (Jacob, Guguen, & Boulbry, 2010). However, other studies have failed to find an effect of prosocial music on tipping behavior (Ruth, 2017). Each study differed in how they established their prosocial and neutral song conditions. The selection of songs for most of these studies (Grietemeyer, 2009; Jacob, Guguen, & Boulbry, 2010; Ruth, 2017) had performed pilot testing that established the chosen songs differed significantly strictly on their prosocial perceptions. These studies did not establish that these songs only differed on prosocial perceptions. Kennedy (2013) had a pilot test to establish the two songs differed significantly on prosocial perceptions and to make sure the songs did not differ on any other quality than their prosocial perceptions. The primary focus of most of these studies was establishing a prosocial and neutral condition focused on choosing songs that significantly differed in prosocial content. Only one study (Kennedy, 2013) focused on the songs being similar in all other perceptions except prosocial. Additionally, only one (Grietemeyer, 2009) matched the prosocial song and neutral song on artist and genre. Although there is evidence that prosocial music has an impact on prosocial behavior, the process through which music affects behavior is unclear. Past research has overlooked investigating what might be the causes of these increased levels of prosocial behavior after 3 listening to a prosocial song. The lyrical content might be the component that influences listeners towards engaging in prosocial behaviors. However, another explanation is that the song influences the mood of the listener. The influenced mood may then predict prosocial behavior. The purpose of this study is to investigate the possible role of mood in consideration to prosocial music and increased prosocial behavior. Positive Mood and Helping Behavior Research on mood and behavior has provided evidence that positive mood can lead to helping behavior. For instance, in Manucia, Baumann, and Cialdini (1984) participants were induced into a positive, negative, or neutral mood, and then told that this mood could or could not change. Those in a negative mood helped more than those in a neutral mood only when they believed their mood could be changed by helping versus when their mood could not change. When participants felt their negative mood could not change, they did not differ from participants in a neutral mood. Positive mood, on the other hand, was unaffected by the fixed versus malleable manipulation. Regardless of if the participant believed their mood was fixed or malleable, participants in a positive mood helped more than participants in a neutral mood. Finally, helping occurred more for those in a positive mood than in a negative mood. Another experiment tested the effects of the helping task's cost and benefit (Weyant, 1978). In this experiment, participants were induced into a positive, negative, or neutral mood and then given the opportunity to sign up to volunteer with the American Cancer Society or Little League baseball team. Also, participants were asked to either volunteer to go door to door to collect donations, or just sit a booth and collect donations. Those in a negative mood, relative to neutral, were only more likely to help when asked to sit at a booth and collect donations for 4 the American Cancer Society (low cost and high benefit). Those in a positive mood helped more than those in a negative mood regardless of the cost or benefit of helping behavior. The mood maintenance hypothesis explains the increase in helping behavior for those in a positive mood (Clark & Isen, 1982; Isen, Shalker, Clark, Karp, 1978; Isen & Simmonds, 1978). The hypothesis poses that when a person is in a positive mood the increase in helping behavior will take place because the person wants to prolong the time that they are in that positive mood. The increase in helping behavior only takes place when the task is pleasant rather than unpleasant (Isen & Simmonds, 1978). However, the increase in helping behavior does not mean that the effects are long-lasting. Tests of longevity reveal that the results disappear quite quickly, after about 20 minutes (Isen, Clark, & Schwartz, 1976). A plausible explanation of the temporary nature of these effects is that while a person engages in helping behavior to prolong their positive mood, the task in and of itself is not guaranteed to prolong that mood. Music and Mood Music can influence mood. Psychology research has shown this with the Musical Mood Induction Procedure (MMIP). The procedure uses a piece of music played to participants with the goal of inducing a specific mood (Clark, 1983; Clark, Teasdale, Broadbent, & Martin, 1983; Matthews & Bradley, 1983; Teasdale & Spencer, 1984). Research supports the procedure's ability to successfully induce desired moods in participants (Sutherland, Newman, & Rachman, 1982; Martin 1990). Also, research finds that the MMIP is comparable to other mood induction procedures (Albersnagel, 1988; Clark, 1983; Kenealy, 1988). Self-report measures (Blaney, 1986; Martin, 1990; Parrott, 1991; Parrott & Sabini, 1990), behavioral measures (Clark, 1983; Clark & Teasdale, 1985; Kenealy, 1988; Stber, 1997; Teasdale & Spencer, 1984, Woods, Saltzberg, & Goldsamt, 1990), and physiological measures (Pigantiello, Camp, Elder, & Rasar, 5 1989) support the MMIP. These results support that the MMIP can induce positive, negative, and neutral moods. However, a glaring issue with the procedure is its subjectivity to demand characteristics (Vstfjll, 2001). Stratton and Zalanowski (1991) made this finding in a study where they had participants listen to happy, sad, or no music while looking at a painting along with being given instructions to come up with a happy story, sad story, or whatever comes to mind. They found that the story instructions affected the mood of the participants more than the music they listened to did. The only condition where the music had an effect was in the neutral condition where participants just made a story of whatever came to mind. Nevertheless, research on the use of music to induce desired moods supports the use of music to induce moods despite its subjectivity to demand characteristics. A review of the effectiveness of the MMIP finds that the procedure has a 75% level of effectiveness in the use of the procedure to induce desired moods (Martin, 1990). Evidence that supports the MMIP displays the power of music to influence the mood of the listener. In cases of explicit mood induction music can influence mood. While the research focuses on experimental desired mood induction, it does not mean that in naturalistic settings that music does not affect mood. Even if it is not explicitly to induce a desired mood state, simply listening to music can influence the mood of the listener. Musical Influence on Mood and Prosocial Behavior Just as there has been research into music's effect on mood, there has been research into music's mood effects as it relates to helping behavior. This research found that music can influence mood and helping behavior. One experiment had participants listen to soothing music, stimulating music, aversive music, or no music. Mood assessments showed that the music produced their desired effects. Those in the stimulating music group showed decreases in 6 tiredness, sadness, and depression. Those in the soothing condition showed changes to indicate they felt more soothed, content, and peaceful. Finally, those in the aversive music group showed changes in mood that indicated decreases in peace, contentment, being soothed, joyful and happy and increases in irritation, annoyance, and anger. Participants who listened to the soothing music engaged in significantly more prosocial behavior relative to participants in all other conditions (Fried & Berkowitz, 1979). Another experiment testing uplifting and annoying music in a gym setting (North, Tarrant, & Hargreaves, 2004) had participants listen to uplifting or annoying music. After listening to the music participants were assessed on their mood and indicated significant differences in the two groups and that the desired mood affects were achieved. Then participants were placed in either a low- or high-cost helping situation. In the low-cost condition, no difference was found between music type and helping. In the high-cost condition, those who listened to the uplifting music helped more than those who listened to annoying music. The studies differed in their music selection but still found observable influences on mood from the music along with increased prosocial behavior based on that influenced mood. The Present Study In this present study, I am interested in taking a deeper look at influences of music on mood in consideration of prosocial songs and prosocial intentions. The study is interested in observing the influence of a prosocial song on mood while also separately observing the influence of a prosocial song on prosocial intentions. The question this research study is interested in answering is if participants who listen to a prosocial song have significantly higher reports of positive affect alongside significantly higher reports of prosocial intentions. This study used a between-subjects design where participants listened to either a prosocial or neutral song. After listening to their assigned song participants reported their affect which indicates the effects 7 the song had on their mood. Separately, a measure was employed to measure the influences the song has on the prosocial intentions of the participants. Prosocial intentions are hypothesized to be higher in participants in the prosocial song condition compared to participants in the neutral song condition. Also, compared to participants in the non-prosocial song condition, participants in the prosocial song condition are expected to have higher reported positive affect. It is expected that the results of the manipulation check will show that participants rate the prosocial song as being significantly more prosocial than the neutral song. This research study intends to bridge findings of past literature and begins to investigate mood along with increases in prosocial behavior observed after participants listen to a prosocial song. Method The experiment compared the participant reactions to a prosocial song to that of the participant reactions to a neutral song. The prosocial intentions of the two groups were measured as well. Participants were randomly assigned to their groups and listened to either a prosocial (experimental) or neutral (control) song. The song used for the prosocial condition was "Imagine" (Lennon, 1971). The use of this song in past research on prosocial music and prosocial behavior (Kennedy, 2013; Ruth, 2017) was the basis for this decision. The song used for the neutral condition was "Changes" (Bowie, 1972). This song was chosen due to its musical similarities to Imagine and lyrics that were considered to be neutral in content compared to Imagine. Tests for participants measured mood, perceptions of prosocial nature of songs (manipulation check), and prosocial intentions. I hypothesized that participants in the prosocial song condition would report higher positive affect than those in the neutral song condition. Also, I hypothesized that those who listened to the prosocial song would have higher reported prosocial intentions than those who listened to the neutral song. 8 Participants The study was run through a Qualtrics online digital survey and participants were recruited using the University of Indianapolis SONA psychology research subject pool. The SONA psychology research subject pool is an online system that lists currently running psychology studies at the university. SONA listed this study along with all other studies that were active at that time. Students who participated in the study received partial credit which could go towards partial credit for a course grade or research participation requirement. A total of 81 participants signed up for the study. This was later reduced to 67 participants (31 in the neutral condition, 36 in the prosocial condition) after removing those who did not complete the study. Non-completion was determined by individuals who did not make it through the manipulation and did not listen to their song or failed to successfully answer the question about their assignment to listen to Changes or Imagine. All individuals included in the final analysis made it through the manipulation, successfully answered a question about their manipulation (if they were assigned to Changes or Imagine) and completed all other measures. The sample consisted of 61 female participants, 4 male participants, and 2 participants who self-identified as a gender other than male or female. Participants came from all grade levels. Most participants were Freshman (n=26) followed by Sophomores (n=18), Seniors (n=12), and Juniors (n=11). Ethics and Confidentiality The participants were informed that participation in this study allowed them to collect partial credit for a course grade or research participation requirement. Multiple studies and alternative assignments were active at the same time. This relayed to potential participants that participation in this study is not required. Participation in other studies or taking part in an 9 alternative assignment could be done instead of this study to receive this credit. Also, this study collected no personal or identifiable information. Procedure After signing up for the study on SONA, the participants received the Qualtrics survey link. The survey welcomed participants and informed them of the purpose of the study. The beginning of the study informed participants of its goal, stating that it was interested in the differences in people's reactions after listening to different songs. After the introduction, participants were then were presented with the informed consent form that was embedded within the survey. Informed consent from participants was collected through the Qualtrics survey. After collecting consent, the survey randomly assigned participants to the prosocial or neutral condition. The songs were embedded within the Qualtrics survey and a timer was placed on that individual question that required them to stay on the question for three minutes before they could proceed forward. The timer was put in place to ensure participants were more likely to have listened to their assigned song. After listening to their song, they had to answer a question about what song they were assigned which assessed if they had read all instructions and paid attention to the written instructions. Next, they were assessed for their positive and negative affect. Positive affect assessed the participants' positive emotions and feelings. Reports of positive affect measured to what extent participants experienced feelings such as being proud, strong, interested, and excited at that moment. Negative affect assessed the participants' negative emotions and feelings. Reports of negative affect measured to what extent participants experienced feelings such as being distressed, upset, scared, and nervous at that moment. Participants then completed a manipulation check which assessed the prosocial nature of each song. Finally, participants completed a measure that assessed prosocial intentions. 10 Upon completion of data collection, participants reported demographic information. Participants reported their academic year, gender, and major. Once demographic information was collected, participants were debriefed of the true intentions of the study. Then participants were thanked for their participation and asked to proceed to the end of the survey. Proceeding to the end of the survey redirected participants back to SONA and automatically granted credit for participation in the study. Experimental Manipulation of Music Participants were randomly assigned to listen to the song "Imagine" (experimental; Lennon, 1971) or "Changes" (control; Bowie, 1972). The similar instrumentals were one factor that influenced the selection of the control song. Both songs feature piano prominently in their instrumental along with accompaniment from other instruments. Also, both songs, despite some minor differences, have similar energy. Both songs are a part of the pop genre and both have been songs that have received notable renown, namely both songs (not the artists) had been inducted into the Grammy Hall of Fame (Recording Academy Grammy Awards, 2020). Measures In this experiment, both positive and negative affect were assessed. The choice to include both types of affect was based on past research assessing both and to better inform influences on positive affect by also observing possible changes in negative affect. The Positive and Negative Affect Scale (PANAS) from Watson, Clark, and Tellegen (1988) was used to measure participant affect. The PANAS consists of 20 items assessing positive and negative affect (e.g. interested, excited, irritable, upset, nervous, scared). Half of the questions assess positive affect while the other half assess negative affect. Participants rate the extent they feel that current emotion using a 5-point Likert-type scale. The 5-point Likert scale ranges from 1 (very slightly 11 or not at all) to 5 (extremely) The following measure has been used in the previous research on this topic (Grietemeyer, 2009; Kennedy, 2013). A hypothetical petition was used to assess prosocial intention. This measure resembled that of past research of North, Tarrant, and Hargreaves (2004). The petition presented is for a hypothetical non-profit organization. Participants were asked about their level of support for organizations such as this one. The level of support was measured using a 5-point Likert-type scale ranging from 1 (very slightly) to 5 (extremely). A semantic differential rating was used for the manipulation check assessing the content of the participant's assigned song. Past research on this topic used this type of measurement (Greitemeyer, 2009; Kennedy, 2013). The semantic differential used comes from Kennedy (2013) and consists of 9 different continuums where participants rate the content of the song (e.g. happy-sad, helpful-unhelpful, active-passive). The continuum scores are on a five-point scale. Participants mark the point closest to the word they feel best fits the song. If the participants feel that neither word fits, then they should mark the middle point. Two items, helpfulunhelpful and cooperativeuncooperative, are the focus of this study's analysis. All other items serve as filler for the survey. A score of 1 was assigned to helpful and cooperative, and a score of 5 was assigned to unhelpful and uncooperative. These two items (helpful and cooperative) served as indicators of prosocial perception of the songs. Data Storage and Handling Responsibility of data storage and handling belongs to myself and my faculty advisor who served as principal investigator of this study. All data was collected digitally and is only accessible by the creator of the survey, me, and my faculty advisor. We collected the data from 12 the password-protected survey host and downloaded it onto secure computers with password protection. Data Ownership The data collected in this study belongs to the University of Indianapolis. The University of Indianapolis IRB controls the sharing of the data. Any results shared by the IRB will be deidentified. Results The data analysis plan consisted of reliability analyses, tests for random assignment, and tests for condition differences. Reliability analyses were run for the PANAS affect scale, and separate tests were run for positive and negative affect. Next, an analysis was run to test for random assignment for condition. Finally, condition differences on outcome variables were analyzed using a mixed model ANOVA and independent samples t-test. A Chi-square test was run to see if the number of participants across conditions significantly differed (i.e. to ensure there was no failure of random assignment). Results of the Chi-square show that the two groups did not differ significantly 2 (1)=.373, p=.541. The results show that there was no failure of random assignment even though there was a difference in the number of participants in the prosocial (n=36) and neutral(n=31) conditions. Next, a test for the reliability of the PANAS scale was run. Separate analyses were run for positive and negative affect. Both positive and negative affect scales had good Cronbach alpha indices: positive affect (= .94) and negative affect (= .70). The PANAS had a composite Cronbach alpha score of = .82. These results suggest that the PANAS was a reliable scale for testing and measuring participant affect. 13 In this experiment, both positive and negative affect were assessed. A mixed model ANOVA was used to look at examine the effect of condition on the intensity of participant affect scores. One independent variable was condition (between subjects: prosocial or neutral), and the other independent variable was affect type (within subjects: positive and negative). There was a significant main effect for affect type, F(1,55)=143.17, p<.001. Positive affect (M=2.75, SD=1.05) was significantly higher than negative affect (M=1.19, SD=.28). However, the effect of condition was non-significant, F(1,65)=1.58, p=.213. The prosocial condition (M=1.89; SD=1.24) did not differ in affect ratings from the neutral condition (M=2.06; SD=1.38). A significant interaction was present between affect type and condition, (F(1,65)=4.63, p=.035). For positive affect ratings, there was a marginally significant difference between conditions, F(1,65)= 3.13, p=.082. Similarly, for negative affect ratings, there was a marginally significant difference between conditions, F(1,65)=3.27, p=.075. See Figure 1 for illustration of the differences of mean positive and negative affect scores by group. The figure presents the interaction. Figure 1 Mean Affect Score by Affect Type and Condition 14 Figure 1: Mean Affect Score by Affect Type and Condition 3.5 Mean Affect Score 3 2.99 2.54 2.5 2 1.5 1.24 1.12 1 0.5 0 Changes Imagine Condition Positive Affect Negative Affect Next, an independent samples t-test was used to test predictions on the outcome of prosocial intentions. The independent samples t-test was run to test if any significant differences in prosocial intentions existed between the prosocial and neutral group. No statistically significant differences were found for the prosocial (M=4.11, SD=.89) and the neutral (M=4.42, SD=.72) conditions, t(65)=1.55, p=.127. Correlations were run among all of the outcomes/ continuous variables. Because of the lack of condition differences across the two conditions, the analyses were run without condition considered. Only three significant correlations were found. A significant negative correlation was present between positive affect and helpful semantic differential item scores, r(65)=.315, p=.009. A significant positive correlation between cooperation semantic differential scores and helpfulness scores was present, r(65)=.401, p=.001. Additionally, a significant, positive correlation was present between cooperation semantic differential scores and prosocial 15 intentions, r(65)=.265, p=.03. See Table 1 for the descriptive statistics for all scale items. See Table 2 to see the Pearson correlations for all correlations run. Table 1 Descriptive Statistics for Scale Items N Minimum Maximum Mean Average Positive Affect 67 1.00 4.80 2.75 Standard Deviation 1.05 Average Negative Affect CooperationConflict Semantic Diff. HelpfulUnhelpful Semantic Diff. Prosocial Intent 67 1.00 2.40 1.19 .281 67 1.00 5.00 2.54 1.17 67 1.00 5.00 2.19 .839 67 2.00 5.00 4.25 .823 Table 2 Pearson Correlation for Correlation of Outcome Variables Average Positive Affect Average Negative Affect Cooperation Conflict Semantic Diff. Helpful Unhelpful Semantic Diff. Prosocial Intent Average Positive Affect 1 Average Negative Affect -.088 Cooperation Conflict Semantic Diff. -.007 Helpful Unhelpful Semantic Diff. -.315** Prosocial Intent -.088 1 .089 .225 -.082 -.007 .089 1 .401** .265* -.315** .225 .401** 1 .059 .152 -.082 .265* .059 1 **. Correlation is significant at the 0.01 level (2-tailed). *. Correlation is significant at the 0.05 level (2-tailed). .152 16 To analyze the manipulation check, an independent samples t-test was run for the semantic differential items. Separate independent samples t-tests were run for the scores on the two items that were the focus of the analysis. The t-tests were run to assess if the prosocial condition significantly differed from the neutral condition on their scores for items assessing prosocial perceptions of the songs. No statistically significant differences in helpfulness scores were found between the prosocial group (M=2.08, SD=.87) and the neutral group (M=2.32, SD=.79), t(65)=1.17, p=.248. Also, no statistically significant differences in cooperation scores were found between the prosocial group (M=2.36, SD=1.15) and the neutral group (M=2.74, SD=1.18), t(65)=1.33, p=.187. These results indicate that the two songs did not differ in prosocial perceptions. This means that the manipulation between the prosocial and neutral condition was not statistically significant in its strength. Discussion The goal of this study was to look into an under researched area of prosocial music and prosocial behavior and to help expand the literature on this topic. I used a between-subjects design that randomly assigned participants to listen to either a prosocial or neutral song. Then participants were asked to report their mood, perform a manipulation check, and report their prosocial intentions. Contrary to expectations, the results of the mixed model ANOVA revealed a main effect for affect type on affect intensity scores, but no main effect for condition on affect scores. There was a significant interaction present between affect type and condition. The interaction suggested that the impact of condition on affect intensity depended on the affect type. The interaction was significant because for positive affect scores the neutral song condition had higher positive affect 17 scores than the prosocial condition, and for negative affect scores the prosocial song had negative affect scores that were just a little greater negative affect scores than the neutral. The independent samples t-test revealed no statistically significant differences in prosocial intentions between the prosocial and neutral group. However, it is notable that the neutral song condition had a higher mean score for prosocial intentions. The mixed model ANOVA showed that the neutral condition was marginally higher in affect intensity than the prosocial condition in positive affect. The means scores of prosocial intentions combined with the results of the interaction revealed in the mixed model ANOVA appear to follow results of other studies (Manucia, Baumann, & Cialdini, 1984; Weyant, 1978) that show an association between positive mood and prosocial behavior. In the case of this study, the neutral group had marginally greater positive affect scores than the prosocial group, and the neutral group had a higher mean score for prosocial intentions. This goes against my prediction of how the prosocial song would have greater positive affect scores. Even though the means of the semantic differential scores indicated that the experimental song was perceived as more prosocial, the prosocial song did not actually make people happier as predicted. The independent samples t-test for the semantic differential scores showed a nonsignificant difference between prosocial perceptions of the songs used. Notably, the mean scores for the helpfulness and cooperation semantic differential items indicated that the song Imagine had been interpreted as more prosocial. These results indicate that there was some difference to indicate the experimental song as being more prosocial than the control song. Yet, the manipulation was not strong enough to be statistically significant. Use of the song "Imagine" as a prosocial song has been done in past research (Ruth, 2017) including research with college students (Kennedy, 2013). The results indicate an issue in the comparison song to the prosocial 18 song. The neutral song was chosen based on its similarity in musical sound and popularity. The weak manipulation of our study was due to unforeseen differences in how participants would perceive the chosen songs. A notable reflection, in retrospect, are that that the songs might not have been as similar each other as initially proposed, and the differences might be more significant than initially thought. The songs Imagine, and Changes were chosen based on their similarity to each other in sound and energy. However, the songs might have been more different than originally believed. The song Imagine (Lennon, 1971) has a consistent mellow and soothing energy throughout the entire song. The song Changes (Bowie, 1972), in comparison, has fluctuations of energy between the different sections (verse, chorus, and bridge) of the song. The verses of the "Changes" are soothing and mellow, similar to the song Imagine. The chorus, however, is more upbeat. Despite the more upbeat and energetic sections of the song "Changes" (Bowie, 1972) it was initially believed believed those changes in energy were not incredibly stark or jarring in their differences. Another discrepancy present was the differences in run-time. Imagine (Lennon, 1971) has a run time of three minutes and three seconds and Changes (Bowie, 1972) has a run time of three minutes and thirty seconds. The differences in run time were also perceived to be non-significant. However, the run time might have been more significant than originally thought, just like differences in song structure. The results of the correlations revealed many non-significant results including a nonsignificant correlation between positive and negative affect. This result is odd as it would be anticipated that a significant correlation would exist between positive and negative affect. These results are likely tied to the PANAS affect scale used in this study. The reliability tests found that both the positive and negative affect scales had good Cronbach alpha indices. However, compared to the Cronbach alpha of positive affect, the Cronbach alpha for negative affect was 19 notably lower. This lower reliability score, compared to that of positive affect, could have contributed to this result. I found one non-significant result for the semantic differential that matched my hypothesis for the participant perceptions of the two songs. The result I found for mood and prosocial intentions somewhat matched my hypothesis, but the result was present in the group opposite of what I hypothesized. While the results were non-significant, they do appear to somewhat follow the results of past studies. In particular, the results appear to follow those of studies suggesting a connection between positive mood and helping (Manucia, Bauman, & Cialdini, 1984; Weyant, 1978) and studies connecting musics ability to influence mood and prosocial behavior (North, Tarrant, & Hargreaves, 2004; Fried & Berkowitz, 1979). Yet, the results go against my prediction that these results for positive affect scores and prosocial intentions scores would present in the prosocial condition. The results were present for the neutral condition rather than the prosocial condition. Despite the chosen prosocial song having a mean score indicating it being more prosocial it did not appear to have any form of connection to higher positive affect scores or greater prosocial intentions as hypothesized. Limitations The biggest limitation of the study was the small sample size. An initial power analysis for a medium effect size informed an initial proposed sample size of 120 participants. A secondary power analysis was run while data collection was occurring. This secondary power analysis assumed a strong manipulation and supported that, if the effect size was strong, a smaller sample size (around 60) would not be an issue. The smaller sample size obtained was due to time constraints of how long the study had to run. Data collection ran from the end of January through the first week of March (5 weeks for data collection). Also, the sample size was 20 affected by the number of participants that had to be removed for not completing the study (i.e. not making it through the manipulation and listening to the song). If the manipulation had yielded a strong effect size, then the smaller sample size could be justified by the secondary power analysis. However, given the weak manipulation found the small sample size became a limitation. The second biggest limitation was the manipulation. The prosocial song selection was made based on its use in past research. The control song, however, was not chosen based on its use as a neutral comparison song in past research. Past research on such subject matter have compared prosocial songs to a neutral songs. The song chosen for this study was not indicated in any reviewed literature to have been used in such studies. The song Changes was chosen for its closeness in release to the song Imagine as well as its similarities in the sound of the songs. The choice to match in the similarity of sound and tone was based on past research that matched the songs on artist and genre (Grietemeyer, 2009). There was one issue with the songs used in Grietemeyers (2009) study. The songs used were not well known. The issue of popularity was addressed in another study, yet the songs were not matched on genre or artist as done in the study whose issues were being addressed (Kennedy, 2013). My study focused on selecting songs that were similar in genre, sound, and tone while using more popular and well-known songs. Such studies on prosocial song compared to neutral songs, focusing on using well-known songs, had not used Changes as a neutral song and had not focused on matching songs based on genre, sound, or tone. In the case of my study, a pilot study should have been conducted to ensure the strength of the manipulation. A pilot study would have allowed for revisions to be made in song selection to ensure that the prosocial and neutral songs strongly differed in their prosocial and neutral perceptions, respectively. 21 Additional factors that could have contributed to this weak manipulation could have been attention of participants. Previous research on prosocial music and prosocial behavior had been conducted in laboratories (Grietemeyer, 2009; Kennedy, 2013). Two notable studies conducted such research in field settings. However, one study found significantly higher tipping behavior in participants who listened to prosocial music (Jacob, Guguen, & Boulbry, 2010) and the other did not find any significant difference in tipping behavior (Ruth, 2017). This study had its own unique difference in that it was conducted online through a Qualtrics survey. Unlike these former studies there was no way for there to be any form of oversight or awareness of the attention participants paid to the songs they had listened to. The Qualtrics survey had a 3-minute timer on the question containing the song which did not allow them to proceed forward until the timer ran out. This was put in place to make the likelihood that they listened to the song higher. Additionally, in the instructions for participants they were told the name of the song they were to listen to and to remember the title of the song. After proceeding forward, they were asked to report the song they had heard. These measures were put in place to help increase the likelihood they listened to the song and paid attention to instructions, however it did not guarantee that participants attentively listened to the song. Participants still could find ways around listening to the song and were also able to stop and pick up the survey whenever they wanted. The ability to stop at any time and continue could also have contributed to the weaker manipulation as the longer interval between listening to the song and reporting their perceptions could have influenced their responses. One final factor that could have contributed to the weaker manipulation could be the age of the songs. Both songs come from the early 1970s. The song Changes was released in 1972 (Bowie, 1972) while the song Imagine was released in 1971 (Lennon, 1971). The cohort 22 studied were undergraduate college students. One study did use the song Imagine with a cohort of undergraduate college students (Kennedy, 2013). However, there is little research to look at the cohort studied could have affected the interpretation. It could have been likely that the age of the songs and the cohort studied had a unique relationship. Freshman and Sophomore students made up more than half of the participants in this study. The younger age of the participants and the use of older songs could have contributed to the weaker manipulation because of how the interpretations of such older songs might differ based on the age of the person listening to them. The limitation of the weak manipulation could have been avoided by using a pilot study to test the experimental manipulation and song choice before going forward with the full experiment. Many studies in the past have used pilot studies to ensure the strength of their manipulation (Grietemeyer, 2009; Kennedy, 2013; Ruth, 2017). However, time constraints limited my ability to conduct pilot testing. The choice of a neutral song was based on my best attempt to pick a neutral song that could be comparable to the prosocial song. Future Directions Future directions would be interested in looking at building upon this study design and looking at the influences of factors not tested in this study. One factor that would be important to test are how the age of the song. The age of the song, as discussed in the limitations, could influence its interpretation by participants. This ties into testing cohorts of different ages to establish a better understanding on how the age of the song relates to the age of the participants. Testing different ages of participants can also indicate if there are differences in the possible differences in results based on the age of participants. Another factor to examine is how the genre of song affects the results. This study used songs that are apart of the pop genre. Future research would be interested to see if the genre of 23 the song influences prosocial intentions of the participants. Additionally, testing different genres of songs could determine if the genre of the song plays a role in prosocial perceptions of the songs. This could further elaborate upon the understanding beyond what was found in this study but also elaborate upon the current research present. One final area that could be important to research is testing different types of prosocial influences. This study used support for a hypothetical organization. Future studies should look at testing different types of prosocial intentions and outcomes. Prosocial influences to consider might be behavioral such as those used in past research such as picking up pens (Kennedy, 2013) or donations to a non-profit (Grietemeyer, 2009). Also, further research might be interested to test prosocial outcomes such as tolerance or acceptance to other individuals. Conclusion The research area of prosocial music and mood has only a small amount of literature present on the topic. Through this study I aimed to contribute to this smaller body of literature. I used a between subjects design which assigned participants to a prosocial or neutral song, then participants reported their prosocial perceptions of these songs, positive and negative affect, and prosocial intentions. A non-significant difference on prosocial perceptions of the songs was found. Also, a non-significant difference for prosocial intentions of the experimental and control group were found. A significant interaction was present for the condition and the affect type for affects scores. Most notable of these findings was that for positive affect those in the neutral condition had marginally different affect scores than those in the prosocial condition. The study largely yielded non-significant results and had a significant limitation of a weaker than expected manipulation. However, by the results of the interaction and the mean scores of prosocial intentions, the results somewhat followed that of past research that indicate a connection between 24 positive mood and prosocial behavior. The biggest difference from my initial hypothesis was that the pattern was found in the control group not the experimental group. Future research should look into the influences of the age of the song and the age of the participant on the results, if the genre of the song affects results, and test different types of prosocial intentions and outcomes than what this study investigated. Reflection An important shortcoming of this study was that of the weaker than expected manipulation. There were present time constraints which limited my ability to pilot test the songs to ensure a strong manipulation. The weak manipulation was mainly due to unforeseen circumstances. This revealed a nuance to research with prosocial music. The perception of music is highly subjective and what might be expected to be a strong manipulation can turn out to be weaker than once hypothesized. Ensuring a strong manipulation is something that would need to require testing and adjustment for the sample that is being studied. It makes it clear that, ideally, as part of any study with prosocial music that pilot test is conducted to ensure a strong manipulation. Additionally, the method for research and conducting studies on the matter of prosocial music should be considered. Online studies, such as the one used here, might not be as ideal as conducting studies in a physical setting with the participants in the room with the researcher. The results could have been affected by the choice of how the study was conducted. It pointed out that in this are of research that there are settings and methods for this research (e.g. laboratory settings) that might be more ideal than others. Personally, developing this project from the initial idea to a formalized proposal to a final project was tough. The proposal stage, in particular, was a trying time. It was difficult to find advisors to help with the project because of its focus. There was a lot of rejection from possible 25 advisors to oversee my project. This personally led to frustration and confusion as the constant rejection started to build up. It was something that was a test of my resilience to keep overcoming that rejection and finally get an advisor to oversee the project. Luckily, after a long time of trials and struggles, an advisor was found and the project was able to continue on my intended subject matter. From there everything was able to move smoothly without a hitch. Professionally, this project was an important introduction to the process of conducting research studies. I got first-hand experiences with writing up an IRB proposal and seeking IRB approval. Additionally, I got the first-hand experience with collecting and cleaning data and interpreting results. This study is extremely beneficial as in my future in education and my career I likely could end up conducting or working on other research studies. This project served as an introduction to the process of developing my own study. It demonstrated, for one, how much adjusting the project occurs and how much revision can occur from the first proposed idea to the final design. It also served as an introduction to the process of drafting an IRB proposal and submission of that proposal for review. The study met some research goals and did not meet some research goals. Namely, the study did serve to investigate an understudied area, but found no significant results and had a weak manipulation. More importantly, the study served as a personally and professionally important project. The struggles in the proposal stage tested personal resilience as the constant rejection of possible advisors made the proposal process significantly more difficult. The conducting of the project itself and the process of proposal stages served to help with professional development. This project was an introduction to formulating research studies and the process to move from idea to finalized study. It gave me valuable experience with composing a project, write-up for proposals and results, and experience with earning IRB approval and 26 ensuring ethical research practice. These personal and professional developments and challenges are ones that will be of benefit for future projects and ventures in the field of psychology. 27 References Albersnagel, F. A. (1988). Velten and musical mood induction procedures: A comparison with accessibility of thought associations. Behavior research and therapy, 26(1), 79-95. Ali, S. O., & Peynirciolu, Z. F. (2006). Songs and emotions: are lyrics and melodies equal partners?. Psychology of Music, 34(4), 511-534. doi:https://doi.org/10.1177%2F0305735606067168 Blaney, P. H. (1986). Affect and memory: a review. Psychological bulletin, 99(2), 229-246. Bowie, D. (1972). Changes. On Hunky Dory [MP3]. London: RCA Records. Clark, D. M. (1983). On the induction of depressed mood in the laboratory: Evaluation and comparison of the Velten and musical procedures. Advances in Behaviour Research and Therapy, 5(1), 27-49. Clark, D.M., Teasdale, J.D., Broadbent, D.E., & Martin, M. (1983). Effect of mood on lexical decisions. Bulletin of the Psychonomic Society, 21, 175-178 Clark, D. M., & Teasdale, J. D. (1985). Constraints on the effects of mood on memory. Journal of Personality and Social Psychology, 48(6), 1595-1608. Clark, M. S., & Isen, A. M. (1982). Toward understanding the relationship between feeling states and social behavior. In A. Hastorf & A.M. Isen (Eds.), Cognitive social psychology. New York: Elsevier. Fried, R. & Berkowitz, L. (1979). Music hath charms and can influence helpfulness. Journal of Applied Social Psychology, 9(3), 199-208. doi:https://doi.org/10.1111/j.15591816.1979.tb02706.x 28 Galizio, M., & Hendrick, C. (1972). Effect of musical accompaniment on attitude: The guitar as a prop for persuasion. Journal of applied psychology, 2(4), 350-39. doi:https://doi.org/10.1111/j.1559-1816.1972.tb01286.x Greitemeyer, T. (2009). Effects of songs with prosocial lyrics on prosocial thoughts, affect, and behavior. Journal of Experimental Social Psychology, 45, 186-190. doi:10.1016/j.jesp.2008.08.003 Gereitemeyer, T. (2011). Exposure to music with prosocial lyrics reduces aggression: First evidence and test of the underlying mechanism. Journal of Experimental Social Psychology, 47, 28-36. doi:10.1016/j.jesp.2010.08.005 Isen, A. M., Clark, M., & Schwartz, M. F. (1976). Duration of the effect of good mood on helping: Footprints on the sands of time. 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Validation of a music mood induction procedure: Some preliminary findings. Cognition & Emotion, 2(1), 41-48. Kennedy, P. E. (2013). The relationship between prosocial music and helping behavior and its mediators: An Irish college sample. Journal of European Psychology Students, 4(1), 115. doi:http://doi.org/10.5334/jeps.av Lennon, J. (1971). Imagine. On Imagine [MP3] New York: EMI. Matthews, A., & Bradley, B. (1983). Mood and the self-reference bias in recall. Behavior Research and Therapy, 21, 233-239. Martin, M. (1990). On the induction of mood. Clinical Psychology Review, 10(6), 669-697. Manucia, G. K., Baumann, D. J., & Cialdini, R. B. (1984). Mood influences on helping: direct effects or side effects?. Journal of Personality and Social Psychology 46(2), 357-364. doi:http://dx.doi.org/10.1037/0022-3514.46.2.357 Mori, K. (2009). The influence of the meaning of lyrics on the expressed emotion of music valence. Systematic Musicology, 53-58. Retrieved from https://www.researchgate.net/profile/Luiz_Naveda2/publication/263851749_Proceedings _of_the_Second_Conference_of_Students_of_Systematic_Musicology/links/02e7e53c1b d8501cee000000.pdf#page=54 North, A. C., Tarrant, M., & Hargreaves, D. J. (2004). The effects of music on helping behavior: A field study. Environment and Behavior, 36(2), 266-275. doi:10.1177/0013916503256263 Parrott, W. G. (1991). Mood induction and instructions to sustain moods: A test of the subject compliance hypothesis of mood congruent memory. Cognition & emotion, 5(1), 41-52. 30 Parrott, W. G., & Sabini, J. (1990). Mood and memory under natural conditions: Evidence for mood incongruent recall. Journal of personality and Social Psychology, 59(2), 321-336. Recording Academy Grammy Awards. (2020). Grammy Hall of Fame. https://www.grammy.com/grammys/awards/hall-of-fame#i Ruth, N. (2017). Heal the world: A field experiment on the effects of music with prosocial lyrics on prosocial behavior. Psychology of Music, 45(2), 298-304. doi:10.1177/0305735616652226 Stratton, V. N., Zalanowski, A. H. (1991). The effects of music and cognition on mood. Psychology of Music, 19, 121-127. doi:https://doi.org/10.1177/0305735691192003 Stratton, V. N., & Zalanowski, A. H. (1994). Affective impact of music vs. lyrics. Empirical Studies of the Arts, 12(2), 173-184. doi:10.2190/35T0-U4DT-N09Q-LQHW Stber, J. (1997). Trait anxiety and pessimistic appraisal of risk and chance. Personality and Individual Differences, 22(4), 465-476. Sutherland, G., Newman, B., & Rachman, S. (1982). Experimental investigations of the relations between mood and intrusive unwanted cognitions. British Journal of Medical Psychology, 55(2), 127-138. Teasdale, J. D., & Spencer, P. (1984). Induced mood and estimates of past success. British Journal of Clinical Psychology, 23(2), 149-150. Vstfjll, D. (2001). Emotion induction through music: A review of the musical mood induction procedure. Musicae Scientiae, 5, 173211. doi:https://doi.org/10.1177/10298649020050S107 31 Watson, D., Lee A. C., Tellegen A. (1988). Development and validation of brief measures of positive and negative affect: The PANAS scales. Journal of Personality and Social Psychology, 54(6), 1063-1070. Weyant, J. M. (1978). Effects of mood states, costs, and benefits, on helping. Journal of Personality and Social Psychology 36(10), 1169-1176. doi:http://dx.doi.org/10.1037/0022-3514.36.10.1169 Withers, B. (1994). Lean on Me. On Greatest hits: Lean on me [MP3]. New York City, New York: Columbia/Legacy. Wood, J. V., Saltzberg, J. A., & Goldsamt, L. A. (1990). Does affect induce self-focused attention?. Journal of personality and social psychology, 58(5), 899-908 32 Appendix A Citi Training Certification 33 Appendix B IRB Submission and Approval Letter 34 Appendix C Positive and Negative Affect Scale (PANAS) The following scale consists of a number of words that describe different emotions and feelings. Read each item and record your answer by writing it in the space next to the word. Indicate to what extent you feel this way right now, that is, at this present moment. Use the following scale to record your answers: 1= Very slightly or not at all 2= A little 3=Moderately 4=Quite a bit Interested ______ Irritable ______ Distressed ______ Alert ______ Excited ______ Ashamed ______ Upset ______ Inspired ______ Strong ______ Nervous ______ Guilty ______ Determined ______ Scared ______ Attentive ______ Hostile ______ Jittery ______ Enthusiastic ______ Active ______ Proud ______ Afraid ____ 5=Extremely 35 Content Semantic Differential Below are a series of scales which describe the content of the songs you have heard. Words are arranged with their opposites for each pair. Please mark the choice that is closest to the word you feel best describes the song for each pair. The closer you place your selection to a word the more it describes that song. If you feel that neither word is relevant to the theme of the song, then mark the middle answer. A B C D E Fantasy _____ _____ _____ _____ _____ Reality Happy _____ _____ _____ _____ _____ Sad Trust _____ _____ _____ _____ _____ Distrust Dependable _____ _____ _____ _____ _____ Cooperation _____ _____ _____ _____ _____ Conflict Active _____ _____ _____ _____ _____ Passive Helpful _____ _____ _____ _____ _____ Unhelpful Love _____ _____ _____ _____ _____ Hate Peace _____ _____ _____ _____ _____ War Not Dependable 36 Hypothetical Non-Profit Organization: Prosocial Intent Measure Please read the selection below about a hypothetical non-profit organization and then answer the following question: Limitless Education is a non-profit organization that aims at helping disadvantaged youth from elementary to high school age. The organization helps to assist families who struggle to cover the costs of school supplies and books. Additionally, for high school-aged students, the organization helps to cover college application fees as well as offering help with ACT and SAT preparation. After reading about this hypothetical non-profit organization what would your level of support for non-profit organizations similar to the one you read about be? Rate your level of support that you would have for non-profit organizations similar to the one described above using the following scale: 1= Very slightly or not at all Level of support: _______ 2= A little 3=Moderately 4=Quite a bit 5=Extremely  ...

... How Statistics and Narrative Influence Attitudes Toward People with Disabilities By Jacqueline Gunther Honors Project Thesis Submitted in Partial Fulfillment Of the Requirements for a Baccalaureate Degree With Distinction In the Ron and Laura Strain Honors College of THE UNIVERSITY OF INDIANAPOLIS May 2020 Faculty Advisor: Kathryn L. Boucher, PhD Executive Director of Honors: James B. Williams, Ph.D. Jacqueline Gunther 2019. Copyright in this work rests solely with the author. Please ensure that any reproduction or re-use is done in accordance with relevant national copyright protection. i Abstract Numerous laws have been enacted to protect the civil rights of individuals with disabilities, including the Americans with Disabilities Act of 1990, yet discrimination against individuals with disabilities continues. Per Girli, Sari, Kirkim, and Narin (2016), unfavorable perceptions are a cause of discrimination. Promoting intergroup contact (Pettigrew & Tropp, 2008) using different kinds of information could change perceptions (Dieckmann, Slovic, & Peters, 2009). This projects purpose was to test whether statistical information, narrative, or a combination were effective in reducing negative stereotyping when compared to no information. Undergraduate students from the University of Indianapolis completed the Attitudes Toward Disabled Persons measure (ATDP) after random assignment to a control or to a treatment where they received only statistics, only narrative, or both types of information about disability discrimination (Yuker & Block, 1986). Treatment conditions did not result in more positive views than the control condition as hypothesized. However, participants having disabilities did have different responses to the conditions than those without disabilities although there was no significant interaction between regular contact and treatment condition. The hope is that healthcare practitioners and disability advocates will use the results of this study to understand what might reduce negative stereotyping of individuals with disabilities. ii Table of Contents Abstract i Acknowledgments iii List of Figures iv Introduction and Literature Review 5 Method 18 Results 23 Discussion 26 Implications and Future Directions 28 Conclusion 30 Reference List 31 Appendices 35 iii Acknowledgments This project would not have been possible without the following generous guidance and assistance: Dr. Kathryn Boucher; I would like to thank her for willingness to be the primary investigator in this research project. She has helped me to develop the project from first submission to the Honors Review Board through the final manuscript. Along the way she has assisted me in developing the necessary research skills to complete a project of this magnitude. Without her assistance, the project would not have been possible. The Ron and Laura Strain Honors College; I would like to thank them for allowing me the opportunity to be in the Honors Program to benefit from its classes and programs and to ultimately pursue this research project. Honors Review Board; I would like to thank them for taking the time to review this project. Dr. Melissa Loria; I would like to thank her for helping me to develop the project topic through her Capstone course. Dr. James Williams; I would like to thank him for coaching and mentoring me throughout my journey at UIndy. His assistance has been invaluable. iv List of Figures Figure 1: This figure shows the significant interaction between experimental condition and disability status on ATDP scores, compared to the non-significant interaction on ATDP scores by condition from no-associated disability status. 5 How Statistics and Narrative Influence Attitudes Toward People with Disabilities The 1970s was a time of great activism regarding the rights of individuals with disabilities, beginning with the passage of the Rehabilitation Act of 1973; its Section 504 was the first disability-related civil rights law to be enacted in the United States. Many other disabilityrelated anti-discrimination laws have been enacted since then including the Americans with Disabilities Act (ADA) of 1990 that expanded protections to private businesses and is the most comprehensive civil rights legislation for individuals with disabilities to date. This law prohibits any public or private entities, that provide public accommodations, from limiting the ability of disabled individuals to use any goods or services provided, in order that disabled individuals may fully participate in the daily life in their respective communities. For example, where it is not cost prohibitive, new construction and renovations are expected to comply with the ADA. Programs and services are supposed to be accessible to individuals with disabilities by using such interventions as Braille, sign language interpreters, or adaptations to the arrangements or the manner in which the program or service is offered. While the ADA and other disability-related legal protections have resulted in greatly expanded access to places and activities for individuals with disabilities, some private business owner/operators continue to hold biases towards individuals with disabilities in their implementation of the ADA (Maroto & Pettinicchio, 2015). These biases are stereotypes because these beliefs are applied to all persons who have disabilities regardless of the specific circumstances or experiences that apply to the individual person with a disability or situation in question. For example, many business owners/operators presume that building modifications to allow access for individuals with disabilities will be very costly when many building 6 modifications could be quite inexpensive like braille in elevators or grab bars in bathrooms (Maroto & Pettinicchio, 2015). Assumptions are often made regarding employees with disabilities. These employees often are not expected to be as productive as their able-bodied peers and this presumption is made without considering past experience related to the specific person being considered for employment (Maroto & Pettinicchio, 2015). Employees with disabilities may also be perceived as being more costly to hire because of greater expenses for health insurance, accommodations in the workplace, or time off due to their disabilities, again without considering the specifics involved in hiring a specific individual with a disability (Maroto & Pettinicchio, 2015). There is wide latitude allowed in interpreting the provisions of the ADA, so the courts have been able to take conservative positions that essentially support the rights of employers to run their businesses as they deem appropriate (Maroto & Pettinicchio, 2015). The courts have tended to rule that businesses should not be encumbered by undue government regulation and should not have to hire individuals with disabilities whom they do not believe can do the job (Maroto & Pettinicchio, 2015). Courts have also tended to rule that businesses should not have to incur what they deem as unreasonable costs in order to provide reasonable accommodations (Maroto & Pettinicchio, 2015). The result has been continued discrimination against individuals with disabilities due, at least in part, to the negative perceptions regarding individuals with disabilities that are held by the business owner/operators charged with complying with the ADA as well as by the general public (Robert & Harlan, 2006). This discrimination is corroborated from the perspective of individuals with disabilities themselves in the 2004 National Organization on Disability Harris Survey of Americans with Disabilities in which only 30% of the individuals with disabilities polled believed that the ADA 7 had made their lives better, while 64% believed it made no difference and 5% did not know (Jackson, Blanck, & Schmeling, 2007). Definitions and Statistics The ADA defines a person with a disability as an individual who has a physical or mental impairment that substantially limits one or more major life activities (ADA, 1990). While major life activities are not defined in the ADA, the regulations of the Equal Employment Opportunity Commission (EEOC) offer examples such as taking care of oneself, the ability to use ones hands, the ability to ambulate, and holding a job (Taylor, 2009). Based on the ADA definition, the latest U.S. Census identified roughly 41.4 million people in the United States civilian, noninstitutionalized population who had a disability in 2016, which constituted 12.8% of Americans (Kraus, 2017). The breadth of this census data provides a better reflection of the types of disabilities and populations being represented by these statistics. The survey instrument used in the Disability Statistics Annual Report portion of the U.S. Census, the source of the data cited above, excludes active military personnel and institutionalized individuals. Military personnel and institutionalized persons are excluded because these groups, by definition, as compared to the total population of persons who have disabilities, have disproportionately large or small numbers of persons in important surveyed characteristics such as employment, which could skew the data. Types of Bias There are many types of negative perceptions or biases that can cause discriminatory behavior, two of which are particularly common. The first is a stigma, which is the act of seeing a physical or character trait in a disproportionately negative light (Goffman, 1963). Stigmas associated with disability characteristics such as paralysis, blindness, or obesity include beliefs 8 that individuals with disabilities are victims, need help, and are defined by their deficits. Stigmas also frequently produce emotions such as fear, pity, or revulsion. These kinds of stigmas can cause individuals to avoid contact with the target of their negative emotion, which can result in discriminatory behavior such as not wanting to hire an individual with a disability (Goffman, 1963; Nario-Remond, 2010). Stigmas also sometimes serve as the basis for stereotyping which is another common type of negative perception. Stereotyping occurs when individuals are assumed to have certain perceived characteristics strictly because they are members of a certain group of individuals (Goffman, 1963). Some people continue to hold negative stereotypes regarding individuals with disabilities, such as the view that individuals with disabilities have less ability than their able-bodied peers (Coleman, Brunell, & Haugen, 2015). Discrimination can include such actions such as omission, exclusion, or removal of a group of people from the ability to exercise the same rights and choices as everyone else based solely on their language, religion, gender or physical differences (Girli, Sari, Kirkim & Narin, 2016). In addition, per Girli et. al (2016), discriminatory actions can result from stigmas and negative stereotyping. Susman (1994) conducted a meta-analysis of past relevant studies regarding stigma and deviance as it relates to the disability experience, where deviance is the perception of a negative difference and stigma is a negative reaction to the perceived difference. In this analysis, negative attitudes about individuals with disabilities were frequently characterized by discriminatory actions such as omission and exclusion from activities or events in which they would have otherwise been included. These negative attitudes were the result of stigmas and associated negatively perceived differences or deviances in such abilities as vision, hearing, and ambulation as compared to people who do not have disabilities (Susman, 1994). It is 9 then the differences between abilities and disabilities, and the perceptions associated with them, which frequently cause discrimination (Green, Davis, & Karshmer, 2005). The main goal of this project was to test ways to reduce bias such as negative stereotyping toward those with disabilities in order to potentially curtail discrimination against these individuals. Determining ways to reduce negative stereotyping is particularly important because efforts to reduce the impact of negative attitudes through law have not been optimal. The welfare of individuals with disabilities, particularly from an employment and economic standpoint, has actually declined since the 1970s in part due to the lack of specificity and absence of concrete definitions in the ADA (Jackson, Blanck, & Schmeling, 2007; Maroto & Pettinicchio, 2015). Although the ADA requires businesses and organizations to make a reasonable effort to offer equal or equivalent access to facilities, programs, and services provided to the general public, reasonable is not defined and is therefore subject to interpretations influenced by negative employer attitudes and judicial rulings (Robert & Harlan, 2006). Discrimination is particularly significant in the areas of employment, education, and recreation. For example, workplace discrimination due solely to the presence of a disability has been reported by both 20% of employers and 20% of employees with disabilities. Per McMahon et al. (2008), discrimination due to the presence of a disability is reflected most notably in poor retention of employees with a disability rather than in hiring practices. In spite of the fact that U.S. children are entitled to a free education through high school graduation ensured by the Federal Education for All Handicapped Children Act, individuals with disabilities continue to encounter numerous barriers to accessing available offerings from elementary through postsecondary educational levels (U.S. Department of Education, 2015). In 2006, the American Psychological Association found that more than twice as many people age 25 to 64 with severe 10 disabilities failed to finish high school when compared to those without disabilities, where severe disability under the ADA is the legal definition of this term found at 41 CFR 511.3.Also, while not covered by a similar educational statutory entitlement, completion of postsecondary educational studies was also affected by whether a student had a disability (Steinmetz, 2006). The U.S. Census Bureau reported in 2015 that more than twice as many people without disabilities age 25 to 64 had a college degree as compared to individuals with severe disabilities in the same age group. Some of the disparity in college educational levels may be affected by the fact that approximately 28% of the total number of individuals with disabilities includes individuals with cognitive, emotional and/or mental disabilities, the vast majority of whom also have a physical disability. Unfortunately, the Disability Statistics Annual Report portion of the Census which is the source of the data above does not collect cross-stratified data on severe versus non-severe disabilities, high school and college graduation rates, and other data detail sufficient to describe potential causes for the failure of individuals with disabilities to earn college degrees. Therefore, it is unknown to what extent cognitive, emotional and mental disabilities may have affected the college graduation rate of individuals with disabilities as a whole (Steinmetz, 2006). Recreation is yet another area where discrimination is encountered. A qualitative study by Yau, Mckercher & Packer (2004) employed in depth interviews and focus groups. The authors determined that limitations on the tourism experiences of individuals with disabilities were complex. The physical accessibility aspects of the travel as well as the accessibility of the travel activities themselves were frequently uneven and were marginal or less than ideal. As a result, a traveler with a disability may have been able to participate in some but not all aspects of a trip and/or may have had to pay a premium price for accessibility. Travelers with disabilities 11 may not have the energy to participate in the same recreational offerings as their non-disabled companions. There are also societal and family barriers to travel such as disapproval of family, irritation of non-family at being inconvenienced by having to provide assistance, or the sense of embarrassment experienced by the individual with a disability at needing assistance (Yau, Mckercher & Packer, 2004). Successful tourism with a disability requires the coordination of the individuals physical, mental, and social capabilities, as well as his or her stamina and preferences, any of which may be adversely affected or compromised by a disability (Yau, Mckercher & Packer, 2004). Even the unintentional limitation of access has the effect of excluding travelers with disabilities, thereby impinging on individuals with disabilities fundamental rights and freedoms to equal treatment under the law and constituting a form of discrimination (Girli, Sari, Kirkim & Narin, 2016). Reducing Stereotypes of Those with Disabilities Using Intergroup Contact Theory Because disability discrimination begins at least in part with the negative biases of others, a great deal of research has been conducted to determine how such biases can be reduced. One long established model for reducing negative biases is intergroup contact theory (Pettigrew & Tropp 2008). Intergroup contact theory posits that prejudice about groups to which people do not belong themselves is reduced after they have meaningful contact with members or objects from these groups (Pettigrew, 1998). Allport (1954) held that the positive effects of intergroup contact can occur only under four conditions: 1) equal group status within the situation where both groups expect and recognize their equal status in that situation; 2) both groups having the same goals for that situation; 3) the willingness of the groups to cooperate together toward the common goal; and 4) the explicit support of the groups by local authorities, law, or custom. 12 The purpose of a study on intergroup contact that was conducted in Turkey in 2016 by Girli, Sari, Kirkim & Narin was to find out how educated, able-bodied students regarded individuals with disabilities. A related purpose of the study was to determine if participant characteristics such as socioeconomic status, geographic location, or previous exposure to individuals with disabilities were associated with positive attitudes toward individuals with disabilities (Girli, Sari, Kirkim & Narin, 2016). The ATDP scale was administered verbally to participating students. Most students agreed that individuals with disabilities were discriminated against more than any other group in Turkey, but that because more disability-based accommodations were being seen, the students believed that discrimination was decreasing over time. Furthermore, students who had spent the most time in small villages, who were more economically disadvantaged, or who had spent more time with individuals with disabilities had more positive attitudes toward this population as shown by their ATDP scores. One of the conclusions of the study was that programs offering exposure to individuals with disabilities should be undertaken more often in an effort to reduce stereotyping and possible subsequent discrimination. Similar findings related to the benefits of intergroup contact were identified in a study conducted with a U.S. sample by Bialka, Brown, Morrow and Hannah (2017). In this study, the Level program was implemented to create a social space and a more level playing field between students who did and did not have disabilities. In this program, the able-bodied students were paired with students with physical disabilities to promote intergroup contact. For the study, the able-bodied students were interviewed before and after their participation in the Level program in order to see whether their attitudes toward individuals with disabilities changed. As predicted, after the exposure of the two student groups to each other, the students without 13 disabilities had a more positive association with and understanding of people with physical disabilities. Students found that their preconceptions about disabilities and concerns regarding interactions with students who had disabilities were not factual assessments and that there were more similarities than differences between the two groups. In an effort to address the need to promote positive attitudes toward individuals with disabilities by healthcare professionals, a longitudinal study was done by Paris (1993) to assess the attitudes of first and fourth year medical students and of healthcare professionals who may have contact with those with disabilities. Paris (1993) used intergroup contact theory as the basis for the design of the study wherein the attitudes of participants toward individuals with disabilities was tested in groupings according to the varying amounts of contact the participants had with this group. The ATDP scale was the measure used to assess group differences by amount of contact as well as across various demographics in regard to attitudes toward individuals with disabilities. The study included how much contact the students and healthcare professionals had with individuals with disabilities and whether the individuals were in a more equal or lower social status. Ultimately, fourth year medical students had more positive attitudes toward individuals with disabilities than the other groups as they had more experience with this population from the beginning through the end of their postgraduate studies (Paris,1993). The attitudes of already practicing healthcare providers fell in between the first and fourth year medical students. The study author believes this is probably due to the fact that while the healthcare professionals had more experience with individuals with disabilities, they did not have the benefit of the new patient-centered medical school training that had been found to promote positive attitudes toward individuals with disabilities. 14 A different but related aspect of intergroup contact theory was pursued in work by Crisp and Turner (2009) on reducing prejudice through simulated (or imagined) social contact. In this work, the authors explored the role of imagination in studies on extended contact theory. In extended contact theory, a person in one group shares with their group the positive experience they had in their direct contact with someone in another group; the sharing of information with the first group allows its members to imagine the positive nature of that contact sufficiently in order to reduce what had been their negative perceptions about the other group to the same extent that actual direct contact does in the intergroup contact theory. Crisp and Turner (2009) also discussed the power of the imagination in the well-studied positive impact of mental imagery. Following their analysis of studies involving imagination, Crisp and Turner (2009) proposed the paradigm of imagined intergroup contact theory as a much needed tool for reducing negative perceptions about others when direct contact is simply not possible such as in multicultural conflicts around the globe. Although imagined social contact produced responses similar in strength to direct social contact, the authors of the authors of this study recommended that further research be done particularly on their proposed paradigm of imagined intergroup contact. More work was also encouraged on the phenomenon of fade out in this paradigm, that is to determine how long the imagined social contact would continue producing strong responses. Schroeder, and Risen (2014) conducted a longitudinal study on intergroup contact theory which included a focus on fade out. Israeli and Palestinian teenagers were followed at annually held three-week summer camps in the United States, over a period of three consecutive summers. The teenagers were interviewed prior to initially meeting the other individuals in the camp setting in the first year and at the end of the session. The campers were then contacted just prior to the camps in each of the two subsequent years. Post tests were administered annually and they 15 continued to be more positive than the intake assessments. There was some fade out of positivity during each intervening year, however, at the end of the three-year period, overall post test results stayed more positive than testing at the original initial intake (Schroeder, & Risen, 2014). In each of the studies just discussed, similar results in support of intergroup contact theory were produced in that association with or knowledge about individuals with disabilities inspired positive attitudes toward the individuals with disabilities by their non-disabled peers. It was also noted in each of these studies that efforts should be made to promote the exposure of these groups to each other as a means to reduce stereotyping and discrimination. Reducing Stereotypes of Those with Disabilities Using Information and Statistics Another means to reduce discrimination that has been studied is the identifiable victim effect (Kogut & Ritov, 2005). In research on this subject, it was found that information about a specific individual can be more persuasive than aggregated statistical information about a group of individuals (Kogut & Ritov, 2005). The identifiable victim effect is based on the study of empathy as it affects helping behaviors (Kogut & Ritov, 2005).. The result of such studies is that the more the victim is identified, the more the recipient of this information is willing to help. The power of a narrative involving the identifiable victim to affect change is the antithesis of an effect known as psychic numbing that produces an absence of emotion where emotion is what motivates people to reduce discrimination and other negative behaviors (Slovic, 2007). In a study by Slovic on genocide (2007), it was found that information about large and impersonal groups did not produce the emotional response needed to evoke a helping response. On the other hand, information about specific individuals produced an emotional response that did spur action (Slovic, 2007). The results demonstrated that, in an extreme case, the absence of individually identifiable information produces the phenomenon of psychic numbing. When the 16 information presented in this study was impersonal, it caused psychic numbing which meant there was no emotional response in the general public so there was no public outcry against the genocide. In the same way impersonal versus individualized information has a differential impact on emotional response, studies have been done regarding the ability of narrative information versus statistical information to affect attitudes. Dieckmann, Slovic, and Peters (2009) examined the use of statistics versus narrative information to persuade its audience there was sufficient cause to make a decision to take precautionary measures in various high risk scenarios. All audiences responded positively to narrative information but depending on participants skills and background, statistical information was persuasive for only some participants. In all three reviewed papers, the more individualized narrative information that is provided, the greater the emotional response and action that is elicited. This is because narratives are a form of contact, and the identifiable victim effect is a form of imagined contact; therefore, per intergroup contact theory, these would be effective ways to reduce stereotyping and discrimination. These studies, then, provided the support for the basis of the current study, which is to determine the effectiveness of using statistics versus a narrative having an identified victim in producing a positive response toward individuals with disabilities. The focus of the current study was on attitude differences among experimental conditions (i.e., having different types of information presented to them) in order to identify how stereotyping can be reduced with different methods rather than investigating a change in attitude over time with one approach. 17 The Current Study The current study was designed to test the types of information that can reduce stereotyping. In prior studies on intergroup contact, including those with imagined intergroup contact and the identifiable victim effect, it has been shown that familiarity with individuals with disabilities fosters appreciation of their accessibility needs. In the current study, statistics or a narrative with an identifiable victim or both were tested to determine the most effective ways to reduce negative stereotyping relative to no additional information. In this experiment, participants were randomly assigned to a statistics-only, narrativeonly, statistics and narrative, or control condition following which they responded to the ATDP measure. This methodology was based upon the work done by Slovic (2007). A combined condition was included to see if receiving more than one type of information would be more effective than each alone. Treatment conditions were expected to have more positive views than the control condition. In addition, participants with disabilities and those who have regular contact with individuals with disabilities were tested for group differences to see if their ATDP responses following the experimental conditions were more or less positive as compared to those without disabilities or without regular contact in secondary analyses. It was hypothesized that individuals who have disabilities or who have regular contact with such individuals would have more positive views of the disabled population than other groups. Groups with prior contact may have predisposed attitudes toward individuals with disabilities and be more or less impacted by the conditions, compared to individuals who do not fall into these subsets. In the testing of group differences, participants having disabilities or regular contact with such persons were expected to have more positive views of individuals with disabilities. The treatment groups within each of 18 these subsets were expected to follow the same patterns hypothesized for persons who had no contact with individuals with disabilities, except at a more positive rate. Method Participants Ninety-two undergraduate students from the University of Indianapolis (UIndy), which was fewer than the 120 participants proposed prior to data collection, participated in this study. The difficulty in obtaining the projected sample size may have been due to the timing of the study in the semester or to other factors that are discussed in the limitations section below. Analyzing the data was still meaningful as the smaller sample size was still large enough to find significant results for a medium to large effect size if condition differences existed; however, the results should be interpreted with some caution in terms of how replicable they might be in future samples due to the smaller size of sample and the potential for a smaller than expected effect size. Of the 92 participants, 80 were female students and 78 were pre-health care professionals. There were 11 individuals who self-identified as having a disability, and 64 individuals who self-identified as being a person with regular contact with individuals with disabilities. All participants, regardless of background, were included in the main data analysis. There was an age exclusion criterion barring individuals who were younger than 18 from participating in the research as those individuals would be unable to consent as legal adults; no other exclusion criteria were included for participants who were drawn from an existing subject pool. As part of secondary analyses, possible differences between those with and without disabilities and those with and without regular contact were explored. 19 Participants volunteered through SONA which requires that they be currently enrolled in a psychology class. SONA is the medium through which the University of Indianapolis School of Psychological Sciences allows students to access surveys for psychology studies. SONA awards students credit for completed studies, which can be used toward extra credit or course credit. The study was presented to participants through Qualtrics, which is a platform within which complex surveys can be created and administered online. All the data were collected anonymously, and Qualtrics was used to store the data securely and confidentially, which was an Institutional Review Board requirement for in this study. Measure Used to Assess Attitudes Toward Individuals with Disabilities The ATDP instrument, which is provided in Appendix A, was selected as the measure of perceptions about individuals with disabilities for this study based on its validation in past work. This instrument was initially introduced in the late 1960s and has been a frequent choice to assess stereotyping in studies about attitudes toward individuals with disabilities. By 1986, there had been approximately 50 other studies that had used the ATDP (Yuker & Block, 1986), and to date, there have been over 500 studies that have used the ATDP. Because of the simplicity of its administration as compared to the time and resources required by other approaches to assessing prejudice, the ATDP is recognized as a practical and effective method of determining biases toward individuals with disabilities. This tool was used successfully to measure attitudes toward individuals with disabilities in two of the studies on intergroup contact discussed in the literature review above: Paris (1993), and Girli, Sari, Kirkim and Narin (2016). In past work, the ATDP has been used to show group differences in attitudes and to show attitude changes as well as differences within participant groups when given after an intervening event that influenced participant attitudes (Yuker, & Block, 1986). For this study, the scale was 20 administered online to measure attitudes once in order to look at group differences by condition and not over time to assess change in attitudes. Specifically, the ATDP Form O was used in this study and has undergone extensive validity and reliability statistical testing (Yuker and Block, 1986). The reliability coefficients ranged from +.75 to +.85 for Form O. Strong coefficients have a range of +/- .7 1. Validity testing of the ATDP was based largely upon construct validity. Construct validity seeks to confirm a series of predictions regarding the relationship of the variable being measured to other variables. The extent to which the majority of predictions are found to be correct, with none yielding results that are diametrically opposed to what was predicted, determines the adequacy of the instrument from the standpoint of validity. The validity of this instrument was demonstrated with comparisons to more than 100 other instruments in the categories of demographics, personality, attitudes and experiential/behavioral considerations. Correlational techniques were the prime method of indicating the relationships between ATDP scores and other theoretically related variables. The resulting correlational coefficients in validity testing with these measures ranged from strong to no correlation, all of which was discussed in detail in the 1986 meta-analysis by Yuker and Block. Ultimately, the strongest, most consistent correlations were with instruments that assessed attitudes toward individuals with disabilities based on the type and amount of experience with this population, which also happens to correspond to findings in studies on intergroup contact theory. Based on this extensive testing, the authors concluded that the ATDP had average validity as compared to other similar tests that measure attitudes toward disabilities. Procedure Participants were given an informed consent page where they were told the research was examining what perceptions and beliefs participants have toward individuals with disabilities. 21 Participants were also informed that if they participated in the study, they would be compensated for their time in terms of course credit or extra credit toward psychology courses. They were informed that they were allowed to leave the study at any point, and that it is by their own free will that they were choosing to participate in the study. Participants were required to give their informed consent electronically before continuing in the study. Participants were then randomly assigned to one of the following conditions via the Qualtrics survey programming. The first treatment group (i.e. statistics only condition) received statistical information about individuals with disabilities in the form of a fact sheet prior to taking the ATDP. These statistics were presented in the form of two online pages with statistics coming from the Disability Statistics Annual Report. The statistics provided information about the disparities experienced by individuals with disabilities as compared to the able-bodied population in areas including employment, education, transportation, recreation, and marital status (Kraus, 2016). These data were used to convey the discrimination faced by individuals with disabilities within multiple domains. Prior to taking the ATDP, the second treatment group (i.e. narrative only condition) received a brief narrative description of life as a person with a disability. The narrative information that was used was a short video called We Carry Kevin, which was embedded into Qualtrics and presented before the survey. A detailed description of this informative film, that I had obtained the rights to use, is attached as Appendix B. The third treatment group (i.e., statistics and narrative condition) received both forms of information with the statistical information first, followed by the narrative, prior to taking the ATDP. The fourth group, the control group, moved from the informed consent page directly to the ATDP survey. This group served as the baseline for disability stereotyping, having received no information regarding the barriers to access experienced by this population. 22 Participants were instructed to respond to each statement on the ATDP survey according to their immediate agreement or disagreement with it. The scale of the ATDP is +3 (strongest agreement) to -3 (strongest disagreement). The length of the ATDP is 30 questions. The ATDP aims to assess perceived attitudes regarding individuals with disabilities in many settings such as work place, family life, and extracurricular activities. An example of a question from the ATDP is Disabled persons are usually friendly. The ATDP has reverse scored items, which are written in a reversed manner so that higher scores mean disagreement whereas most of the rest of the questionnaire is worded that higher scores mean agreement with the statement. The ATDP uses a scoring methodology that provides a summed score of all items for each participants responses after changing the signs on the reverse scored responses. A constant of 60 points is then added to each participants score to compensate for the reverse scoring so that all total scores are positive values. This allows conclusions to be drawn regarding how positive or negative the views of the participants are toward individuals with disabilities. After completing the ATDP, participants were asked to provide demographic information such as age, gender, and race/ethnicity. Participants were also asked for their academic major and year in school in order to determine if there were any related trends in responses; however, no analyses were conducted given the lack of diversity in respondents on these dimensions. The last two questions of the survey allowed for the exploration of group differences based on condition and participants self-identification of disability status or contact with others who have disabilities. The first question allowed the participant to self-identify on a strictly voluntary basis that he or she had a disability. The question used the definition of disability from the ADA: Do you regard yourself as having a mental or physical impairment which substantially limits one or more activities of daily living? Respondents could answer with: Yes, I have a disability, No, 23 I do not have a disability, or I do not wish to answer. Secondly, a multiple choice question inquired about the amount of contact with individuals with disabilities that the participant had on a regular basis where for the purposes of this study, regular contact was to be based on the participants own self-definition. The response options were Yes, I do have regular contact with individuals with disabilities, No, I do not have regular contact with individuals with disabilities, or I do not wish to answer. The full study took each participant less than fifteen minutes. They were automatically credited through SONA upon completion. Having these responses fall in to three categories, allowed the data to be analyzed in a more succinct way that produced a clear pattern. Results Data Analysis Plan The first statistical analysis run was a test of the reliability of the ATDP measure. The items of the ATDP were tested for their reliability by computing a Cronbachs alpha for the measure. Cronbachs alpha is a measure of internal reliability and consistency that denotes how well the items on a measure such as the ATDP relate to one another to determine whether they measure the same thing and if they can be combined into one index. In this case, the single index is the overall ATDP score. Opposite items were reverse-scored prior to this analysis, and each item was examined to see how frequently the responses were recorded and how well each related to the rest. These items did not combine well ( = .499), meaning that the items had some variability in what they were tapping and were not as consistent in assessing the same construct as hoped. After reliability testing was complete, the main test was a one-way ANOVA using the experimental condition as the independent variable and the average attitude scores on the ATDP 24 as the dependent variable. Post hoc tests would have been run to see which groups differ from one another if the one-way ANOVA was statistically significant. Next, previous contact with people with disabilities and self-identification of disability status were also included as factors in separate between-subjects factorial ANOVAs. The first between-subjects factorial ANOVA was run with condition and disability status as independent variables and the ATDP as the dependent variable. The second between-subjects factorial ANOVA was run with condition and contact as the independent variables and with the ATDP again as the dependent variable. ATDP Scores by Condition The treatment conditions were expected to have more positive ATDP scores than the control condition. A one way ANOVA was conducted for the manipulation of condition and resulted in non-significant differences among conditions on ATDP scores, F(3,92)=.117, p=.950, with the control condition (M=74.208), statistics only condition (M=73.364), narrative only condition (M=72.423), and both information condition (M=73.950) having similar scores on the ATDP. Impact of Participants Disability Status and Contact Two separate between-subjects factorial ANOVAs were then run to test for any interactions between the manipulation of condition and whether participants had a disability or whether participants reported having regular contact with individuals with disabilities. For the regular contact analysis, there was not a significant main effect of the extent of contact, F(2,92)= .565, p=.454, such that participants who reported having regular contact (M=73.77) did not perceive individuals with disabilities more positively than those who did not (M=71.768). There was also not a significant main effect of condition, F(3,92)= .215, p=.866, such that the control condition (M=73.250) did not differ from the statistics only condition (M=71.427), the narrative 25 only condition (M=72.154), or both information condition (M=74.227). Furthermore, there was not a significant interaction between condition and regular contact, F(3,92)= 1.219, p=.308. These results do not substantively change when only including those without disabilities in the analysis. For the disability status analysis, there was a not significant main effect of selfidentification of having a disability, F(1,92)= .234, p=.783, such that participants who selfidentified as having a disability (M=73.042) had an ATDP score similar to participants who did not (M=73.188). There was also not a significant main effect of condition, F(3,92)= 1.912, p=.134, such that the control condition (M=79.199) did not differ from the statistics only condition (M=65.593), the narrative only condition (M=69.278), or both information condition (M=74.356). There was, however, a significant interaction between condition and disability status, F(3,92)= 2.748, p=.024. For disability status, there were significant differences between conditions for participants who self-identified as having a disability, F(3,92)=3.926, p=.011, such that those in the control condition (M=85.667) and both information condition (M=83.000) scored higher than those in the statistics only condition (M=55.50) and narrative only condition (M=68.0). There were not significant differences between the conditions for participants who did not self-identify as having a disability F(3,92)=1.272, p=.972, , such that those in the control condition (M=72.571) and both information condition (M=71.071) scored similarly to those in the statistics only condition (M=76.278) and narrative only condition (M=72.833). 26 Figure 1: This figure shows the significant interaction between experimental condition and disability status on ATDP scores compared to the non-significant interaction on ATDP scores by condition from no-associated disability status. Discussion The main hypothesis was that there would be a difference in positive responses based on condition. However, there were no significant differences among the four conditions, so the hypothesis was not confirmed based on the findings above. The secondary hypotheses were that groups having previous regular contact with people with disabilities or self-identified as having a disability would have more positive outcomes on the ATDP. There were no significant effects of the conditions on those with regular contact with individuals with disabilities and those without regular contact across the four conditions. This did not support the hypothesis that individuals who had regular contact with individuals with disabilities would have more positive outcomes on the ATDP across all conditions. As for disability status, there was a significant interaction when comparing the self-identification of having a disability or not across the conditions of the manipulation. Specifically, for those individuals who self-identified as having a disability, there were differences in condition such that the both group and the control group responded more positively than the statistics groups and narrative group. However, for those individuals who did not have a disability, there were no significant differences by condition. These results of this 27 were not consistent with the current literature in that there was no difference in the perceptions of participants among the treatment conditions. There is a strong case in all current literature for why differences should have been found among the treatment groups as compared to the control condition and between disability and disability-contact groups in this study based on intergroup contact theory and types of information used. Limitations A major limitation of the study was that the Cronbachs alpha for the measure of attitudes toward disability, the ATDP, was low suggesting that the internal reliability or consistency of this measure was low. Moving forward, using a different version of the ATDP or another measure for assessing perceptions of individuals with disabilities may result in improved internal reliability for this outcome. In addition, since the ATDP is an older measure, its questions may be too dated for use in the current world causing participants to interpret them in different ways than would have been normative 50 years ago when the measure was created and tested. The studys sample size was also smaller than proposed because of time constraints outside of the researchers control: the study had to be conducted during the beginning and middle of the semester when most participation comes toward the end of the semester and at a time that students did not yet require the compensation of extra credit they would receive in return for participation. To address this limitation, spanning the research across multiple semesters or on multiple platforms may increase the sample size. The study is still relevant despite the small sample size as it was created to start to address missing pieces in the current literature. In addition, the smaller sample size was still large enough to find significant results if they existed although some caution should be used in interpreting the results for future replicability. The group that participated in the study that self-identified as having a disability 28 was a small proportion of the total respondents and they were very unevenly distributed by condition. The results of each group by condition were compared, but could benefit from more participants in this category participating in future research so that the data is more reflective of the whole population. Lastly, a manipulation check was not included in the questionnaire. The use of a manipulation check was overlooked in creating the demographic questions. In the future, a test question regarding what condition the participant was exposed to may provide the ability for researchers to disregard data based on the inability to respond to this question correctly. This would allow researchers to eliminate participants that did not pay attention to the conditions. Implications and Future Directions This study was intended to evaluate perceptions regarding individuals with disabilities and to highlight the importance of conducting future research on this topic, because of the researchs potential to improve perceptions about individuals with disabilities. In this study, only individuals who self-identified as having disabilities and who were exposed to a combination of statistics and narrative and the control group prior to taking the ATDP were positively influenced, when compared to individuals who self-identified as having a disability in the other treatment conditions, and to those who did not self-identify as having a disability. These results were statistically significant, and disability status should be used as a variable that is taken into account in future research that is working toward increasing positive perceptions of individuals with disabilities. Since there were not significant findings when comparing the treatment conditions to the control condition without looking at disability status or when including contact as an additional independent variable, one possible conclusion is that the information presented was not 29 persuasive enough to produce the expected differences in perceptions without participants having a disability themselves. In the future, having imagined scenarios where individuals without a disability undergo an imagined perception activity prior to taking the ATDP in order to increase empathy with individuals with disabilities may change the outcomes. An imagined perception activity is one where the participant would try to put themselves in someone elses place in order to better appreciate the others point of view. Another way to change the narrative into a more influential condition that produces more positive outcomes than the use of a video may be to have an in-person narrative, whether it be a forum or speech prior to taking the ATDP. Using more measures before or after administering the ATDP measure also might be beneficial as the secondary assessments could be run and validity could be cross-referenced with the ATDP. The ATDP could also be introduced to each participant before and after the conditions to track changes instead of comparing differences between groups. In addition, individuals could be measured in a longitudinal manner to assess long term changes. The overall ATDP score was just slightly positive as compared to standardized scores obtained from compiled average data as part of testing of the ATDP (Yuker & Block, 1986). The means obtained demonstrated that the individuals who participated in the study overall did not have an overly positive view of individuals with disabilities. This finding suggests that there could be more work done in the future to determine what can effectively change perceptions with a different model or different conditions for such a study. Future studies could look at other ways to change perceptions like the examples mentioned above. The results of this study indicate that, in spite of findings in the literature, the conditions of having direct or imagined contact with individuals with disabilities may not be enough to create more positive attitudes toward individuals with disabilities. In this study, the group that 30 had the most positive perceptions of individuals with disabilities were the participants who selfidentified as having a disability. This demonstrates that self-identification as having a disability may improve the perceptions of this group of its own value. This finding may aid in future research models. Conclusion The goal of this study was to conduct research, to provide results that could be used by healthcare professionals and disability advocates in understanding how to reduce negative attitudes toward individuals with disabilities. The hope was that by reducing negative perceptions, discrimination against the individuals with disabilities they serve could also be reduced. Another aim was to gain more awareness of the applicability of intergroup contact theory in promoting more positive attitudes toward individuals with disabilities for further research. While the data did not produce significant results across the conditions, it did provide information about the population of persons who currently identified themselves as having disabilities compared to those who do not. It is hoped that this research will be continued and expanded as recommended in the current literature and by the findings of this research. Intergroup contact theory in its varying forms presents exciting opportunities for improving relations between minority and majority populations such as individuals with disabilities and those without. Research into this approach needs to be pursued and refined as it can not only improve perceptions of individuals with disabilities and myriad other minority groups but also reduce discrimination toward these groups and improve their real world treatment. 31 References Allport, G. W., Clark, K., & Pettigrew, T. (1954). The nature of prejudice. Americans With Disabilities Act of 1990, 42 USC 12101, Public Law 101-336, 104 Stat. 328 (1990). Americans With Disabilities Act of 1990 as amended. 42 USC 12101, Public Law 110-325, Publication Number ADA-0001 (2000). Bialka, C. S., Brown, K. S., Morro, D., & Hannah, G. (2017). On their LEVEL: How participation in a university student group shapes members perceptions of disability. Journal of Diversity in Higher Education, 10(2), 110-117. 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Stigma: Notes on the Management of Spoiled Identity. Englewood Cliffs, NJ: Prentice Hall Green, S., Davis, C., Karshmer, E., Marsh, P., & Straight, B. (2005). Living stigma: The impact of labeling, stereotyping, separation, status loss, and discrimination in the lives of individuals with disabilities and their families. Sociological Inquiry, 75(2), 197-215. Hafer, M., Wright, W. R., & Godley, S. H. (1983). Dimensionality of the attitudes toward disabled persons scale revisited. Educational and Psychological Measurement, 43(2), 459-463. Jackson, P. M., Blanck, P., & Schmeling, J. (2007). The Impact of the Americans with Disabilities Act: Assessing the Progress Toward Achieving the Goals of the ADA. Washington, DC: National Council on Disability. Kazoleas, D. C. (1993). A comparison of the persuasive effectiveness of qualitative versus quantitative evidence: A test of explanatory hypotheses. Communication Quarterly, 41(1), 40-50. Kogut, T., & Ritov, I. (2005). 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Research with the Attitude Toward Disabled Persons scales (ATDP) 1960-1985: Hofstra University. 29-31. 35 Appendix A The Attitudes Toward Disabled Persons (ATDP) Survey (Hafer, Wright & Godley, 1983) Instructions: Participants are to respond according to their initial agreement or disagreement with each statement. +3 I agree very much +2 I agree pretty much +1 I agree a little -1 I disagree a little -2 I disagree pretty much -3 I disagree very much 1) Disabled persons are usually friendly. 2) People who are disabled should not have to pay income taxes. 3) Disabled persons are no more emotional than other people. 4) Disabled persons can have a normal social life. 5) Most physically disabled persons have a chip on their shoulder. 6) Disabled workers can be just as successful as other workers. 7) Very few disabled persons are ashamed of their disabilities. 8) Most people feel uncomfortable when they associate with disabled people. 9) Disabled people show less enthusiasm than non-disabled people. 10) Disabled people do not become upset any more easily than non-disabled people. 11) Disabled people are often less aggressive than normal people. 12) Most disabled persons get married and have children. 13) Most disabled persons do not worry more than anyone else. 14) Employers should not be allowed to fire disabled persons. 15) Disabled persons are not as happy as non-disabled persons. 16) Severally disabled persons are harder to get along with than are those with minor disabilities. 17) Most disabled persons expect special treatment. 18) Disabled persons should not expect to live normal lives. 19) Most disabled persons get discouraged easily. 20) The worst thing that could happen to a person would be for him to become to be severely injured. 21) Disabled children should not have to compete with severely disabled children. 22) Most disabled people do not feel sorry for themselves. 23) Most disabled persons prefer to work with other disabled people. 24) Most severely disabled people are not as ambitious as other people. 25) Disabled persons are not as self-confident. 26) Most disabled persons do not want more affection and praise than other persons. 27) It would be best if a disabled person would marry another disabled person. 28) Most disabled people do not need special attention. 29) Disabled persons want sympathy more than other people. 30) Most physically disabled persons have different personalities than normal persons. 36 Appendix B The Narrative Film We Carry Kevin The narrative information that will be used is a video called We Carry Kevin. This informative film is about a young man with severe physical disabilities whose friends carry him when necessary throughout a once in a lifetime vacation journey. Without his friends help, Kevin would not have been able to access the places he visited. He talks about what a freeing experience it was. Although the video emphasizes disability more than abilities, it is the limitations of the disability that cause the lack of access, the negative attitudes, and ultimately the discrimination toward persons with disabilities in the first place so it is appropriate for the information provided to participants to emphasize the impact of the disability and its limitations from a personal point of view. The video is also unique in that it gives the perspectives of the able-bodied individuals who are providing disability access in a very personal way. The video is approximately three minutes long, and the rights to use this video in the study have been obtained. 37 Appendix C IRB Approval Letter 38 Appendix D CITI Training Certificate  ...