Berry Summer Thesis Institute Proceedings 2018 by University of Dayton Honors Program

Berry Summer Thesis Institute

Proceedings 2018

Berry Summer Thesis Institute

University of Dayton Honors Program Publication Alumni Hall 124-125 300 College Park Drive Dayton, Ohio 45469-0311 937.229.4615 Copy Editor: Nancy Martorano Miller, Ph.D. Designer and Production Manager: Ramona R. Speranza Printer and Binder: Globus Printing and Packaging, Minster, Ohio Cover Photographs and Illustrations: See Pages 78-79 for credits Copyright ÂŠ 2018 University of Dayton Honors Program. All rights reserved. Reproduction or translation of any part of this work beyond that permitted by the United States Copyright Act without the permission of the copyright owner is unlawful. The copyright of each article is held by the author. Requests for permission or further information should be addressed to the University of Dayton Honors Program. ISSN: 2332-9890 eISSN: 2332-9912 Printed in the United States of America

FROM THE DIRECTOR

Dear Reader: The faculty, staff, friends and family who attended the annual Berry Summer Thesis Institute (BSTI) symposium in August 2018 witnessed an impressive group of emerging scholars presenting the early stages of Honors thesis research. The projects included in this volume of the Proceedings range from an investigation of how youth sports coaches manage concussions and concussion protocols, to the sustainability of the food retail industry, to an analysis of the pedagogical uses of comic books in Catholic religious education. This collection once again reflects the Honors Program’s goals for the BSTI: (1) to introduce rising juniors to peer review and publication; and (2) to begin the process of contributing to knowledge or producing original creative works. As the director of the Honors Program, I am especially grateful for the friends and alumni that continue to support the Berry Summer Thesis Institute. In particular, let me express my sincere gratitude to Mr. John W. Berry, Jr., Mrs. Shirley Berry and the entire Berry family for their continuing support for the University of Dayton Honors Program. I also want to extend my thanks to Dr. Nancy Martorano Miller for her leadership of the BSTI, to Ramona Speranza for designing and producing the Proceedings manuscript and to Maria Ollier Burkett for facilitating the connections with community partners that allowed our students to give back to the Dayton community when they weren’t conducting research. Please enjoy the work of some of UD’s talented Honors students! Best, John P. McCombe, Ph.D. Director University Honors Program University of Dayton

ACKNOWLEDGEMENTS

Thanks to a gift from the Berry Family Foundation and the Berry family, the UHP offered eight rising juniors the opportunity to participate in the 2018 Berry Summer Thesis Institute. First initiated in the summer of 2012, the Institute introduces students with a proven record of academic success and interest in intensive research, scholarship opportunities and professional development while earning Honors credits towards their Honors Program diplomas. Students selected for the Institute were competitively selected for participation by the University Honors Program review committee. Each student pursued a 12-week summer thesis research project under the guidance of a UD faculty mentor. In coordination with the Center for Social Concern, Campus Ministry and the Fitz Center for Leadership in Community, the students also learned about civic engagement and servant leadership by volunteering with local community partners.

TABLE OF CONTENTS Page ENGINEERING Industry 4.0 in the Retail Sector: Sustainability of Food Retail Industries by Katrina A. Coleman . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

TEACHER EDUCATION Capes and Catechesis: Using Comic Books to Catechize Catholic Youths by Lindsey M. Bronder . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

HEALTH AND SPORT SCIENCE The Effect of Remote Ischemic Preconditioning on Hypoxic (Low Oxygen) Cognitive Function and Exercise Performance by Jenna N. Sorensen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

Youth Sport Concussion Management: A Review by Cordell J. Stover . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

LIFE AND PHYSICAL SCIENCES The Human Gut Microbiome, Microbial Metabolites and the Development of Analytical Techniques of SCFA Analysis by Ashton N. Dix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

Finding the Switches That Activate Animal Genes Through a Combined in Silico and in Vivo Approach by Chad M. Jaenke . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

Effect of Bacterial Membrane Composition on Antibiotic Susceptibility by Samantha L. Neanover . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

Tyrosine Kinase Inhibitors and Their Use as Drugs in Glioblastoma Chemotherapy: A Review by Jordan M. Terschluse . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

Photograph and Illustration Credits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76

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Industry 4.0 in the Retail Sector: Sustainability of Food Retail Industries Katrina A. Coleman1,2,3 University of Dayton, 300 College Park, Dayton, OH, 45469 1. Department of Engineering Management, Systems and Technology 2. Berry Summer Thesis Institute 3. University Honors Program

Mentor: Sean Falkowski, M.S. Department of Engineering Management, Systems and Technology Corresponding Author: Sean Falkowski, M.S. Email: sfalkowski1@udayton.edu

Abstract Digital transformation is becoming an important part of manufacturing and the service industries worldwide. Europe is leading the push for this in manufacturing under the umbrella of Industry 4.0. This is a relatively new phenomena in the United States in the manufacturing sector. The service industries are also embracing this and transforming how companies are doing business. One area of transformation is in the retail sector, specifically, food retail. This involves technical as well as managerial developments. With the advent of Industry 4.0, the fourth wave of the industrial revolution, the food retail industry has been revolutionizing at an unprecedented rate. Stores have become stocked with a large variety and quantity of goods, leading to an increase in waste accumulation. Although there have been advancements in large scale agriculture, not as many advancements have been made in efficiently distributing produce to areas of lower incomes mainly or minimizing waste in the retail space. Stores are overordering on some items, while underordering on others. Customers are expecting items in real time and with a high degree of quality. This push for customer satisfaction has led to the advancement and an increase in online shopping for groceries where the customer can track their items with absolute transparency of their purchased products. While there have been some improvements to ensure customer satisfaction and the minimization of waste, it is only in the beginning stages for food retail industries. Waste of transportation time, product, labor and other waste-causing factors 2

can all be minimized. Further data analysis is required; however, the information gathered thus far into research suggests that a new method to reducing waste in grocery stores can be implemented while also helping to feed those in lower-income areas who do not have the means to obtain fresh produce.

Introduction Industry 4.0 is described as the “marriage of digital and physical systems” [8]. Industry 4.0 was first introduced in 2011 and was defined as: “A paradigm shift . . . made possible by technological advances which constitute a reversal of conventional production process logic. Simply put, this means that industrial production machinery no longer simply ‘processes’ the product, but that the product communicates with the machinery to tell it exactly what to do.” [7] (Figure 1)

Industry 4.0 ties into retail, more specifically, food retail, because the satisfaction of the customer is an important factor on which Industry 4.0 focuses [6] (Figure 2). Customers are wanting items faster than ever before, and everything should be attainable to them. When a customer goes to a grocery store, there should not be any items out of stock that they have to wait to get back in stock. Schmaus et al. described Industry 4.0 in relation to food retail as:

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Figure 1. The Different Waves of the Industrial Revolution [7]. Courtesy of Deloitte University Press, DUPress.com.

â&#x20AC;&#x153;The Industry 4.0 digital revolution is mobilizing a new type of R&C (Retail and Consumer goods) consumer, who wants a seamless, fast, efficient shopping experience and who is looking for products that are more personalized than ever before. For retailers, these preferences can be addressed through multichannel offerings, a combination of an online and physical outlet shopping experience with channels that consumers can switch between with ease, depending on their schedule or preferences on any given day. In a multichannel environment, enhanced product tracking and transparency lead to improved consumer services.â&#x20AC;? [6] (Figure 2)

Online grocery shopping is a direction in which food retail is headed. Currently, most grocery stores have an online pickup service, such as Meijer Curbside, or Kroger ClickList [2,4]. This is where one can order their groceries online Proceedings 2018

and pay for them, and then pick the groceries up at whatever time the customer chooses. Online food retail is still in its beginning stages, but it is already significantly popular for consumers.

ENGINEERING Aside from Industry 4.0 and how that relates to food retail online, one of my main focuses is waste in grocery stores overall, mainly produce. I am researching the highest areas of waste in food retail, and how to decrease that amount. I am also interested in how sustainable food retail industries are, and how to create a more sustainable environment in each company without decreasing profit significantly. Along with making the grocery store chains sustainable, I am researching how these companies educate their shoppers on sustainability. For example, I am looking to see if resources are available at the grocery store (advertisements, signs, brochures, charts, etc.) to encourage people to live a more sustainable lifestyle by standing with their local grocery to help create a healthier and more organic environment.

Methods Participants During my research, I did field work where I visited 12 different grocery stores within the Dayton, Ohio, area. These grocery stores were in three different areas of incomes: lower class, lower-to-middle class and middle-to-higher class (Table 1). I visited four grocery stores in each different-incomed area and the common grocery store in every city was Kroger. Table 1. Grocery Stores Visited. Courtesy of Katrina Coleman 2018.

Procedure While in each grocery store, I had a specific plan of what I would look for upon entering. My main focus was the products, and more specifically, the produce. I looked at the variety of goods, quantity, quality and pricing. Further than that, I wanted to look if each grocery store contained special areas for dietary restrictions such as: an organic section, a “dairy-free” section, etc. If the grocery store contained such areas, I further analyzed that specific area to see its variety, quantity, quality and price, as well as the overall size of the specialized section. After looking at the products, I would observe the advertisements and information given around the grocery store. I was curious about if there were signs that said anything about local farmers and nutrition. I was also curious if these grocery stores contained any educational resources on sustainability, even if that means a small sign that says, “Go Green!” to encourage sustainability and make the shopper wonder what exactly that means. After the observations of all 12 grocery stores, I decided to have an informal interview with a manager from a Kroger in a lower income area. My goals were to obtain some samples on their recorded highest areas of waste and figure out what Kroger’s plans were to reduce that amount of waste, on a corporate level as well as a local level. I was also curious about what Kroger’s goals were overall with sustainability and where they plan to go as a company in the next five to ten years. Lastly, I wanted to look more into the cyber-side of things, from the perspective of Industry 4.0 and learn about online grocery shopping and where it is currently at. This led me to having another informal interview but this time with a user of Meijer Curbside. I wanted to learn more about the exact process of online grocery shopping, as well as figure out what customers like and what they do not like about the process.

Following the grocery store visits, I had an informal interview with a manager at the most common grocery store in the Dayton area, Kroger, which was located in a lower-class area. I also had an informal interview with a user of the online and pick-up option (Meijer Curbside) at the grocery store, Meijer, and this user was from a middle-to-higher class area.

Results Due to Kroger being the common grocery store in all three different areas, I chose to mainly use that information to compare the differences. After entering the grocery store, I noticed different things at each location. At the lowerincome Kroger, the most common and largest signs read, “Low Price!” and were placed around Proceedings 2018

ENGINEERING the entire grocery store (Figure 3). In the middle and middle-to-higher income areas, I saw many signs about Kroger going local and organic. The signs were a lot greener and had more of a clean look to them that made the grocery store look â&#x20AC;&#x153;high endâ&#x20AC;? (Figure 4).

much lower and not much to choose from (Figure 6). Due to there not being a large produce section in the lower income areas, I continued to observe the other sections of the grocery and noticed there were many non-produce items that were high in fats, calories, carbs or anything else that

When looking at the produce, the higher income areas have a much larger variety of goods (Figure 4) when compared to the lower income areas. The section itself in the higher income area is much larger. In some grocery stores, the produce section was at least double in size. The produce at the higher income grocery stores was very fresh and was fully stocked with many options to choose from (Figure 5). Looking at tomatoes and avocadoes, for example, there were multiple brands and types of each item, whereas at the lower income grocery stores, there was only one type and kind of produce items, and the stock was

Figure 5. Produce from a Middle-to-Higher Income area. Courtesy of Katrina Coleman 2018.

Figure 3. Largest advertisement in Lower-Income area. Courtesy of Katrina Coleman 2018.

Figure 4. Part of the produce section at Middle-to-Higher Income location. Courtesy of Katrina Coleman 2018.

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Figure 6. Produce from a Lower-Income area. Courtesy of Katrina Coleman 2018.

ENGINEERING is filling. This food was also significantly less expensive. When analyzing organic and dietary restriction sections, almost every grocery store had an organic section. The higher-income stores also had a “non-dairy” refrigerated area which contained a wide variety of goods that seemed to replace most dairy items. At the lower income stores, the organic section was usually a fourth of the size, sometimes smaller and did not have a large variety. After visiting the grocery stores, I chose to speak to a manager from one of the Krogers to obtain a more personal and local point of view of the sustainability methods and goals to eliminate waste. After the informal interview, I researched further into Kroger’s sustainability goals online and everything seemed to line up from a corporate perspective as well. I achieved only half of my initial goal due to the fact that the manager was not willing to share specific information on the highest areas of waste, so we mainly discussed how waste is aiming to be reduced at Kroger. This manager discussed their “Shrink Care” with me, which is what Kroger calls their “sustainability commitment” and “was developed to take steps in further reducing waste” [3]. Kroger has been making an effort to go local, but local is defined differently for each company, due to the large size of some companies, like Kroger. Kroger corporately is committed to zero food waste by 2025 and is very confident in this goal [3]. The manager stated that Kroger is committed to eliminating plastic shopping bags by 2025. As far as what Kroger is currently doing, they have created a bag recycle barrel, which is where

customers can bring their own recycling so it is not wasted. Kroger also takes food that is still edible to food banks and puts produce on sale if it is near expiration. This Kroger, I am not sure about every Kroger, has created a “clearance rack” for produce where they will bundle different produce items together and the bundle will only cost around $1 to also help produce near expiration to be sold. Lastly, I had an informal interview with a user of Meijer Curbside. We discussed the pros and cons of the online pickup option (Table 2). The main thing the user liked about Meijer curbside was its convenience. They loved that they could sit at home and order their groceries while watching their children and choosing whatever time they wanted to pick the groceries up. The user stated that they receive all the same sales as inside the grocery, and the website is extremely user-friendly, even for someone who is not very experienced with computer use. The user is someone who is very picky about their produce, but they stated that the staff at Meijer picks out exactly what is desired and you can even specify the ripeness of your produce in the notes section. Changes can be made to the order up to three hours before pickup, and there is only a $10 fee. The user said they are actually saving money in the long run because when they normally physically go to the grocery, there are many items they will see in the aisles and grab them because in the moment they sound good [2]. There were a few things the user disliked about the website as well. When you are ordering, the website does not always say if things are out of stock. Sometimes, you can order an item and Meijer will not notify you the item is out of stock

Table 2. Perspective on the Pros and Cons of Meijer Curbside. Courtesy of Katrina Coleman 2018.

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ENGINEERING up until an hour before or even at pickup time. There is an option to substitute if an item is out of stock, but the user said they never do this due to the fact that the item that is substituted could be nothing like what you originally wanted. The user also stated that many people enjoy going to the grocery and use it as exercise and also to get out of the house, and this user specifically will still go to the grocery store physically when they want to take their time and just browse the aisles. Another downside to using something online is the system could glitch. This has not happened to the user yet, but they said it is a potential cause to not use this shopping option. Lastly, the user also brought up that not every location currently has this option, so mainly in lower-income areas, it is not accessible to everyone [2].

Data Analysis and Discussion The observations made in each area of income were almost parallel to one another. For example, the Kroger in the lower income area had similar results to the other three grocery stores in the lower income area, and the same pattern repeated throughout. Even though the grocery store changed, the advertising techniques and variety and quantity of products remained the same. The only variable that changed anything was the income of the area I was observing. The fact that the produce was lower in quality, quantity and variety at the low-income grocers makes it almost impossible for people to want to purchase the produce. The consumers already do not want to purchase produce because it is less filling and more expensive than other foods, and now the consumers really will not purchase the produce because it is poor quality on top of all of that. Consumers of all incomes should be encouraged to eat healthy and live sustainably. This should not be an idea solely for “the higher class.” As well as having organic and non-dairy sections, there are all kinds of people who have food allergies or people who just choose to eat a no-dairy or no-meat diet, and someone of a low income should not have to travel to a completely different grocery in a different area to purchase foods that meet their wants and needs. After taking into account all research done thus far, I have created my own personal ideas that I believe grocers can implement into their stores that are small ideas but will make a big difference Proceedings 2018

in the long run. First, I believe taking food to food banks, which most grocers already do, is extremely important. The way things currently work with that process in the Dayton area is very disorganized and needs to be redone in a more efficient way. But more than food banks, more fresh food can be taken to many different areas other than food banks. For example, perishable items such as produce can be composted if it is no longer edible, or if it is edible, it can be taken to houses where people living with homelessness stay. Last, I believe shoppers should be educated on sustainability. Shoppers are going to make the majority of the difference in the food retail industry, because the consumers are who keep the grocery stores in business. The grocery stores can only do so much, but once the consumers take the groceries home what they do with them is completely out of the stores’ control. Most people do not even know what is recyclable and what is not or how to compost. If grocery stores even had simple advertisements that said “Go Green!” and then in a smaller font, “Ask us for more information or go to our website,” I believe the shoppers would highly consider going more in depth and learn about living more sustainably.

Future Direction In the future, I have many different directions I could potentially elaborate on with my research. My ultimate goal is to create a method for reducing waste in a more organized and economically sound way. I have not figured out exactly what that method will be, but I have a few ideas on what it will contain. First, I would like to repeat my procedure at more that just one location. I spoke with a manager at one Kroger and spoke with one user of online grocery shopping at Meijer. I would like to discuss similar topics with multiple different online grocery shopping users from multiple different grocery chains and speak to managers from different grocery chains, as well as from the same chain but in different locations. After speaking with the manager at Kroger, I learned that the method that is already set in place to deliver food to local food banks is not at its full potential and could be reorganized. I would like to research further into what that distribution looks like and how it could be altered 7

ENGINEERING to be more organized and be more beneficial to Kroger, along with other grocery store chains, as well as the food banks. Furthermore, I would like to look deeper into online grocery shopping and its future. I believe online shopping in general is increasing daily and online grocery shopping is going to take off within the next five years. I would like to incorporate online grocery stores in my overall method of sustainability, but I have to make sure the method is proven to be more sustainable than physically going to the grocery store. Another potential area of study could be looking further into online grocery shopping at chains that do not have a physical store, specifically more along the catalog route to places such as Schwan’s. I am interested to see if their methods of delivery and transportation are more sustainable than what physical stores are already doing and if we could implement their methods into my sustainability method. While grocery stores are the main focus of my study, I think it would be interesting to look at the shoppers and their contribution to waste and sustainability revolving around grocery stores. While I cannot change people, I can encourage grocers to educate their shoppers and create more of an awareness of sustainability. More than just looking at food waste, I would like to look at other areas of waste that are also involved with grocery stores as a whole. Two factors that I specifically am interested in are the packaging used, as well as the transportation involved, whether that is delivering groceries to buyers’ homes or transporting to each store. Most packaging used at large chain grocers are still not the most sustainable method of use. However, the packaging used is the cheapest route, which is why they are continuing to use it because companies receive the most profit that way.

issue in the Dayton area by providing those of lower incomes with fresh produce at a more affordable price while also being sustainable [5]. Another organization which grocery stores can partner with is Homefull, which is similar to MOMF, but Homefull also has other services such as housing those living in homelessness [1]. I believe that if grocery stores partner with organizations such as these, it will help resolve the food desert issue, as well as increase profit because those with lower incomes who do not normally purchase produce would start to. It would also be beneficial to the nonprofits because the larger chain grocery stores could donate their compostable items to these organizations, which would be more sustainable as well as give groups like MOMF and Homefull compost to use.

Acknowledgements I would like to thank my mentor and professor at the University of Dayton, Sean Falkowski, for leading and guiding me through my research. I would also like to thank the Department of Engineering Management, Systems and Technology for their guidance, encouragement and support. Thank you also to the management at Kroger for allowing me to include them in my research. I also would like to express appreciation to the Berry Family Foundation, as well as the Berry Summer Thesis Research Institute and the University Honors Program. None of this would have been possible without all of their knowledge, kindness, support, scholarship, time and effort. Also, I want to thank Mission of Mary Farm for creating such a welcoming environment for me to be a part of. I would like to also show gratitude towards my fellow Berry Summer Thesis researchers for building a great community within our cohort this summer. Lastly, I would like to thank all of my family and friends for their support, mainly: John, Alana, Tori, Jonathan, Preston, Malena and Daniel.

Last, I want a portion of my sustainability method to include helping to feed those less fortunate. One way that could be done is by grocery store chains partnering with Community Supported Agriculture (CSA) groups. CSA “allows city residents to have direct access to high quality, fresh produce grown locally by regional farmers” [9]. One farm which participates in CSA is Mission of Mary Farm (MOMF) which was founded by lay Marianists from the Dayton area [5]. MOMF is a nonprofit and strives to help the food desert 8

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ENGINEERING Works Cited 1.

Community Supported Agriculture Program (2018). Homefull. www.homefull.org/homefull-solutions/urbanagriculture-2/csa/.

Curbside Pickup, Meijer.com (2018). Deli Meijer.com, www.meijer.com/content/content.jsp?pageName= curbside.

Krogerâ&#x20AC;&#x201D;2018 Corporate Sustainability Report. Kroger 2016 CSR, sustainability.kroger.com/planet-zero-waste. html.

Kroger, www.kroger.com/topic/clicklist-select-your-store.

Our Programs. Mission of Mary Cooperative, www. missionofmary.org/our-programs/.

Schmaus, Benedikt, et al. Industry 4.0: Opportunities and Challenges for Consumer Product and Retail Companies. Strategy&â&#x20AC;&#x201D;the Global Strategy Consulting Team at PwC, 24 Oct. 2016, www.strategyand.pwc.com/ reports/retail-industry4.0.

Sniderman, Brenna, et al. Industry 4.0 and Manufacturing Ecosystems. Deloitte United States, 22 Feb. 2016, www2.deloitte.com/insights/us/en/focus/industry-4-0/ manufacturing-ecosystems-exploring-world-connectedenterprises.html.

Taliaferro, Alan, et al. Industry 4.0 and Distribution Centers. Deloitte United States, 12 Sept. 2016, www2. deloitte.com/insights/us/en/focus/industry-4-0/ warehousing-distributed-center-operations.html.

What Is CSA? (2018). Just Food, www.justfood.org.

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TEACHER EDUCATION

Capes and Catechesis: Using Comic Books to Catechize Catholic Youths Lindsey M. Bronder1,2,3,4 University of Dayton, 300 College Park, Dayton, OH, 45469 1. Department of History 2. Department of Teacher Education 3. Berry Summer Thesis Institute 4. University Honors Program

Mentors: Caroline Merithew, Ph.D., Department of History Susan Ferguson, M.S., Department of Teacher Education Corresponding Author: Caroline Merithew, Ph.D. Email: cmerithew1@udayton.edu

Abstract Since the 1930s, comic books starring superheroes have been popular among youth in America. Recently, comic books and graphic novels have gained popularity and greater recognition as a valid literary form both in American culture as well as the classroom. This original archival research analyzes the extent to which comic books can serve as an effective means to catechize American Catholic youth. The scope of this investigation includes research into the origins and history of comic books, especially in the United States, to analyze how they have influenced American culture and American youth. Using George Pflaumâ&#x20AC;&#x2122;s comic series, Treasure Chest of Fun and Fact (19461972), this study then examines and analyzes the images, texts and stories of Catholic comic books in order to create a more modern example of a Catholic comic strip. For my research, I also use many secondary sources as well as official Roman Catholic documents and teachings to inform the creation of my Catholic comic strip so that it may serve as a tool for catechesis.

Introduction Since the late 1930s, comic books starring superheroes have been popular among youth in America. With popular comic series like Batman1 and Superman2, many American youth looked up to these superheroes as examples of justice and American patriotism. As described in Jill Leporeâ&#x20AC;&#x2122;s The Secret History of Wonder Woman (2014), William Moulton Marston originally created Wonder Woman3 as feminist propaganda so that girls had a female superhero to look up to amongst all the

Batman first appeared in Detective Comics #27 in 1939 and continues to appear in DC comics and movies today. Batman is the alias of Bruce Wayne, a millionaire who uses his wealth to fight crime with advanced technology. (Batman, n.d.; Lepore, 2014, p. xi)

Superman first appeared in Action Comics #1 in 1938 and continues to appear in DC comics and movies today. Known also as Clark Kent, Superman is originally from the planet Krypton and on Earth has super capabilities including speed, strength, X-ray vision and the ability to fly. He uses these super abilities to fight for truth and justice. (Superman, n.d.; Lepore, 2014, p. xi)

Wonder Woman first appeared in All-Star Comics #8 in 1941 and continues to appear in DC comics and movies today. Wonder Woman is Princess Diana of the Amazons, mythical Greek women-warriors who lived in isolation from the rest of human society. After entering modern human society, Diana must adapt to American life while fighting evil with her super strength and Lasso of Truth. (Wonder Woman, n.d.; Lepore, 2014, p. xi)

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TEACHER EDUCATION male superheroes. These figures like Superman and Wonder Woman were considered so influential on American youth that in the 1940s and 1950s efforts were made to censor such comics, such as the National Organization for Decent Literature (NODL) by American Catholic bishops (Cadegan, 2001, p. 258). In response, Pflaum of Dayton, Ohio, created the Treasure Chest of Fun and Fact (1946-1973) comic series to promote justice, American patriotism and Roman Catholic teachings in a way in keeping with NODL. Primarily sold and distributed through Catholic grade schools, Treasure Chest was clearly intended as a teaching tool, even including a teacher’s edition with lesson plans from 1959-1965 (The Catholic University of America, n.d.). So the purpose of this study is to evaluate the extent to which comic books, like Treasure Chest, can serve as an effective means to catechize, or teach the Catholic faith to American Catholic youth today.

Research Questions This paper will seek to answer the research questions below.

Figure 1. Research Questions. Courtesy of Lindsey Bronder 2018.

These questions are relevant due to the recent rise in popularity of comic books and graphic novels with the release of movies and TV series based on superheroes, whose stories were originally told through comics.4 Roman Catholicism was chosen as the religion of focus since it is interesting to examine how the catechesis employed has evolved from generation to generation. In order to discuss and answer these questions, I will define “youth” for the purpose of this study as any individual between the ages of 13 and 18. This age range was chosen because teenagers are often the target audience of superhero comic books and movies which are typically rated PG-13.5 As a future high school and junior high teacher, I am also interested in this age range because most junior-high and high-school students are teenagers. The first question seeks to understand the use of comic books and graphic novels, or book-length stories in a comic book style, in classrooms as a teaching tool. Since there is a significant amount 4.

For example, the movie Wonder Woman (2017) tells the origin story of Wonder Woman and who Princess Diana is as previously seen in the comics (Wonder Woman biography, n.d.).

For example, the movie Wonder Woman was rated PG-13 “for sequences of violence and action, and some suggestive content” (Wonder Woman biography, n.d.).

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TEACHER EDUCATION of literature related to comic books as a teaching tool, I plan to answer this question in a synthesis of literature. While there has been research into comic books and graphic novels as teaching tools, there is limited research on its use in the present and past as a tool for catechesis. The second question seeks to understand how comic books have been used in the past as a catechetical tool and could serve as examples for me to then create my own more modern example. Thus, my study will primarily involve original historical research into the Treasure Chest comic series as an example of how comic books have been used in the past to catechize American Catholic youth. This essay will seek to answer questions 1 and 2. The answers to questions 1 and 2 will provide crucial background information necessary to begin evaluating the effectiveness of comic books in catechizing American Catholic youth today as part of question 3. To answer the third question, I plan to develop at a later date my own more modern example of a Catholic comic informed by my analysis of Treasure Chest and official Roman Catholic documents and teachings. In order to determine its effectiveness as a catechetical tool, I will also develop my own lesson plan where my comic can be used as a teaching aid that I can teach as part of my Religion Methods Lab (EDT 331L).

Synthesis: Pictures Are Worth A Thousand Words The idiom “a picture is worth a thousand words” conveys the potency of a visual depiction in communicating ideas, especially since the human brain processes images faster than words and images are more salient in memory (Baker, 2015, p. 2). Recently, comic books and graphic novels have also gained greater recognition as a valid literary form both in American culture as well as the classroom. While initially viewed as inferior and childish, graphic novels or book-length stories in a comic book style, have become increasingly more recognized as having the same content level of books with the added benefit of a visual component (Brozo, Meyer, & Moorman, 2014, p. 5). Comic books and graphic novels can be a useful tool for visual learners and struggling readers such as those with learning disabilities like dyslexia or English Language Learners (ELLs). They can aid students in their reading by presenting the content in context with the use of visual aids alongside the printed text. Thus, graphic novels could be used as a presentational accommodation for students because it allows students to gain access to instructional materials in ways that do not require them to read standard print presented in a standard visual format. So graphic novels should not be seen as a modification because the learning demands are still the same with the same content level being presented through multiple modes. Comic books and graphic novels can also be useful as a tool for developing students’ literacies, including multimodal literacy, visual literacy, critical literacy and disciplinary literacy.

Multimodal Literacy Multimodal literacy refers to how language can be presented in a way that combines two or more modes or means of meaning making. According to Brozo, Meyer and Moorman (2014), graphic novels can be used to address the Standard 7 of the Common Core State Standards (CCSS) Anchor Standards for reading, which states: “Integrate and evaluate content presented in diverse formats and media, including visually and quantitatively, as well as words” (p. 10). In this way, multimodal literacy challenges students to go beyond word-based literacy to see how the different modes are interrelated thereby allowing students to see how “written-language modes of meaning are part and parcel of visual, audio, and spatial patterns of meaning” (Cope & Kalantzis as cited in Jacobs, 2007, p. 21). As outlined by Darby Orcutt (2010), “religious experience is similarly multimodal” with “religious traditions include[ing] beliefs, rituals, texts, morals, and more, expressed by means of speaking, writing, performance, prayer, meditation, music, art, and so on” (p. 94). In a religion classroom, the development of multimodal literacy with comic books is a great way to illustrate the complex relationship between the means of religious expression.

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Figure 2. Literacy Types. Courtesy of Lindsey Bronder 2018.

Visual Literacy This also suggests the importance of developing multiple literacies in addition to the traditional book and print literacy such as visual literacy, which involves the ability to read or understand information presented through pictures and graphic images. In terms of comic books and graphic novels, students can engage in multimodal literacy by seeing the relationship between the words and images that illustrate the text also called the visual text. According to Michael Cromer and Penney Clark (2007), this is seen in how the words and images are co-dependent in that “the words are interpreted through the images and the images through the words” (p. 579). In comics, visual literacy includes the use of icons, colors, perspective and the cartoon vocabulary (e.g. dashes to indicate rain and a cloud with dots from a head to indicate that character’s thoughts). Catholic tradition is also rich with the use of icons, or visual symbols, such as the cross and the ichthys6 or fish. Thus, Orcutt (2010) claims that the development of visual literacy with comic books can help promote student’s ability to think symbolically and make connections in religion.

Critical Literacy As outlined by Dale Jacobs (2007) as well as Cromer and Clark (2007), multimodal literacy is needed because an understanding gained from either the print or visual text is incomplete. For Jacobs (2007), this promotes students being “active creators, rather than passive consumers, of meaning” because they have to actively produce meaning based on all of the different modes available (p. 24). In reading graphic novels, the students have to connect the print text with the visual text as well as fill in the blanks and make connections between panels. Although the students will be able to understand the gist of the story from just the illustrations, Jacobs (2007) argues that graphic novels should not be seen as a way for students to avoid reading text. In this way, multimodal literacy can also promote critical literacy, or the ability to analyze texts, because they must analyze and make connections between the different modes.

Icthys is the Greek word for fish and forms an acrostic with the first letters from the phrase “Jesus Christ, Son of God, Savior” (Hassett, 1909). In this way, the fish has come to symbolize Jesus for Catholics.

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TEACHER EDUCATION Disciplinary Literacy In addition to critical literacy, graphic novels and comic books can promote disciplinary literacy, or literary practices pertinent to the academic discipline. The use of art and visuals to teach the Catholic faith is deeply rooted in Catholic tradition and history. During the Medieval Ages, many European Catholics were illiterate and so religious art was used as a visual aid to help catechize them. For example, stained glass windows were used by priests to illustrate and reflect on the faith topics covered in the day’s mass readings and homilies (Jones, 2008, p. 19; Orcutt, 2010, p. 96). The Church still uses visuals like comics and coloring books to teach children today (Blankenship, 2010, p. 64). This tradition and history seems to validate the use of comic books and graphic novels as a catechetical tool and as promoting disciplinary literacy in a religion classroom.

Comic books and graphic novels also have the potential to develop disciplinary literacy by promoting skills pertinent to reading religious texts, including the Bible. This is similar to how in a history classroom comic books and graphic novels can promote the methodology and analytical framework historians use in studying historical documents. For example, this can include analysis of political cartoons for both meaning behind the image and text as well as how the image and text can combine to enhance or create new meaning. This connects to the kind of multimodal, visual and critical literacy skills necessary for reading comic books and graphic novels. Comic books and graphic novels can also support disciplinary literacy by being read as primary or secondary documents to be analyzed in terms of source or authorship and the context (e.g. audience and purpose) they were written in (Matthews, 2011, p. 418). Considering graphic novels like Art Spiegelman’s Maus7 and Marjane Satrapi’s Persepolis8, students can get an insight into Spiegelman’s and Satrapi’s experiences and interpretations of events similar to a kind of visual journalism (Cromer & Clark, 2007). According to Jeffrey Kunkel, Spiegelman’s In the Shadow of No Towers describing his 9/11 experience can: “...provide high school students the opportunity to delve into the different ways in which events are recorded and interpreted....It is a biased work, without question but one of Spiegelman’s points seems to be that any attempt to record history is inherently subjective and driven by ideological intent.” (cited in Cromer and Clark, 2007, p. 584)

So graphic novels and comic books can support students’ disciplinary literacy by giving them insight into how history is written and the methodology used to analyze historical documents. Similarly, graphic novels and comic books could promote disciplinary literacy in religion such as biblical exegesis, which involves the study or examination of biblical texts to discover its meaning and derive an interpretation (Maas, 1909). The Bible is unique because the Catholic Church teaches that the Bible is true as God’s Word divinely revealed and written by humans inspired by the Holy Spirit (Catholic Church, 2012, para. 105-106). In this way, the Bible is understood by Catholics to have both a literal sense, or the verbal meaning or that intended by the human author, as well as a typical (also called spiritual) sense, which is based on the literal sense and is the truth God intended to convey through the inspired human authors (Prior, 2001, p. 53-5). In “Dei Verbum (On Divine Revelation)”, Pope Paul VI (1965) claims that: “…since God speaks in Sacred Scripture through men in human fashion, the interpreter of Sacred Scripture, in order to see clearly what God wanted to communicate to us, should carefully investigate what meaning the sacred writers really intended, and what God wanted to manifest by means of their words.” (Article 12)

Spiegelman’s Maus: A Survivor’s Tale chronicles the Spiegelman’s father’s tale of surviving the Holocaust using powerful imagery of a cat-and-mouse game in depicting Nazis as cats and Jews as mice (Spiegelman, 2011).

Satrapi’s Persepolis (2004) is a graphic autobiography of Satrapi’s childhood in Iran during the Iranian Revolution as the Islamic Republic came to power and led to a more conservative and oppressing Iranian culture.

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TEACHER EDUCATION In order to discern these intentions, Pope Paul VI (1965) emphasizes the importance of understanding and analyzing the author’s use of contemporary literary forms, as well as: “...the customary and characteristic styles of feeling speaking and narrating which prevailed at the time of the sacred writer, and to the patterns men normally employed at that period in their everyday dealing with one another.” (Article 12)

Exegesis in biblical studies can involve the use of the historical critical method (HCM) for interpreting the Bible in order to determine the literal sense of a biblical text. According to Joseph Prior (2001), “The literal sense roots the text in its original historical context” (p. 85). So HCM seeks to understand the historical context by considering historically what was going on when the text was composed and so what the text meant in and for the ancient communities that produced it. Just like reading historical documents, HCM would include considering who the author and intended audience was, the literary styles and devices used, the genre or form, as well as any historical facts or context found in the text. In this way, the use of comic books and graphic novels as a tool for catechesis connects to the Catholic Church’s tradition of using visual aids and promotes disciplinary literacy in terms of going beyond the surface meaning of the text to a deeper meaning, such as the literal sense. According to Charles Carraher (1975), comic books can also serve as a useful teaching tool because the interest in the genre can make them motivating and help connect the material with the students (p. 30). Rae Hancock (2015) agrees with Jacobs that reading graphic novels and comic books is a personal experience requiring students to be ‘active creators’. By showing the characters’ inner thoughts and outward actions, Hancock (2015) claims that comic books are important in religious education because they can help show the relationship between belief and practice (p. 31). For Rachel Marie Crane Williams (2008), comic books and graphic novels then have the potential to generate a sense of empathy and human connectedness among students” by helping them to visualize the characters and conflicts they undergo (p. 15). In this way, comic books and graphic novels have been increasingly used to discuss difficult and complex topics such as war, peace and human rights as seen in Persepolis and Maus.

Comics, Americanization and Anti-Catholicism As comic books starring superheroes first gained popularity during the late 1930s, World War II (WWII) was being waged from 1939-1945. During this time, there was a push for a national unity among American citizens in the face of war. In 1941, Wonder Woman debuted just months before the United States (U.S.) officially declared war on Germany and entered WWII. As described in Lepore’s The Secret History of Wonder Woman (2014), Marston originally created Wonder Woman with the intent “to fight fascism with feminism” (Lepore, 2014, p. 200). For example, Wonder Woman fought her nemesis Dr. Psycho, who was a fascist depicted as a mutant with the heads of Hitler, Mussolini, and Hirohito (Lepore, 2014). In addition to Wonder Woman, superheroes like Hawkman and all the other members of the Justice Society joined the armed services to fight in WWII (Lepore, 2014, p. 204). In this way, comic book superheroes and their adventures focused on promoting American patriotism and fighting fascist ideals during this time period. Although intended to promote American ideals, superheroes were accused of being fascist. A 1945 Time magazine article asked “Are Comics Fascist?” and featured critiques from Walter J. Ong, a Jesuit priest and professor of English at Regis College. Ong compares Superman to the ubermensch (overman), a superior human proposed by German philosopher Friedrich Nietzsche and later appropriated by the Nazi Party in their pursuit of the Aryan race (Lepore, 2014, p. 255). According to Ong, “Everything is centered on one man—the leader, the hero, the duce, the Fuhrer” (Time, 1945, p. 67). Similarly, psychiatrist Frederic Wertham also condemned the Batman-Superman-Wonder Woman comics for racism. Wertham called attention to how the comic book heroes are typically “an athletic, pure American white man” and “the villains on the other hand, are foreign-born, Jews, Orientals, Slavs, Italians, and dark-skinned races” (as cited in Lepore, 2014, p. 267). Marston countered these arguments and defended his Wonder Woman claiming that: Proceedings 2018

TEACHER EDUCATION “And the two wishes behind Superman are certainly the soundest of all; they are, in fact, our national aspirations of the moment-to develop unbeatable national might, and to use this great power, when we get it, to protect, innocent, peace-loving people from destructive ruthless evil.” (Cited in Lepore, 2014, p. 185)

In other words, Marston argues that superheroes like his Wonder Woman are not fascist because with their power comes the responsibility to protect and care for those who may be weaker and innocent. This calls to mind the words “With great power comes great responsibility” that would later come to be associated with superheroes but was not yet popular (Spider-Man, 2002). In addition to concerns about superheroes being fascist, there were increasing concerns regarding the morals, especially violence, presented in comic books and the potentially negative impact on young readers. Ong also disapproved of Wonder Woman for her paganism as seen in her prayers to Aphrodite and use of swears like “By Zeus” (Time, 1945, p. 68). Both Wertham and Josette Frank9 condemned Wonder Woman for being scantily clad and repeatedly being chained or bound in some way as sexually exploiting women plus promoting bondage and sadism (Lepore, 2014, p. 236-237). Furthermore, Wertham argued that the violence and crime depicted in comics had a negative effect on the psychology of American youth and could lead to juvenile delinquency (Lepore, 2014, p. 264-265). While there were many defenders of comic books, Ong’s and Wertham’s critiques show that comic books were controversial during this time period. The concern regarding fascism and communism was considered so pressing that American Catholic bishops highlighted this in their 1941 pastoral letter called the “Crisis of Christianity.” In addition to emphasizing the threats posed by Nazism and communism, the bishops also outlined their concern “about the future of supernatural religion in our country” threatened by the growing “evils of ‘false doctrine, immorality, disbelief and reborn paganism’” (Mooney, et al., 1941, p. 376). Although they condemned communism, they addressed the rising consumer culture and “condemned the evils of unrestrained capitalism” (Mooney, et al., 1941, p. 376). According to the bishops, these evils threatened youth the most and there is a need to “encourage youth to realize the constructive need of Christian doctrine and Christian discipline” (Mooney, et al., 1941, p. 376). In their 1947 pastoral letter “Secularism,” the bishops outlined how the increasing secularization creates a moral decay with the denial of God and the Church’s decreasing influence on the moral formation of youth (Dougherty, et al., 1947, p. 404). The follow-up pastoral letter “The Christian in Action” outlines constructive efforts that Catholics could undertake to combat secularization and emphasizes the relationship between religion and American citizenship in promoting national morality (Dougherty, et al., 1948, p. 411-12). In their 1961 pastoral letter “Unchanging Duty in a Changing World”, the bishops claim that: “[t]he evidences of our moral decline are everywhere to be seen: in the alarming increase in crime, particularly among the young; in the sensational treatment of violence and sexuality in literature, on the stage, screen and television…” (Spellman, et al., 1961, p. 536).

These statements seemed to show how there was a growing fear of the American Catholic youth falling away or losing their faith and morals as they became increasingly more part of and surrounded by secular society. When confronted with overwhelming culture, they were concerned over the seeming little influence they had in shaping the minds of their children and blamed modern media for contributing to the apparent moral decline in the U.S. Movies and comic books were considered to be so influential on American youth that in the 1940s and 1950s efforts were made to censor them by the Legion of Decency and National Organization for Decent Literature (NODL), which was founded by American Catholic bishops (Cadegan, 2001, p. 258). While the Legion of Decency was established in 1934 to censor movies, NODL was established in 1938 to censor paperback literature, especially comic books (Cadegan, 2001, p. 252, 257). NODL sought to promote good morals and steer readers away from reading material that was considered morally objectionable or indecent. NODL created a code that could be used to evaluate whether a

Frank was the former director of and advisor on the Children’s Book Committee of the Child Study Association of America. (Lepore, 2014, p. 236)

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TEACHER EDUCATION publication was indecent and, therefore, place it on their list of publications that were disapproved. For example, NODL put Sensation Comics featuring the adventures of Wonder Woman on its list of “Publications Disapproved for Youth” because it violated Point 4 with Wonder Woman “not sufficiently dressed” (Lepore, 2014, p. 203).

Figure 3. NODL Code. Cadegan, 2001, p. 258-259. Courtesy of Lindsey Bronder.

NODL’s mission reflected the fears of youth falling away from the faith and of secularization leading to moral decay and totalitarianism as outlined in the pastoral letters. NODL was even founded and headed by John F. Noll, who was Bishop of Fort Wayne, Indiana, and listed as an author of many of the pastoral letters after World War II (Cadegan, 2001, p. 256)10. In the 1957 pastoral letter “Censorship,” the bishops praise NODL and explained that the need for censorship is seen in the moral decay of the U.S. (Mooney, et al., 1957, p. 501). According to Catholic historian Una Cadegan (2001): “While left-wing defenders of American democracy sought to maximize American freedom as a safeguard against the growing threat of totalitarianism, conservative defenders of the same system hoped to temper freedom—or ‘license,’ as they would have said—with moral safeguards against the relativism that they believed permitted totalitarianism to take root.” (p. 254)

In other words, the goal for many conservatives, often including Catholics, was to combat fascism and communism by preventing moral relativism. Although NODL intended to combat fascism and communism, many criticized their efforts as fascist and communist. In 1956, John Fischer wrote an editorial that claimed NODL was “conducting a shocking attack on the rights of their fellow citizens…an un-American activity which is as flagrant as anything the Communist party ever attempted” (as cited in Cadegan, 2001, p. 267). In their 1957 “Statement on Censorship by Private Organizations and the National Organization for Decent Literature,” the American Civil Liberties Union (ACLU) accepted NODL’s goal of informing Catholics but objected to the threat of boycotts by NODL members as well as the use of NODL lists by civil authorities (Cadegan, 2001, p. 265). In response to Fischer, John Courtney Murray wrote that he agreed that NODL’s lists were misused by civil authorities but affirmed the validity of their 10. Noll was listed as an author on the pastoral letters “The Crisis of Christianity” (1941), “Secularism” (1947), “The Christian in Action” (1948), “The Child Citizen of Two Worlds” (1950), etc. Noll, however, was not listed as an author of “Censorship” (1957).

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TEACHER EDUCATION mission. According to Murray, NODL was working towards the Catholic duty “to inject the Catholic tradition of rationality into a mass democracy that is rapidly slipping its moorings in reason” (as cited in Cadegan, 2001, p. 273). Although called a censor, America editors emphasized that NODL was not: “...a legislative body; it can lay down no laws. The purpose of its ratings is to remind Catholics of obligations they already have by virtue of the natural moral law.” (cited in Cadegan, 2001, p. 272)

In order to distance themselves from the negative connotation of censorship, NODL emphasized how the obligations to read ‘decent’ materials in keeping with the code was “freely chosen, not imposed” because NODL members voluntarily joined (Cadegan, 2001, p. 274). Although they exerted a significant influence during the 1940s and 1950s, NODL’s influence by the early 1960s, as the Second Vatican Council occurred, decreased and Catholic censorship was no longer perceived as a significant threat. The pushback to NODL’s efforts also reflects the anti-Catholic sentiments prevalent in the United States. Since the founding of the United States, Catholics were a minority with Protestants as the dominant religion in the U.S. As a minority, Catholics faced discrimination as an un-American group because the Catholic Church was seen as being authoritarian and having monarchical ties in Europe (PBS, 2010). As the Catholic population grew in the 1840s and 1850s, Protestants feared Catholics taking over American institutions and anti-Catholic sentiments grew. According to historian John McGreevy, this led to “a big anti-Catholic political party, lots of discussion about what should the role of Catholics in politics and Catholics in society be, whether or not Catholics could make good citizens” (PBS, 2010). McGreevy describes how Protestants saw Catholics as “more or less like sheep; they’re going to do exactly what they’re told” (PBS, 2010). After World War I and World War II, Americans were especially against dictators and mindlessness on the part of citizens. In addition to the comparison between Catholic and communist censorship, comparisons were drawn between the pope as head of the Catholic Church and fascist dictators. As outlined by Michael McConnell (2011), Protestants viewed the Pope as having been too sympathetic towards Mussolini and not respecting the separation of church and state (p. 1641). Paul Blanshard, arguably the leader of the anti-Catholic movement post-World War II, opposed NODL’s censorship and the efforts of the Catholic bishops in politics claiming it went against the separation of church and state that is crucial to democracy and thereby made Catholicism un-American (Cadegan, 2001, p. 269). In this way, Catholics not only faced discrimination but also had to reconcile their Catholic and American identity to show you could be a Catholic American citizen.

Searching for Treasure: Why Treasure Chest Is a Catholic Treasure In response, Pflaum of Pflaum Inc. in Dayton, Ohio, created the Treasure Chest of Fun and Fact (1946-1973) comic series to promote justice, American patriotism and Roman Catholic teachings in a way in keeping with NODL. Primarily sold and distributed through Catholic grade schools, Treasure Chest (TC) was clearly intended as a teaching tool, even including a teacher’s edition with lesson plans from 1959-1965 (The Catholic University of America, n.d.). For the majority of the time they ran, TC was published every two weeks from September through May in an effort to parallel the traditional school year. Although Catholic school enrollment peaked in the early 1960s, the decline of Catholic school enrollment in the 1970s led also to the decline of TC, whose readership depended on Catholic schools (NCEA, 2018; Blankenship, 2010, p. 65). Despite not being the first of its kind, TC is considered to have been the most successful Catholic comic series because it ran longer and produced more issues over its 27 volumes than other comics (Blankenship, 2010, p. 65). In this way, TC seems to be the leading example of how comic books have been used in the past to catechize American Catholic youth. Pflaum (1946a) created and marketed TC as “the new, approved-type of comic magazine” (p. 32). TC was to be “a worthwhile comic magazine as a counteractive to the objectionable type” (Pflaum, 1946a, p. 32). This language reflects the language used by NODL, who used terms like ‘indecent’ and ‘objectionable’ rather than ‘immoral’ or ‘obscene’ that were legal terms and could lead to 18

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TEACHER EDUCATION prosecution for violating First Amendment rights (Cadegan, 2001, p. 260-261). In the story series “The Cost of a Lie”, a child named Arthur goes to see a movie despite his mother telling him he could not because it was rated ‘B’, or objectionable in part, by the Legion of Decency (Pflaum, 1946c-d). After choosing to see the movie and lie about it, Arthur must deal with the guilt and negative consequences leading him to conclude that “a lie is just like a snowball” that continues to grow and cost him even more (Pflaum, 1946d, p. 19). While these ties are not explicit, the Figure 4. Image from back cover of Treasure Chest of Fun and Fact, Volume 1, Issue No.1 (Pflaum, language used and references 1946a). Courtesy of The Catholic University of America Digital Collections. to censorship efforts suggest that Pflaum Inc. was aware of and working to create TC so that it was in keeping and promoted the mission of the major Catholic censors like the Legion of Decency and NODL. As NODL’s influence declined, comic book publishers formed the Comics Magazine Association of America (CMAA) and created a regulatory code that was used to grant a seal of approval in 1954 (Nyberg, n.d.). This seal of approval was even sought by comic giants Marvel and DC, as well

Figure 5. The first TC issue to bear the CMAA seal of approval in the top right-hand corner on Volume 10, Issue No. 20 (Schaller, 1955). Courtesy of The Catholic University of America Digital Collections.

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Figure 6. The last TC issue to bear the CMAA seal of approval in the top right-hand corner on Volume 22, Issue No. 20 (Langdon, 1967b). Courtesy of The Catholic University of America Digital Collections.

TEACHER EDUCATION as Pflaum, Inc., with TC bearing the seal of approval starting with their Volume 10 Issue No. 20 (Schaller, 1955). TC issues would bear this seal of approval until 1967, with the last issue being the Volume 22 Issue No. 20 (Langdon, 1967b). Although it is not clear why TC issues no longer bore this seal, it is clear that Pflaum Inc. was concerned with ensuring his comics truly were an “approvedtype of comic magazine” since the beginning of TC. In addition to censorship, the creation of TC was also informed by the fear of youth falling away from the faith and predominant anti-Catholic sentiments, such as Catholicism being called un-American. As outlined by historian Anne Blankenship (2010), TC’s principal theme was American citizenship and emphasized that children could be good Catholic American citizens. She claims that TC stories modeled behaviors and gave explicit ‘prescriptions’ or instructions on both civil and religious duties in an effort to show how they can overlap and connect. Through my archival research, I have identified key prescriptions or characteristics of a good American Catholic citizen like being anticommunist, being patriotic and defenders of democracy, having and sticking to your morals, as well as being part of a "melting pot."

Figure 7. Part of a Melting Pot. Courtesy of Lindsey Bronder 2018.

Part of a Melting Pot In order to determine what makes a good Catholic American citizen, Blankenship (2010) claims that TC had to first address the question “What is America?” (p. 65). According to Blankenship (2010), TC “regularly defined America by freedom, democracy, and multiculturalism” (p. 64). She offers as an example the Volume 5 series, “It Happens Here: A Story of Young Citizens,” which shows Catholic immigrants as they come to the U.S. and learn more about being American citizens (Blankenship, 2010, p. 66). Blankenship (2010) also offers as an example the Volume 10 series, “We Built America,” that was sponsored by the Catholic University of America’s Commission for American Citizenship (p. 67). The series follows a group of students as they befriend two new students whose national origins are a mystery. After a girl remarks that “it may take them a little time to get used to the American way of life,” a boy reminds her that it will not take them long because “remember it was people who came here from foreign countries who made the American way of life” (Bren, 1954, p. 20). In an effort to welcome the new students, the civic club plans to introduce them to the ‘American way 20

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TEACHER EDUCATION of life’ or American culture and get to know them better by inviting them to have dinner with their families to show them the kinds of people who built America, since many of their older family members were immigrants or children of immigrants. At the dinners, the families explain “the fact that true Americanism is a matter of spirit, not of birth” and tell stories of the contributions to American culture by their ethnicity, including Spanish, Italian, German, British, Chinese, Irish, Polish and Norwegian Americans (Eaglin, 1954, p. 16). The new students remark that “understanding different people is just a matter of getting to know them” and eventually bring home the civic club members for dinner to reveal that they are Native American (Eaglin, 1955b, p. 31). The series emphasized how Native Americans or “those who were already here have also helped to weave the American pattern” and even showed newly arrived immigrants to “point out that the pattern is not finished. There are people coming even in our times who will add new designs to the pattern” (Eaglin, 1955d, p. 28; Eaglin, 1955c, p. 13). The emphasis on these contributions seems to be to show readers that American culture is very complex and dynamic. Similarly, the later Volume 18 series, “America: The Melting Pot,” highlights the contributions of different ethnicities to American culture because “sometimes we forget that our country is a great melting pot of people of many different races, working together

Figure 9. Civics Club Meeting. (Ostendorf, 1950, p. 13.) Courtesy of The Catholic University of America Digital Collections.

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Figure 8. We Built America. The students discuss how none of them are pure English and that American culture is a “mixture” of different ethnic contributions. (Eaglin, 1955a, p. 31.) Courtesy of The Catholic University of America Digital Collections.

and contributing to America’s greatness” (Pehowski, 1962a, p. 18-19). What distinguished “America: The Melting Pot” from “We Built America” is the more direct statements addressing the discrimination and prejudice that ethnicities have faced in America. Considering how the series ran during Civil Rights movement, their statement that “Laws protected [blacks’] rights but couldn’t always stop prejudice” and that “time proved [blacks’] to be good neighbors” was revolutionary (Pehowski, 1962b, 18-19). Similarly, the Volume 6 series, “Where Do We Stand?,” sponsored by the Commission on American Citizenship that highlighted Catholic social teachings included a story on racial injustice and prejudice. Key points were summarized at the end of the story such as in Figure 9 where the Catholic Church condemned racial prejudice because “there is no such thing as ‘race’ as most people understand it” and “there are no such things as races that are born ‘inferior’ or ‘superior’” (Ostendorf, 21

TEACHER EDUCATION 1950, p. 13). Another “America: The Melting Pot” highlighted how Jewish immigrants faced discrimination and prejudice similar to Catholics in America (Ostendorf, 1951, p. 19). As seen in Figure 10, readers were encouraged to interact with and befriend immigrants like Jews in order to help them assimilate into the ‘melting pot’ and because this exposure would also decrease prejudice (Ostendorf, 1951, p. 19). These call to mind the “the fact that true Americanism is a matter of spirit, not of birth” and what unites citizens is their American identity, not their religion or race (Eaglin, 1954, p. 16). In this way, TC worked to promote tolerance, justice and acceptance of diversity. Although TC encouraged assimilation into American culture, they acknowledged the contributions by different ethnicities and encouraged Americans to preserve their cultural traditions. Blankenship (2010) contrasts TC’s version of a melting pot with the story of Wonder Woman, who upon coming to America completely assimilates as seen in how she only speaks English and never refers to her Greek heritage or former country, except to claim America is superior to Amazonia (p. 68). According to Blankenship (2010), TC “modified the usual interpretation of the melting pot metaphor” in their promotion of tolerance and multiculturalism as characteristics of America (p. 68).

Figure 10. America, The Melting Pot. (Ostendorf, 1963, p. 18-19.) Courtesy of The Catholic University of America Digital Collections.

Anti-Communism Although TC shows America as tolerant and accepting of diversity, TC shows no tolerance towards communist ideals and takes a strong anti-communist stance. In addition to promoting multiculturalism, the Volume 5 series, “It Happens Here: A Story of Young Citizens,” in telling the story of displaced persons (DPs) from communist countries also contrasts American democratic ideals with communist ideals. As outlined by Blankenship (2010), the DP Paul’s behavior and mistrust of authority figures reflects the fears of those living in communist countries (p. 66). A key moment in the series is Paul’s realization that unlike in his home country the police are ‘friends’ that need not be feared (Schaller, 1949a, p. 30). Another is his defense of an honest election for the school’s civic club president claiming, “that only the best person for the job should be elected” as seen in Figure 11 (Schaller, 1949b, p. 34). Figure 11 also shows a priest-teacher affirming Paul’s statements, reminding students before voting that “an honest election is an obligation of good citizenship” (Schaller, 1949b, 22

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TEACHER EDUCATION p. 34). Similarly, a “Where Do We Stand?” story (Figure 12) on Catholics as politicians emphasize that, “no one should vote for [a candidate] simply because [they’re] a Catholic,” which was a fear prevalent among non-Catholics of the time (Ostendorf, 1951b, p. 4). Figure 12 and the rest of the story also shows the Catholic, political candidate having to make moral decisions and goes on to emphasize the importance of citizens voting and having a moral conscience to prevent totalitarianism from undermining democracy. In this way, TC seems to address the Church-State question and suggests the need to combat fascism and communism by preventing moral relativism. Another “Where Do We Stand?” story outlined more the dangers of communism and how it is the opposite of American democracy and freedom. The scene opens with a girl being tricked into signing her support for “The Stockholm Peace Proposal,” which her friends recognize as a communist scheme. When confronted by her friends for seeming to support communism, she defends herself claiming that “communism would be all right if it didn’t fight against religion and belief in God” (Ostendorf, 1951a, p. 9). A friend that is a DP then tells of his personal experiences living under a communist regime and his motivations for escaping to America. While he does highlight the lack of religious freedom, his story also focuses on how communism deprives an individual of the Four Freedoms: freedom of speech, freedom of worship, freedom from want and freedom from fear. In 1941, President Franklin D. Roosevelt outlined these freedoms in his Annual Message to Congress, which was to serve as the aims of the U.S. in WWII and later as the basis for the Atlantic Charter and Universal Declaration of Human Rights (FDR Library and Museum, 2018). This story conveys to readers that Catholics should oppose communism not only because it lacks religious freedom but because it limits all other freedoms as well. For example, the DP explains how the Four Freedoms were violated when a farmer was punished with imprisonment and the burning of his farm for speaking up against the communists for not letting him practice his religion and for taking his farm

Figure 11. Only the Best Person Should be Elected. (Schaller, 1949b, p. 34.) Courtesy of The Catholic University of America Digital Collections.

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Figure 12. “Where Do We Stand?” Story. (Ostendorf, 1951b, p. 4.) Courtesy of The Catholic University of America Digital Collections.

TEACHER EDUCATION animals as government property (Ostendorf, 1951a, p.12). The story as seen in Figure 13 prompts the revelation by the girl that “[Communists] destroy everything society is built on-belief in God, the family, rights of the individual and the Four Freedoms. What kind of morals do they have?” (Ostendorf, 1951a, p. 13). The following panels in Figure 13 show acts of subversion believed to be used by communists to infiltrate American society. In light of how the girl was tricked into supporting the communists in the beginning of the story, readers are encouraged to be cautious of simply being followers because Lenin claimed that “it is necessary to use any ruse, cunning, unlawful method, evasion, and concealment of the truth” (as cited in Ostendorf, 1951a, p. 9). In the bottom of the last panel on Figure 13, there is the quote by Marxist Anatoly Lunarcharsky “We must learn to hate, and it is only then that we shall conquer the world” (as cited in Ostendorf, 1951a, p. 13). The inclusion of this quote suggests that communism is spread by hate as well as a lack of care for one’s neighbor. The comic ends in Figure 13 with the girl saying they “must first be strong in [their] own faith” as well as “pray and lead better lives” (Ostendorf, 1951a, p. 13). So readers are charged with the task of protecting their freedoms from communism by strengthening their faith in an effort to find the truth and to prevent hate and moral relativism. Similarly, the Volume 17 series, “This Godless Communism,” explicitly instructs readers about the dangers of communism and how to prevent it. Considering that the Cold War was being waged between the U.S. and the Union of Soviet Socialist Republics (U.S.S.R.), “This Godless Communism” was a dystopian story of if the U.S.S.R. won and the U.S. was taken over and made into a communist country called the Union of Soviet States of America (U.S.S.A.). The series showed the U.S.S.A. as having closed churches and people imprisoned for practicing their religion, secret police, government ownership of all private sectors, families broken up with parents forced to work and censorship with only one official government news source (Crandall, 1961a). The series opens with

Figure 13. Subversion. (Ostendorf, 1951a, p. 13.) Courtesy of The Catholic University of America Digital Collections.

Figure 14. Prayers and the Spreading of Truth. (Crandall, 1961a, p. 8.) Courtesy of The Catholic University of America Digital Collections.

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TEACHER EDUCATION a letter from J. Edgar Hoover, who was the first Director of the Federal Bureau of Investigation (FBI). In the letter, Hoover (1961) claims that “Communism represents the most serious threat facing our way of life” and that the responsibility to protect the freedoms of democracy belongs to all American citizens, including children (p.1). According to Hoover (1961), “The most effective way for you to fight communism is to learn all you can about it” because “it helps us recognize and detect the communists as they attempt to infiltrate the various segments of our society” (p. 1). Hoover is also shown in the last panel of the story as seen in Figure 14 echoing the words of his letter. Figure 14 also shows a man lamenting “If I and my fellow Americans had only realized how horrible communism really is…” (Crandall, 1961a, p. 8). Hope is given, however, with the words that “it is a story that will never happen if we stay alert and guard our freedoms” (Crandall 1961a, p. 8). A priest is shown in Figure 14 telling Figure 15. Marx and Hegel Philosophy. (Crandall, 1961b, p. 31.) Courtesy readers that “our prayers and the spreading of of The Catholic University of America Digital Collections. truth” will end communism and Hoover is also shown, echoing the words of his letter, saying that learning more is the best way to fight communism (Crandall, 1961a, p. 8). This echoes the message in “Where Do We Stand?,” including the end message that communism is spread by hate and lack of care for one’s neighbor so a solution is prayer and the spreading of truth. The series, “This Godless Communism,” also traces the history of communism to the communism of the day, including telling the story of Karl Marx whose writings began communism. A key moment is Marx debating with his friends over their teacher Georg Hegel’s philosophy that there is no freedom and no God in Figure 15. A student explains that, “We Catholics know that no ideas are forced on our minds. God has given us a mind with which we make free choices every day” (Crandall, 1961b, p. 31). This seems to again be an effort to address anti-Catholic sentiments and the fear of Catholics being “more or less like sheep; they’re going to do exactly what they’re told” (PBS, 2010). In this way, TC seems to emphasize that Catholics are just as free as any other American citizen. Overall, TC seems to contrast Catholics and communists in order to emphasize the differences between them in an effort to reduce anti-Catholic sentiments of the time. Furthermore, they seem to take a hard anti-communist stance in order to distance themselves and not seem sympathetic towards fascism and communism. To this extent, TC was largely influenced and informed by the anti-Catholic sentiments and fears of a communist and or Catholic takeover of American institutions that characterized this time period. George Pflaum worked to enable TC readers to defend against anti-Catholic sentiments and to defend democracy from the threat of communism.

Patriotism As part of encouraging readers to defend democracy, Pflaum and TC worked to instill in readers a sense of pride in and a sense of duty to their country. In the teacher issues for Volume 20, teachers were informed that the TC issues for the 1964-65 school year were designed with eight goals in mind, including to “6. create in [students] a sense of history” (Wischmeyer, 1964a, p. 37). To accomplish this goal, TC included on the last page of each issue a “Pages of History” section that told what happened historically 200, 100, 50, 10, 1, etc. years ago in both American and Catholic history (Wischmeyer, 1964a, p. 35). Since the beginning, TC has also included stories about historical events and historical figures. The Volume 1 series, “The Ark and the Dove: Founding of Maryland,” Proceedings 2018

TEACHER EDUCATION tells of how Maryland came to be established as a colony for Catholics (Pflaum, 1946a, p. 13-16). The focus on how Catholics faced discrimination in the colonies and the development of the Toleration Act, which granted freedom to practice all religions in Maryland also reminds readers that people came to America for religious freedom, which would later become one of the Four Freedoms identified by Franklin Delano Roosevelt (Pflaum, 1946a, p. 13-16; Pflaum 1946b, p. 12-15). Blankenship (2010) claimed that TC “regularly defined America by freedom,” and I argue that religious freedom was considered by TC as the most important (p. 64). The story of Maryland and others like it remind Catholics of their place in American history and how key religious freedom was to American democracy. Considering anti-Catholic sentiments of the time, TC suggests that there is not something un-American about Catholicism like Blanshard charged and that there should not be discrimination because America was founded on principles of religious freedom.

Figure 17. Dr. Pat Smith serves and has been commended for her work as a doctor to the people and American soldiers in Vietnam (Grady, 1967, p. 34). Courtesy of The Catholic University of America Digital Collections.

Figure 16. Colonists celebrating governor Dongan’s acts and his creation of a “Charter of Liberties” preventing religious persecution (Klosterman, 1965a, p. 4). Courtesy of The Catholic University of America Digital Collections.

Another goal for the 1964-65 school year was to “3. inspire [students] toward lives of service to their country and their fellow man” (Wischmeyer, 1964a, p. 37). To accomplish this, TC ran a series of stories on the lives of 10 ‘great’ American Catholic lay people: Margaret Haughery, Dr. Thomas Dooley, Thomas Dongan, John F. Kennedy, Margaret Brent, Charles Carroll, Admiral John Barry, Pierre Toussaint, Philip Sheridan and Babe Ruth (Wischmeyer, 1964a, p. 39-40). During the late 1600s, Dongan served as the governor of New York while overcoming antiCatholic sentiments and even drafted a “Charter of Liberties” as seen in Figure 16 that prevented religious persecution, which would later be a basis for the U.S. Constitution (Klosterman, 1965a). Despite Catholics being unallowed to participate in politics, “Colonial Patriot” tells of how Carroll would not be silenced as he wrote in support of the Patriots and later went on to sign the Declaration of Independence (Klosterman, 1965c). The purpose of including these stories and others like it seems to be to remind readers that Catholics have overcome discrimination and shaped American history, especially in promoting religious freedom. Barry’s story tells of his patriotism and service to his country as part of the Navy during the Revolutionary war and commends his efforts to establish a peacetime navy making him the “Father of the American Navy” (Klosterman, 1964b, p. 8). In “‘Little Phil’ Sheridan: Hero of the Civil War,” TC describes how Sheridan bravely led Union troops into battle and Proceedings 2018

TEACHER EDUCATION was later commended by the Cardinal Gibbons as a “‘hero who helped seal the unity of our country’” (Klosterman, 1965b, p. 31). Although their Catholic identity was not stressed, these stories show how Catholic, American citizens have throughout American history served their country and been defenders of democracy and the freedoms of democracy. These stories were key in conveying to readers that one could be both Catholic and patriotic. The inclusion of stories on more modern lay Catholics showed readers how they could serve both their country and their neighbor in their time. In “Tom Dooley, Valiant American Doctor,” TC tells of how Tom Dooley served in the U.S. Navy helping refugees flee from communist North Vietnam and later returned to Laos as a doctor to selflessly serve the sick, despite suffering from cancer (Klosterman, 1964a). Similarly, a Volume 22 story told of how American Dr. Patricia Marie Smith serves the people of Vietnam as seen in Figure 16 (Grady, 1967). A panel of Figure 17 shows an American soldier saying “Dr. Pat and her staff are winning the hearts and minds of the people! That can only help us do our job of protecting freedom!” (Grady, 1967, p. 34). Stories like this convey that there are many ways to combine service to one’s country and neighbor beyond serving in the military or politics. TC emphasized this further in the career advice given by Monsignor James Conroy, editor of the “Our Sunday Visitor”11 youth section (Wischmeyer, 1964a, p. 40). The series was formatted as “Msgr. Conroy Asks - Would You Like to Be a [insert occupation here]?”, with Conroy proceeding to describe how one could serve their country, neighbor and even God through this occupation. For example, “Msgr. Conroy Asks- Would You Like to Be a Doctor?” outlines how doctors continue God’s work in their care for others and reverence for the human body (Conroy, 1964b, p. 28). On lawyers, Conroy (1964a) writes that “A lawyer really does the work of God on earth by helping carry out the laws of his state and country. These are only extensions of God’s laws for us” (p. 16). Conroy (1964c) argues that journalists have a serious duty because “[They] are the inspiration of uncounted multitudes who are looking for the Truth all over the world. You can be Communism’s most formidable opponent” (p. 21). The advice given in this series on various occupations conveys that one’s identity and duties as a Catholic and an American citizen can overlap and connect. In this way, TC emphasized that children could be good Catholic American citizens.

Morality After answering “What is America?,” Blankenship (2010) claims that TC then seeks to answer “How do we keep America this way?” (p. 65, 69). In addition to outlining the need to defend democracy from communism, TC also encouraged readers to be good Catholic, American citizens by developing good morals and serving others in their community. Blankenship (2010) offers as an example an “It Happens Here” story on students in a civic club searching for an answer to the question of “Who is a good citizen?” (Blankenship, 2010, p. 69; Schaller, 1950, p. 25). The DP Paul’s answers reflects his experience under a communist regime in his explanation that good citizenship is “doing whatever the government says to do, without question” (Schaller, 1950, p. 25). In an interview with the mayor, the students are told that “A good citizen is someone who obeys the laws, pays taxes, and votes at all elections” (Schaller, 1950, p. 26). Finding this answer lacking, the students ask the editor of the newspaper, who answers that a good citizen “pays his bills, sweeps the snow from his sidewalk, and joins clubs that help the community” (Schaller, 1950, p. 26). While they found merit in both of these answers, the students believing something to be missing then consult their priest. The priest tells them that the missing link is so simple that they have overlooked it: love, also known as charity (Schaller, 1950, p. 27). Pointing to a painting of the story of the Good Samaritan, the priest goes on to define citizenship in terms of loving one’s neighbor as shown “not by words alone, but by deeds” (Schaller, 1950, p. 27). As seen in Figure 18, he emphasizes that one’s neighbor is all people and so good citizenship involves showing “justice to everyone; charity to all who need it” (Schaller,

11. “Our Sunday Visitor” was started by Bishop Noll for the purpose of publishing a weekly newspaper and has become the “largest English Catholic publisher in the world" (Our Sunday Visitor Inc., 2018).

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TEACHER EDUCATION 1950, p. 27). The imagery of the Good Samaritan reaffirms this message and reminds readers of the importance of showing mercy and kindness to all, which Blankenship (2010) identifies as the essence of all the other actions described as good citizenship (p. 70). Before telling the parable of the Good Samaritan, Jesus also gives what is known as "The Greatest Commandment": “You shall love the Lord, your God with all your heart, with all your being, with all your strength, and with all your mind, and your neighbor as yourself.” (Luke 10:27, New American Bible Revised Edition)

This is reflected in the priest’s message at the bottom of Figure 18 that “first and last, a good citizen obeys the laws of God. He loves God and loves his neighbor” (Schaller, 1950, p. 28). Blankenship (2010) also emphasizes the importance of not only the answers given but also who the students seek as an authority on the subject (p. 70). While the politician and the journalist would seem like authorities on the community and citizenship, the priest is shown as having the key answer thereby also affirming the supremacy and authority of the Catholic Church.

Figure 18. A priest tells students what good citizenship is in terms of loving one’s neighbor (Schaller, 1950, p. 28). Courtesy of The Catholic University of America Digital Collections.

(Blankenship, 2010, p. 70). Blankenship (2010) argues that this suggests that Catholics “hold the key to a more perfect America” (p. 70). In this way, TC portrays the focus on charity and common good found in Catholic social teachings as what qualifies Catholics as good American citizens. In light of the fear of communism and Catholic youth falling away from the faith, TC also sought to instill good morals in readers and serve as an alternative to the comics considered ‘objectionable’ by NODL. TC stories often contained moral teachings and characters served as models of good moral behavior. The “Chuck White” series that ran through all the issues of TC showed the protagonist, White, facing common moral dilemmas that readers of the time may experience and could relate to. For example, the second volume follows Chuck White as he gets arrested for robbery and “cleans up his act,” including joining the school’s football team. After losing his temper and starting a fight on the field during a football game, Father Carroll, the school priest and football coach, tells White a lesson on how “humility is a virtue” as seen in Figure 19 (Pflaum, 28

Figure 19. Humility Is a Virtue. (Pflaum, 1946e, p. 22.) Courtesy of The Catholic University of America Digital Collections.

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TEACHER EDUCATION

Figure 21. After telling the story of St. Agnes, the sister tells a girl “What better model could you follow if you want to be pure and good?” (Ostendorf, 1955, p. 8). Courtesy of The Catholic University of America Digital Collections.

Figure 20. A religious sister tells a girl that she should pray to St. Agnes (Ostendorf, 1955, p. 3). Courtesy of The Catholic University of America Digital Collections.

1946e, p. 22). Another example is the previously mentioned series, “The Cost of a Lie,” which tells the story of Arthur lying and going to see a movie in defiance of his mother’s wishes (Pflaum, 1946c-d). Arthur must deal with the guilt and negative consequences leading him to conclude that “a lie is just like a snowball” that continues to grow and cost him even more (Pflaum, 1946d, p. 19). Although TC stories did not always show characters acting with perfect moral behavior, the mistakes these characters made were to serve as lessons for readers to help keep them from making the same mistakes. In contrast to the popular superheroes of ‘objectionable’ comics, TC offered the saints and biblical figures as role models for their readers. This is established during the first issue of the first volume in the telling of the story of “Joseph Anchieta S.J.: Apostle of Brazil”. Rather than look up to figures like Christopher Columbus, his mother points at a cross and tells him that “if you want to be a hero, Joseph, take the greatest hero as your model” (Pflaum, 1946b, p. 2). This was a very key message for readers that as Catholics they should hold paramount Jesus as a role model and look to other saints who strove to serve and follow Jesus as examples of how to live. For example, Figure 20 shows a girl telling a religious sister that “I’d like to offer my prayers to some special saint who will help me to be the kind of person my parents and God want me to be” (Ostendorf, 1955, p. 3). The sister suggests St. Agnes then proceeds to tell St. Agnes’ life story, ending in Figure 21 with how “naturally she has become the patron saint of young girls. What better model could you follow if you want to be pure and good?” (Ostendorf, 1955, p. 8). Another goal outlined in the teacher issue for the 1964-65 school year was to “1. interest [students] in the Old Testament” through a series of stories called “Heroes of the Old Testament” (Wischmeyer, 1964a, p. 37). In the first story on Abraham, TC prefaces the series claiming that “many events in the Old Testament are as exciting as a modern adventure story” (Ostendorf, 1964, p. 3). In a note to parents on the story of Daniel, TC notes that these stories are not necessarily historical fact but that: Proceedings 2018

TEACHER EDUCATION “The author of the Book of Daniel used these stories to encourage the Jews of the second century B.C. to be brave in the face of persecution. Daniel and his three companions triumphed over difficulties with the help of God. The persecuted second-century Jew could easily apply these stories to himself.” (Wischmeyer, 1965, p. 35)

This note to parents reflects the use of historical critical method (HCM) in understanding the historical context of the story of Daniel and that the intended audience was persecuted. The inclusion of this note also reflects that TC was aware of anti-Catholic sentiments of this time and that they included the story with the purpose of encouraging readers who may be facing discrimination to lean into their faith. The goal was for readers, like the persecuted second-century Jews, to be able to apply these stories to their own lives and use them as inspiration. So by including many stories of saints and using terms like “heroes” and “adventure” to describe them, TC suggests that saints and biblical figures should be looked to as role models for their readers rather than superheroes and that these stories can be just as interesting.

Conclusion In conclusion, the answer to my first research question is that comic books and graphic novels can be useful as a teaching tool in a religious education classroom. This is reflected in the first teacher issue for Volume 20 of Treasure Chest (TC), which starts with the statement that: “Teachers know that when the printed word and illustration are combined effectively, the impact is much stronger than when one or the other is used independently.” (Wischmeyer, 1964a, p. 37)

The literature I have found supports this claim that comic books and graphic novels can aid students in their reading by presenting the content in context with the use of visual aids alongside the printed text. This statement also reflects how comics promote the development of multimodal literacy because an understanding gained from either the print or visual text is incomplete (Jacobs, 2007; Cromer & Clark, 2007). Multimodal literacy challenges students to go beyond word-based literacy to see how the different modes are interrelated. As outlined by Orcutt (2010), the development of multimodal literacy with comics is a great way to illustrate the complex relationship between the various means of religious expression. Since comics use icons and cartoon vocabulary, the development of visual literacy with comic books can help promote student’s ability to think symbolically and make connections that is important in religion (Orcutt, 2010). Furthermore, Jacobs (2007) claims that comics promote critical literacy with students being “active creators, rather than passive consumers of meaning” because they must analyze and make these connections (p. 24). In addition to critical literacy, graphic novels and comic books can promote disciplinary literacy, or literary practices pertinent to religion. Catholic tradition and history are rich with the use of icons and art, which have often been used to teach the faith. Just as TC did, the Church still uses visuals like comics and coloring books to teach children today (Blankenship, 2010, p. 64). In this way, the tradition and history of using visuals to teach the Catholic faith seems to validate the use of comic books and graphic novels as a catechetical tool. Comic books and graphic novels also have the potential to develop disciplinary literacy by promoting skills pertinent to reading religious texts, including the Bible. As a future high school religion and history teacher, my goal is to create a developmentally appropriate comic that will utilize the historical critical method (HCM) and help Catholic high-schoolers begin to understand the historical context in which scripture emerged. The United States Conference of Catholic Bishops (USCCB, 2008) outline as first of the core curriculum themes for high schoolers “The Revelation of Jesus Christ in Scripture” with a focus on understanding what Scripture is and how to interpret Scripture. Under this, students are to learn about how the Bible is truly God’s Word divinely revealed and written by humans inspired by the Holy Spirit (USCCB, 2008, p. 3; Catholic Church, 2012, para. 105-106). This involves explaining the senses of scripture: the spiritual sense and the literal sense discovered by exegesis (USCCB, 2008, p. 3). Furthermore, Pope Paul VI’s “Dei Verbum (On Divine Revelation)” (1965) is listed as a key source to be covered in the curriculum (USCCB, 2008, p. 3). 30

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TEACHER EDUCATION This is significant because the focus of this encyclical is on the need to read Scripture considering “what meaning the sacred writers really intended, and what God wanted to manifest by means of their words” (Paul VI, 1965, article 12). This message is further reflected as number two listed under criteria for interpreting Scripture to be taught (USCCB, 2008, p. 3). These criteria also include: “3. Take into account the conditions of the time when it was written and the culture where it was written.” (USCCB, 2008, p. 3)

This connects to how HCM seeks to understand the historical context of a religious text by considering historically what was going on when the text was composed and so what the text meant in and for the ancient communities that produced it. In TC, this was most evident in the note to parents on the historical context of the story of Daniel and that the intended audience experienced persecution. TC seemed to include this story because of how anti-Catholic sentiments were prevalent during that time, and, therefore, may be relatable to readers. In the development of my comic, I plan to also choose biblical stories that connect to the theme or lesson and include a similar note prefacing the story on the historical context, so my students can then use HCM to interpret the story and gain a deeper understanding. In order to answer my overall research question on the effectiveness of comic books in catechizing American Catholic youth today, I focused my second research question on Treasure Chest (TC) as an example of how comic books have been used in the past to catechize American Catholic youth. My archival research of TC even involved using some of the historical analysis skills I want my students to develop. For example, I analyzed the comics in terms of the source, Pflaum Inc., and the context they were written in, such as how TC was written in keeping with NODL and as an alternative to the considered ‘objectionable’ comics of the time. In addition to censorship, the creation of TC was also informed by the fear of youth falling away from the faith and predominant anti-Catholic sentiments, such as Catholicism being called un-American. Through my archival research, I have identified key characteristics of a good American Catholic citizen like being anti-communist, being patriotic and defenders of democracy, having and sticking to your morals, as well as being part of a ‘melting pot’. TC repeatedly emphasizes that one could be a good American Catholic citizen. According to Blankenship (2010): “Contradicting common stereotypes about Catholics, Treasure Chest declares that American Catholic children have more grounds and capacity than anyone to be the best American citizens.” (p. 76)

This is because the key to improving America and the core of citizenship was understood as charity and loving one’s neighbor that was prominent in Catholic social teachings (Blankenship, 2010, p. 70). TC conveyed this message by offering connections to current events and history in a way that was enticing to youth of the day. This seems to have been successful as seen in the letters written by readers to the editor of TC, which were printed with responses in the “Backtalk” section that began appearing in Volume 22. A reader named Valerie Groncki wrote in: “Dear Editor: I am a great fan of TC. I hope your fabulous magazine will carry on its entertainment for children for many more years!” (Langdon, 1967a, p. 34)

Short letters expressing appreciation for TC and or telling jokes continued to appear at the end of each issue for the remainder of TC’s running. These reflect the idea of how comic books can serve as a useful teaching tool because the interest in the genre can make them motivating and help connect the material with the students (Carraher, 1975, p. 30). TC’s success is also evidenced in how it ran longer and produced more issues than any other Catholic comic series (Blankenship, 2010, p. 65). In this way, TC seems to be the leading example of how comic books have been used in the past to catechize American Catholic youth.

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TEACHER EDUCATION Future Research These answers to questions 1 and 2 have provided crucial background information necessary to begin evaluating the effectiveness of comic books in catechizing American Catholic youth today as part of question 3. To answer this overall question, I plan to develop, at a later date, my own more modern example of a Catholic comic informed by my analysis of TC and official Roman Catholic documents and teachings. While anti-communism is not as prevalent today, how TC “regularly defined America by freedom, democracy, and multiculturalism,” as outlined by Blankenship (2010) is still relevant (p. 64). TC’s focus on issues of immigration, racism, tolerance, religious freedom, loving one’s neighbor and debates over who qualifies as an American citizen are issues still prominent in American politics and culture today. As I move forward with the development of my own comic, I plan to include some of these issues, especially religious freedom and tolerance. I plan to implement a similar framework and include style elements similar to those found in TC. I plan to include a history story, a saint’s story, a biblical story, as well as a more modern story like Chuck White showing character’s navigating moral dilemmas. If I focus on religious freedom, I may retell in a more modern way the story of the founding of Maryland for religious freedom and tolerance for Catholics. Since TC was designed for grade schoolers, I intend to make my comic more age appropriate for high schoolers by including primary sources such as excerpts from Scripture and Catholic social teachings from the Catechism, encyclicals and/or pastoral letters. This may look similar to the Volume 6 “Where Do We Stand?” series, which asked questions of the readers throughout the story and summarized key points at the end as seen in Figure 9 (Ostendorf, 1950, p. 13). Readers also had to match Catholic social teachings found in the dialogue with the statements and sources listed at the end of the story (Ostendorf, 1950, p. 14). To this extent, the development of my comic will use TC as an example and be based upon it. The development of such a comic would then allow me to determine its effectiveness as a catechetical tool. In keeping with the lesson plan outline format used by the Forum for Young Catechetical Leaders, I plan to develop a lesson plan centered on the use of the comic strip as a teaching tool. In my Religion Methods Lab (EDT 331L), I plan to then use this comic strip as a teaching aid in the high school religious education classroom I will be assigned. By teaching at least one class with the comic strip and another without it, I plan to evaluate the effectiveness of comics as a tool for catechesis based on any significant differences between the two classes test or quiz scores for that unit. If there is a correlation between the use of the comic strip and higher scores, religion teachers might attempt to increase the use of comics as a tool for catechesis. Overall, the results of this project potentially hold implications for religious education teachers and others who are working to catechize American Catholic youth.

Acknowledgements I would like to thank the Berry Family and the Berry Summer Thesis Institute for funding my research and giving me this opportunity to focus on my passion this summer. Thank you to the University Honors Program and those who worked with BSTI: Dr. Nancy Miller, Maria Burkett, Laura Cotten, Dr. John McCombe, Ramona Speranza and Jill Talley. Thank you for giving us your expertise, providing us with direction and showing us the joys of research and academia. I would also like to thank the College of Arts and Sciences Dean’s Summer Fellowship program for their funding and support of my research. Special thanks to my mentors Dr. Caroline Merithew and Susan Ferguson for your honesty and wisdom. Thank you for helping me to see myself as a writer and encouraging me to grow as a researcher. I would also like to thank Dr. Una Cadegan for her expertise and guidance in writing as a Catholic historian. I would like to thank Stephanie Shreffler and the U.S. Catholic Special Collection of Roesch Library for their guidance in archival research and use of the Treasure Chest of Fun and Fact comic series. Finally, I would like to thank my parents, Joshua Krile, my roommates and the rest of my cohort for their love and support. I am so grateful for BSTI allowing me to grow as a person and for everyone who helped make this experience so impactful and positive for me.

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TEACHER EDUCATION References Are Comics Fascist? (1945, October 22). Time, 46(17), 67-67. Retrieved from http://time.com/vault/issue/1945-10-22/page/69/. Baker, L. (2015). How many words is a picture worth? Integrating visual literacy in language learning with photographs. English Teaching Forum, 53(4), 2-13. Retrieved from https://eric.ed.gov/contentdelivery/servlet/ERICServlet?accno=EJ1084470. Batman. (n.d.) Retrieved from https://www.dccomics.com/characters/batman. Blankenship, A. (2010). Catholic American citizenship: Prescriptions for children from Treasure Chest of Fun and Fact (194663). In A. D. Lewis and C. H. Kraemer (Eds.), Graven Images: Religion in Comic Books and Graphic Novels (p. 63-77). New York, NY: The Continuum International Publishing Group. Bren, J. (Illustrator.). (1954, September 23). We built America [Cartoon]. In Treasure Chest of Fun and Fact (2nd ed., Vol. 10, p. 20-22). Dayton, OH: George A. Pflaum, Publisher, Inc. Retrieved from https://cuislandora.wrlc.org/islandora/object/ cuislandora%3A15409#page/22/mode/1up. Brozo, W. G., Meyer, C. K., & Moorman, G. B. (2014). Wham!: Teaching with Graphic Novels Across the Curriculum. New York: Teachers College Press. Cadegan, U. (2001). Guardians of democracy or cultural storm troopers? American Catholics and the control of popular media, 1934-1966. The Catholic Historical Review, 87(2), 252-282. Retrieved from http://www.jstor.org/stable/25025928. Carraher, C. (1975). Comics: No-nonsense classroom aids. The Science Teacher, 42(9), 30. Retrieved from http://www.jstor. org/stable/24125744. Catholic Church. (2012) The Profession of Faith. In 2nd ed., Catechism of the Catholic Church (1262). Vatican City: Libreria Editrice Vaticana. Conroy, J. (1964a, September 10). Would you like to be a lawyer? Treasure Chest of Fun and Fact (1st ed., Vol. 20, p. 15-16). Dayton, OH: George A. Pflaum Publishing. Retrieved from https://cuislandora.wrlc.org/islandora/object/ cuislandora%3A22882#page/15/mode/1up. Conroy, J. (1964b, October 8). Would you like to be a doctor? Treasure Chest of Fun and Fact (3rd ed., Vol. 20, p. 27-28). Dayton, OH: George A. Pflaum Publishing. Retrieved from https://cuislandora.wrlc.org/islandora/object/cuislandora%3A22964#page/26/ mode/1up. Conroy, J. (1964c, December 3). Would you like to be a journalist? Treasure Chest of Fun and Fact (7th ed., Vol. 20, p. 20-21). Dayton, OH: George A. Pflaum Publishing. Retrieved from https://cuislandora.wrlc.org/islandora/object/ cuislandora%3A23112#page/19/mode/1up. Crandall, R. (1961a, September 28). This godless communism [Cartoon]. Treasure Chest of Fun and Fact (2nd ed., Vol. 17, p. 3-8). Dayton, OH: Geo. A. Pflaum, Publisher, Inc. Retrieved from https://cuislandora.wrlc.org/islandora/object/ cuislandora%3A20654#page/2/mode/1up. Crandall, R. (1961b, October 26). This godless communism [Cartoon]. Treasure Chest of Fun and Fact (4th ed., Vol. 17, p. 29-34). Dayton, OH: Geo. A. Pflaum, Publisher, Inc. Retrieved from https://cuislandora.wrlc.org/islandora/object/ cuislandora%3A20732#page/28/mode/1up. Cromer, M., & Clark, P. (2007). Getting graphic with the past: Graphic novels and the teaching of history. Theory And Research In Social Education, 35(4), 574-591. Retrieved from http://www.socialstudies.org.libproxy.udayton.edu/publications/ theoryandresearch. Dougherty, D., Mooney, E., Stritch, S. A., Spellman, F.J., McNicholas, J. T., Lucey, R. E., … Ready, M. J. (1947). Secularism. In H. J. Nolan (Ed.), Pastoral Letters of the American Hierarchy, 1792-1970 (p. 403-408). Huntingon, IN: Our Sunday Visitor. Dougherty, D., Mooney, E., Stritch, S. A., Spellman, F.J., Keough, F. P., McNicholas, J. T., … Ready, M. J. (1948). The Christian in action. In H. J. Nolan (Ed.), Pastoral Letters of the American Hierarchy, 1792-1970 (p. 409-415). Huntingon, IN: Our Sunday Visitor. Eaglin, T. (Illustrator.). (1954, December 16). We built America [Cartoon]. Treasure Chest of Fun and Fact (8th ed., Vol. 10, p. 11-16). Dayton, OH: Geo. A. Pflaum, Publisher, Inc. Retrieved from https://cuislandora.wrlc.org/islandora/object/ cuislandora%3A15631#page/10/mode/1up. Eaglin, T. (Illustrator.). (1955a, January 12). We built America [Cartoon]. Treasure Chest of Fun and Fact (10th ed., Vol. 10, p. 31-34). Dayton, OH: Geo. A. Pflaum, Publisher, Inc. Retrieved from https://cuislandora.wrlc.org/islandora/object/ cuislandora%3A15705#page/30/mode/1up. Eaglin, T. (Illustrator.). (1955b, February 10). We built America [Cartoon]. Treasure Chest of Fun and Fact (12th ed., Vol. 10, p. 27-31). Dayton, OH: Geo. A. Pflaum, Publisher, Inc. Retrieved from https://cuislandora.wrlc.org/islandora/object/ cuislandora%3A15779#page/26/mode/1up. Eaglin, T. (Illustrator.). (1955c, April 7). We built America [Cartoon]. Treasure Chest of Fun and Fact (16th ed., Vol. 10, p. 13-17). Dayton, OH: Geo. A. Pflaum, Publisher, Inc. Retrieved from https://cuislandora.wrlc.org/islandora/object/ cuislandora%3A15927#page/17/mode/1up. Eaglin, T. (Illustrator.). (1955d, June 2). We built America [Cartoon]. Treasure Chest of Fun and Fact (20th ed., Vol. 10, p. 27-30). Dayton, OH: Geo. A. Pflaum, Publisher, Inc. Retrieved from https://cuislandora.wrlc.org/islandora/object/ cuislandora%3A16075#page/26/mode/1up. FDR Library and Museum. (2018). FDR and the Four Freedoms Speech. Retrieved from https://fdrlibrary.org/four-freedoms.

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TEACHER EDUCATION Grady, M. E. (1967, June 1). Viet-Nam’s ‘Ya Pogang’ the story of Dr. Pat Smith [Cartoon]. Treasure Chest of Fun and Fact (20th ed., Vol. 22, p. 29-34). Dayton, OH: Geo. A. Pflaum, Publisher, Inc. Retrieved from https://cuislandora.wrlc.org/islandora/ object/cuislandora%3A25083#page/28/mode/1up. Hancock, R. (2015). More credo, less capes: Why and how we should use comics in the Religious Education classroom. Studies in Comics, 6(1), 25-41. doi:10.1386/stic.6.1.25_1. Hassett, M. (1909). Symbolism of the Fish. The Catholic Encyclopedia. New York: Robert Appleton Company. Retrieved from New Advent http://www.newadvent.org/cathen/06083a.html. Hoover, J. E. (1961, March 13). Letter. In Treasure Chest of Fun and Fact (20th ed., Vol. 22, p. 1). Dayton, OH: Geo. A. Pflaum, Publisher, Inc. Retrieved from https://cuislandora.wrlc.org/islandora/object/cuislandora%3A20654#page/1/mode/1up. Jacobs, D. (2007). More than words: Comics as a means of teaching multiple literacies. English Journal, 96(3), 19-25. Retrieved from http://www.ncte.org.libproxy.udayton.edu/journals/ej/issues. Jones, C. A. (2008). Lights of faith: Stained glass windows as tools for catechesis. Journal of the Institute for Sacred Architecture, (14), 17-20. Retrieved from http://www.sacredarchitecture.org/images/uploads/volumesPDFs/Issue_14_2008.pdf. Klosterman, B. (1964a, October 22). Tom Dooley, valiant American doctor [Cartoon]. Treasure Chest of Fun and Fact (4th ed., Vol. 20, p. 21). Dayton, OH: Geo. A. Pflaum, Publisher, Inc. Retrieved from https://cuislandora.wrlc.org/islandora/object/ cuislandora%3A23001#page/20/mode/1up. Klosterman, B. (1964b, December 17). Courageous Captain [Cartoon]. Treasure Chest of Fun and Fact (8th ed., Vol. 20, p. 3-8). Dayton, OH: Geo. A. Pflaum, Publisher, Inc. Retrieved from https://cuislandora.wrlc.org/islandora/object/ cuislandora%3A23149#page/2/mode/1up. Klosterman, B. (1965a, February 11) Great white father [Cartoon]. Treasure Chest of Fun and Fact (12th ed., Vol. 20, p. 3-8). Dayton, OH: Geo. A. Pflaum, Publisher, Inc. Retrieved from https://cuislandora.wrlc.org/islandora/object/ cuislandora%3A23301#page/2/mode/1up. Klosterman, B. (1965b, March 17) “Little Phil” Sheridan, hero of the Civil War [Cartoon]. Treasure Chest of Fun and Fact (14th ed., Vol. 20, p. 27-31). Dayton, OH: Geo. A. Pflaum, Publisher, Inc. Retrieved from https://cuislandora.wrlc.org/islandora/ object/cuislandora%3A23375#page/26/mode/1up. Klosterman, B. (1965c, June 3) Colonial patriot [Cartoon]. Treasure Chest of Fun and Fact (20th ed., Vol. 20, p. 22-27). Dayton, OH: Geo. A. Pflaum, Publisher, Inc. Retrieved from https://cuislandora.wrlc.org/islandora/object/cuislandora%3A23601#page/21/ mode/1up. Langdon, J. J. (Ed.). (1967a, February 23). Backtalk. Treasure Chest of Fun and Fact (13th ed., Vol. 22, p. 34). Dayton, OH: Geo. A. Pflaum, Publisher, Inc. Retrieved from https://cuislandora.wrlc.org/islandora/object/cuislandora%3A24824#page/34/ mode/1up. Langdon, J. J. (Ed.). (1967b, June 1). Dr. Pat Smith in Vietnam [Cartoon]. Treasure Chest of Fun and Fact (20th ed., Vol. 22, p. 1-32). Dayton, OH: Geo. A. Pflaum, Publisher, Inc. Retrieved from https://cuislandora.wrlc.org/islandora/object/ cuislandora%3A25083#page/1/mode/1up. Lepore, J. (2014). The Secret History of Wonder Woman. New York City, NY: Random House. Maas, A. (1909). Biblical Exegesis. The Catholic Encyclopedia. New York: Robert Appleton Company. Retrieved from New Advent http://www.newadvent.org/cathen/05692b.htm. Mathews, S. A. (2011). Framing preservice teachers' interpretations of graphic novels in the social studies classroom. Theory And Research in Social Education, 39(3), 416-446. Retrieved from http://www.socialstudies.org.libproxy.udayton.edu/ publications/theoryandresearch. Mooney, E., McNicholas, J. T., Stritch, S. A., Murray, J. G., Spellman, F.J., Gannon, J. M., … Duffy, J. A. (1941). The crisis of Christianity. In H. J. Nolan (Ed.), Pastoral Letters of the American Hierarchy, 1792-1970 (p. 372-377). Huntingon, IN: Our Sunday Visitor. Mooney, E., Stritch, S. A., Spellman, F.J., McIntyre, J. F., Keough, F. P., Ritter, J. E, … Gilmore, J. M. (1957). Censorship. In H. J. Nolan (Ed.), Pastoral Letters of the American Hierarchy, 1792-1970 (p. 498-503). Huntingon, IN: Our Sunday Visitor. National Catholic Educational Association (NCEA). (2018). Catholic School Data. Retrieved from https://www.ncea.org/NCEA/ Proclaim/Catholic_School_Data/Catholic_School_Data.aspx. Nolan, H. J. (1971). Pastoral Letters of the American Hierarchy, 1792-1970. Huntingon, IN: Our Sunday Visitor. Nyberg, A. K. (n.d.) Comics Code History: The Seal of Approval. Retrieved from http://cbldf.org/comics-code-history-the-seal-ofapproval/. Orcutt, D. (2010). Comics and Religion: Theoretical Connections. In A. D. Lewis and C. H. Kraemer (Eds.), Graven Images: Religion in Comic Books and Graphic Novels (p. 93-106). New York, NY: The Continuum International Publishing Group. Ostendorf, L. (1950, November 23). Where do we stand? [Cartoon]. Treasure Chest of Fun and Fact (6th ed., Vol. 6, p. 9-14). Dayton, OH: Geo. A. Pflaum, Publisher, Inc. Retrieved from https://cuislandora.wrlc.org/islandora/object/ cuislandora%3A12593#page/9/mode/1up. Ostendorf, L. (1951a, January 18). Where do we stand? [Cartoon]. Treasure Chest of Fun and Fact (10th ed., Vol. 6, p. 9-14). Dayton, OH: Geo. A. Pflaum, Publisher, Inc. Retrieved from https://cuislandora.wrlc.org/islandora/object/ cuislandora%3A12741#page/8/mode/1up.

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TEACHER EDUCATION Ostendorf, L. (1951b, February 15). Where do we stand? [Cartoon]. Treasure Chest of Fun and Fact (12th ed., Vol. 6, p. 9-14). Dayton, OH: Geo. A. Pflaum, Publisher, Inc. Retrieved from https://cuislandora.wrlc.org/islandora/object/ cuislandora%3A12815#page/2/mode/1up. Ostendorf, L. (1955, January 13). St. Agnes [Cartoon]. Treasure Chest of Fun and Fact (10th ed., Vol. 10, p. 3-8). Dayton, OH: Geo. A. Pflaum, Publisher, Inc. Retrieved from https://cuislandora.wrlc.org/islandora/object/cuislandora%3A15705#page/2/ mode/1up. Ostendorf, L. (1963, May 23). America: The melting pot… Part 10 [Cartoon]. Treasure Chest of Fun and Fact (20th ed., Vol. 18, p. 18-19). Dayton, OH: Geo. A. Pflaum, Publisher, Inc. Retrieved from https://cuislandora.wrlc.org/islandora/object/ cuislandora%3A22052#page/17/mode/1up. Our Sunday Visitor, Inc. (2018). About Our Sunday Visitor. Retrieved from https://www.osv.com/More/AboutUs.aspx. Paul VI. (1965). Dei Verbum: On Divine Revelation. Retrieved from http://www.vatican.va/archive/hist_councils/ ii_vatican_council/documents/vat-ii_const_19651118_dei-verbum_en.html. Pehowski, M. (1962a, September 13). America: The melting pot… Part 1 [Cartoon]. Treasure Chest of Fun and Fact (1st ed., Vol. 18, p. 18-19). Dayton, OH: Geo. A. Pflaum, Publisher, Inc. Retrieved from https://cuislandora.wrlc.org/islandora/object/ cuislandora%3A21370#page/18/mode/1up. Pehowski, M. (1962b, December 6). America: The melting pot… Part 4 [Cartoon]. Treasure Chest of Fun and Fact (7th ed., Vol. 18, p. 18-19). Dayton, OH: Geo. A. Pflaum, Publisher, Inc. Retrieved from https://cuislandora.wrlc.org/islandora/object/ cuislandora%3A21600#page/17/mode/1up. Pflaum, G. (Ed.). (1946a, March 12). Lent [Cartoon]. Treasure Chest of Fun and Fact (1st ed., Vol. 1, p. 1-32). Dayton, OH: Geo. A. Pflaum, Publisher, Inc. Retrieved from https://cuislandora.wrlc.org/islandora/object/cuislandora%3A9586#page/1/mode/1up. Pflaum, G. (Ed.). (1946b, April 9). In this issue: Joseph Anchieta, S.J. … [Cartoon]. Treasure Chest of Fun and Fact (3rd ed., Vol. 1, p. 1-32). Dayton, OH: Geo. A. Pflaum, Publisher, Inc. Retrieved from https://cuislandora.wrlc.org/islandora/object/ cuislandora%3A9652#page/1/mode/1up. Pflaum, G. (Ed.). (1946c, September 3). The cost of a lie [Cartoon]. Treasure Chest of Fun and Fact (1st ed., Vol. 2, p. 25-27). Dayton, OH: Geo. A. Pflaum, Publisher, Inc. Retrieved from https://cuislandora.wrlc.org/islandora/object/cuislandora%3A9785#page/1/ mode/1up. Pflaum, G. (Ed.). (1946d, September 17). The cost of a lie – Part II [Cartoon]. Treasure Chest of Fun and Fact (2nd ed., Vol. 2, p. 17-19). Dayton, OH: Geo. A. Pflaum, Publisher, Inc. Retrieved from https://cuislandora.wrlc.org/islandora/object/ cuislandora%3A9818#page/4/mode/1up. Pflaum, G. (Ed.). (1946e, December 10). Chuck White [Cartoon]. Treasure Chest of Fun and Fact (8th ed., Vol. 2, p. 20-27). Dayton, OH: Geo. A. Pflaum, Publisher, Inc. Retrieved from https://cuislandora.wrlc.org/islandora/object/ cuislandora%3A10016#page/19/mode/1up. Prior, J. G. (2001). The Historical Critical Method in Catholic Exegesis. Rome, Italy: Gregorian University Press. Public Broadcasting Services (PBS). (2010, October 11). Interview: John McGreevy. Retrieved from http://www.pbs.org/ godinamerica/interviews/john-mcgreevy.html#ursulineconvent. Raimi, S. (Director). (2002). Spider-Man [Film], New York City: Marvel Entertainment, LLC. Satrapi, M. (2007). Persepolis. Paris: L'Association. Schaller, J. G. (Ed.). (1949a, September 20). It happens here: A story of young citizens [Cartoon]. Treasure Chest of Fun and Fact (2nd ed., Vol. 5, p. 29-34). Dayton, OH: George A. Pflaum Publishing. Retrieved from https://cuislandora.wrlc.org/ islandora/object/cuislandora%3A11704#page/28/mode/1up. Schaller, J. G. (Ed.). (1949b, October 18). It happens here: A story of young citizens [Cartoon]. Treasure Chest of Fun and Fact (2nd ed., Vol. 5, p. 29-34). Dayton, OH: George A. Pflaum Publishing. Retrieved from https://cuislandora.wrlc.org/islandora/ object/cuislandora%3A11778#page/28/mode/1up. Schaller, J. G. (Ed.). (1950, April 4). It happens here: A story of young citizens [Cartoon]. Treasure Chest of Fun and Fact (16th ed., Vol. 5, p. 25-28). Dayton, OH: George A. Pflaum Publishing. Retrieved from https://cuislandora.wrlc.org/islandora/object/ cuislandora%3A12222#page/24/mode/1up. Schaller, J. G. (Ed.). (1955, June 2). Jesus, meek and humble of heart [Cartoon]. Treasure Chest of Fun and Fact (20th ed., Vol. 10, pp. 1-32). Dayton, OH: George A. Pflaum Publishing. Retrieved from https://cuislandora.wrlc.org/islandora/object/ cuislandora%3A16075#page/1/mode/1up. Spellman, F.J., McIntyre, J. F., Cushing, R., Muench, A., Meyer, A., Ritter, J., … Zuroweste, A. R. (1961). Unchanging duty in a changing world. In H. J. Nolan (Ed.), Pastoral Letters of the American Hierarchy, 1792-1970 (p. 536-541). Huntingon, IN: Our Sunday Visitor. Spiegelman, A. (2011, October 20). Why mice? New York Review Daily. Retrieved from https://www.nybooks.com/ daily/2011/10/20/why-mice/. Superman. (n.d.) Retrieved from https://www.dccomics.com/characters/superman. Treasure Chest of Fun and Fact Comic Book Collection. (n.d.). American Catholic History Research Center and University Archives. Retrieved from The Catholic University of America http://archives.lib.cua.edu/findingaid/treasurechest.cfm.

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TEACHER EDUCATION United States Conference of Catholic Bishops (USCCB). (2008). Doctrinal Elements of a Curriculum Framework for the Development of Catechetical Materials for Young People of High School Age (p. 1-5). Washington, D.C.: United States Conference of Catholic Bishops. Williams, R. (2008). Image, text, and story: Comics and graphic novels in the classroom. Art Education, (6), 13. Retrieved from http://www.jstor.org/stable/27696303. Wischmeyer, R. A. (Ed.). (1964a, September 10). Treasure Chestâ&#x20AC;&#x2122;s 1964-1965 program. Treasure Chest of Fun and Fact (1st ed., Vol. 20, p. 37-40). Dayton, OH: George A. Pflaum Publishing. Retrieved fromhttps://cuislandora.wrlc.org/islandora/object/ cuislandora%3A22882#page/37/mode/1up. Wischmeyer, R. A. (1964b, November 19). John F. Kennedy: 35th President. Treasure Chest of Fun and Fact (6th ed., Vol. 20, p. 9-14). Dayton, OH: George A. Pflaum Publishing. Retrieved fromhttps://cuislandora.wrlc.org/islandora/object/ cuislandora%3A23075#page/8/mode/1up. Wischmeyer, R. A. (1965, April 22). Parents corner. Treasure Chest of Fun and Fact (17th ed., Vol. 20, p. 35). Dayton, OH: George A. Pflaum Publishing. Retrieved from https://cuislandora.wrlc.org/islandora/object/cuislandora%3A23486#page/34/ mode/1up. Wonder Woman (n.d.). Retrieved from https://www.dccomics.com/characters/wonder-woman. Wonder Woman biography (n.d.). Internet Movie Database. Retrieved from https://www.imdb.com/title/tt0451279/.

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TEACHER EDUCATION

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HEALTH AND SPORT SCIENCE

The Effect of Remote Ischemic Preconditioning on Hypoxic (Low Oxygen) Cognitive Function and Exercise Performance Jenna N. Sorensen1,2,3 University of Dayton, 300 College Park, Dayton, OH, 45469 1. Department of Health and Sport Science 2. Berry Summer Thesis Institute 3. University Honors Program

Mentor: Anne R. Crecelius, Ph.D. Department of Health and Sport Science Corresponding Author: Anne R. Crecelius, Ph.D. Email: acrecelius1@udayton.edu

Introduction Blood flow regulation and oxygen demand are vital concepts in the discussion of physiological performance. Blood flows down a pressure gradient, and flow is determined primarily by resistance. Arterioles are considered the resistance vessels of the body, and changes in arteriole diameter affect resistance, thereby affecting flow. Resistance can change in two ways – through vasodilation or vasoconstriction. When vasodilation occurs, the arterioles dilate and resistance decreases, increasing blood flow through those vessels. When vasoconstriction occurs, the arterioles constrict and resistance increases, limiting blood flow through those vessels. Both processes can be impacted by systemic factors including epinephrine and norepinephrine, respectively, from the sympathetic nervous system. Blood flow to the tissue and the amount of oxygen carried by the blood determines the tissue’s oxygen supply. Oxygen demand is the metabolic consumption of oxygen by a given tissue. When oxygen demand increases, oxygen supply will increase to match the demand through an increase in blood flow to the tissue (Andersen & Saltin, 1985). During exercise, there is an increased metabolic demand that is matched by an increase in skeletal muscle blood flow (exercise hyperaemia). Initially increasing rapidly, blood flow can increase up to 100 times greater than resting values during intense exercise (Casey & Joyner, 2011). When arterial oxygen content is decreased – such as during systemic hypoxia – blood flow increases, 38

through vasodilation, at a greater magnitude to match the increased metabolic demand of the tissue (Heistad & Abboud, 1980). The purpose of this review is to examine and summarize the current literature related to the control of blood flow during acute hypoxia, hypoxic exercise, and cognitive function. A brief review of hypoxia and its impact on exercise and cognitive function will be given, followed by a discussion of remote ischemic preconditioning and its potential role as an intervention.

Hypoxia — Influence on Exercise Systemic isocapnic hypoxia occurs when there is a decreased amount of oxygen in the blood while carbon dioxide levels remain constant. Decreased oxygen in the blood occurs from either a decrease in percent oxygen of inspired air, such as can be experimentally controlled in a laboratory used for testing hypoxia (Banzett, Garcia, & Moosavi, 2000; McMorris, Hale, Barwood, Costello, & Corbett, 2017), or a decrease in atmospheric pressure, such as at high altitudes. Both lead to a decline in the partial pressure of oxygen (PaO2) and therefore the driving gradient to load oxygen on to blood cells at the level of the lung. While normally hypoxia is accompanied by hyperventilation which subsequently decreases partial pressure of carbon dioxide (pCO2), often when examining hypoxic conditions experimentally carbon dioxide levels are held constant, such that the impact of the change in oxygen alone can be examined. Proceedings 2018

HEALTH AND SPORT SCIENCE Hypoxic conditions can lead to a decrease in oxygen supplied to muscle tissues due to less oxygen binding as a result of decreased PaO2. At rest, exposure induces vasodilation and augmented blood flow in skeletal muscles in healthy individuals (Casey & Joyner, 2011). During exercise, oxygen demand increases and blood flow increases (Anrep & von Saalfeld, 1935). In a state of hypoxia, when the oxygen supply in the blood is decreased, exercise hyperaemia is enhanced and blood flow must increase at an even greater magnitude to compensate for the increased oxygen demand. The “compensatory” vasodilation and augmented blood flow in contracting skeletal muscles is similar to the process that occurs in normoxia, but exceeds predicted values (Casey & Joyner, 2011). Studies have been done to determine the mechanisms driving hypoxic exercise hyperaemia and have shown that nitric oxide (NO) plays a major role in hypoxic exercise hyperaemia, prostaglandins play a minor role, and any unaccounted-for response can be attributed to endothelium-derived hyperpolarizing factor (EDH), specifically KIR channels and Na+/K+/ ATPase (Crecelius, Kirby, Hearon, Luckasen, Larson & Dinenno, 2015; Crecelius, Kirby, Voyles, & Dinenno, 2011; Crecelius, Luckasen, Larson, & Dinenno, 2014; Crecelius, Richards, Luckasen, Larson, and Dinenno, 2013). According to a study done by Wilkins, Pike, Martin, Curry, Ceridon & Joyner, 2011, the contribution of these mechanisms, as opposed to sympathetic increase in vasoconstriction in response to hypoxic vasodilation, is dependent on exercise intensity. Most prominently, vasodilation prevails over the vasoconstrictor response caused by the sympathetic nervous system during acute hypoxia (Casey & Joyner, 2011). Despite this additional signaling for an increase in blood flow, exercise in hypoxia can be limited by its exaggerated activation of the sympathetic nervous system and augmented signals for fatigue (Crecelius, Kirby, & Dinenno, 2015).

Hypoxia — Influence on Exercise: Methodology In these types of studies, blood flow is often quantified to provide information regarding the influence of a condition. Many current studies utilize a Doppler Ultrasound to collect blood flow data and measure the hypoxia-induced vasodilation. The measurements typically collected Proceedings 2018

are mean blood flow velocity (MBV) and brachial artery diameter. These measurements can then be used to determine forearm blood flow (FBF). These measurements are taken during rest and during exercise trials. Calculated FBF indicates augmented blood flow to the skeletal muscles during normoxic rest and exercise trials and during hypoxic rest and exercise trials. While overall blood flow values are collected, muscle oxygenation is also of interest. An example of exercise is rhythmic handgrip exercise at 10 percent maximal voluntary contraction (MVC) to be performed by the subject to assess performance. A Moxy Monitor can be used to measure muscle oxygen saturation and relative total hemoglobin. These values can be taken during rest and during exercise trials. The Moxy uses near-infrared spectroscopy to obtain local measurements when it is placed on the skin over a muscle. A study done by Crum, O’Connor, Van Loo, Valckx and Stannard, (2017), tested the validity and reliability of the Moxy Monitor in its measurement of muscle oxygenation during cycling and concluded that the device was reliable for low to moderate exercise intensities. At higher intensities, however, the device presented greater variance in measurements, most likely due to ischemia on increased movement (Crum, O’Connor, Van Loo, Valckx & Stannard, 2017). At low and moderate intensity exercise however, this intensity-dependent variation is not a concern. The Moxy Monitor is able to record a difference in the oxygen supply of the muscle during sham and experimental conditions.

Hypoxia — Influence on Cognitive Function Hypoxic conditions also impact cognitive functioning. McMorris, Hale, Barwood, Costello and Corbett’s 2017 review of acute hypoxia on cognition concluded that arterial partial pressure of oxygen (PaO2) was the key predictor of cognitive performance and that a decrease in PaO2 resulted in a negative effect on executive and non-executive tasks (with no significant difference between the two task forms). Like with exercise during hypoxia, there is an increase in cerebral blood flow to the brain (Figure 1) which, under normoxic conditions, improves cognition. Under hypoxic conditions, however, arterial desaturation attenuates cognitive improvements 39

HEALTH AND SPORT SCIENCE Hypoxia — Influence on Cognitive Function: Methodology A method to assess cognitive function is a cognitive testing procedure using the program CNS Vital Signs. CNS Vital Signs (CNSVS) is a computerized neurocognitive test battery that can be used to detect subtle changes or deficits in most neuropsychiatric conditions before and after a clinical condition or procedure (cnsvs.com). CNSVS has been evaluated for its validity and reliability in screening and diagnostic assessments due to its implementation of well-known neuropsychological tests, such as the Stroop Test, verbal and visual memory tests, and the finger tapping test. It has been found that CNSVS is suitable for use as a screening instrument but not for formal neuropsychological testing as a diagnostic tool (Gualtieri & Johnson, 2006). Many studies are not concerned with diagnostics or even screening to some extent, and subject’s scores can be compared before and after an experimental protocol rather than scores being compared across subjects. CNSVS provides accurate representation as it is sensitive enough to detect subtle change in neurocognitive status (Gualtieri & Johnson, 2006).

Figure 1. A) Middle cerebral artery mean velocity (MCAVmean) during cognitive tasks B) Cerebral oxygen delivery during cognitive tasks Komiyama, T., Katayama, K., Sudo, M., Ishida, K., Higaki, Y., & Ando, S. (2017). “Cognitive function during exercise under severe hypoxia.” Sci Rep, 7(1), 10000. Courtesy of http://creativecommons.org/licenses/by/4.0/.

(Komiyama, Katayama, Sudo, Ishida, Higaki & Ando, 2017). Blood flow to the brain is concentrated to the vertebral arteries, which serve regions of the brain concerned with cardiorespiratory control, with less emphasis on the internal carotid arteries, which supplies the areas of the brain involved in cognition. The organism places importance on control of vital survival systems during bouts of hypoxia. Attention, verbal abilities, and executive functioning decline with exposure to hypobaric hypoxia, with considerable individual variability (Griva, Stygall, Wilson, Martin, Levett, Mitchell, et al., 2017; Turner, Barker-Collo, Connell & Gant, 2015).

As studies aim to compare scores within subject trials, subjects will be required to take the CNSVS test multiple times. This leads to the concern for a test-retest learning curve in subsequent testing trials, especially as the tests will be administered in brief test-retest intervals. In past studies (Figure 2), practice effects have been shown to occur significantly between first and second test administrations, with small, nonsignificant improvements observed between subsequent test administrations (Collie, Maruff, Darby, & McStephen, 2003). Due to these practice effects, the experimental protocols will need to be tailored so as to ensure the difference in test scores observed is due to experimental conditions only, with no influence from practice and learning in brief test-retest intervals.

Remote Ischemic Preconditioning — Mechanisms, Protocol, and Intervention Remote ischemic preconditioning (RIPC) refers to brief periods of exposure to ischemia, or a lack of blood flow, performed in an organ different from the target organ to protect against injury in subsequent hypoxic exposures. Proceedings 2018

HEALTH AND SPORT SCIENCE

Figure 2. Within and between assessment practice effects revealed by analyses of trial by trial data for the Simple Reaction Time (SRT) test. Power curves are fitted to the group mean data plotted by trial number. Regression equations and r2 values are displayed on the chart. SRT1_1 5 SRT Test 1 in Assessment 1; SRT1_2 5 SRT Test 1 in Assessment 2; SRT1_3 5 SRT Test 1 in Assessment 3; SRT1_4 5 SRT Test 1 in Assessment 4. Collie, A., Maruff, P., Darby, D. G., & McStephen, M. (2003). “The effects of practice on the cognitive test performance of neurologically normal individuals assessed at brief test-retest intervals.” Image from the Journal of the International Neuropsychological Society, 9(3), 419-428 © 2003, The International Neuropsychological Society, published by Cambridge University Press.

This technique arose in response to ischemia- 220 mmHg to completely occlude flow and in reperfusion injury, particularly after coronary sham conditions is only increased to 15 mmHg so artery surgeries, as a possible intervention to as not to interrupt arterial flow. Inflation occurs decrease the occurrence and extent of injuries for 5 minutes, followed by 5 minutes of deflation. (Joung, Rhim, Chin, Kim, Choi, Lee, et al., 2013). This is repeated multiple times to increase The underlying mechanisms that produce RIPC protection are widely under debate and many components remain unknown. Nitric oxide (NO) and adenosine are thought to be early phase mediators and reactive oxygen species (ROS) play a pivotal role as endogenous free radicals in RIPC-mediated protection. Erythropoietin, adenosine, prostaglandins, and other bloodborne mediators are undergoing study to determine their role in RIPC-induced protection (Berger, Figure 3. Schematic illustration of the two phases of preconditioning. An early phase (dark gray) Macholz, Mairbäurl, & Bärtsch, of protection develops within minutes from the initial ischemic stimulus and lasts for a few hours. 2015). A typical RIPC protocol begins with participants in a semirecumbent position. Cuffs are then placed around both legs at the mid-thigh level and inflated with a rapid cuff inflator. For RIPC, pressure is increased to roughly Proceedings 2018

The protection of this phase is probably more powerful than the protection of the second late phase (light gray), which becomes apparent 12 to 24 hours later and lasts three to four days. Both phases are separated by a window where no protection occurs. The different time courses of protection are explained by the different mechanisms underlying both phases: whereas the early phase is assumed to be caused by rapid post-translational modification of pre-existing proteins, the late phase is most likely caused by the synthesis of new, protective proteins. Berger, M. M., Macholz, F., Mairbäurl, H., & Bärtsch, P. (2015). “Remote ischemic preconditioning for prevention of high-altitude diseases: Fact or fiction?” Image from the Journal of Applied Physiology, © 2015.

HEALTH AND SPORT SCIENCE the RIPC effect (Rieger Hoiland, Tremlblay, Stembridge, Bain, Flück, et al., 2017). RIPC protection occurs in two phases, an early phase and a late phase (Figure 3). The early phase occurs within a few minutes of the ischemic stimulus and lasts a few hours. A second late phase occurs 12 to 24 hours after the initial stimulus and lasts three to four days. The phases are separated by a period of no protection. Additionally, multiple stimuli are more effective than a single stimulus (Cunniffe, Sharma, Cardinale, & Yellon, 2017; Thijssen, Maxwell, Green, Cable, & Jones, 2016). Remote ischemic preconditioning has been shown to increase exercise performance and reduce the risk of high-altitude diseases such as acute mountain sickness (AMS), high altitude cerebral edema (HACE), and high altitude pulmonary edema (HAPE). RIPC attenuates the signaling cascade of these conditions and stimulates intrinsic defense mechanisms, which leads to a short-term state of hypoxic/ischemic tolerance (Berger, Macholz, Mairbäurl & Bärtsch, 2015).

Figure 4. Brachial artery FMD before (pre) and after (post) the intervention (RIPC or sham) as well as post-5-km TT in healthy volunteers during the sham (black squares) and IPC (white squares) interventions. Error bars represent 95 percent confidence interval. Data from the generalized estimated equation (GEE) were included in the figure, meaning that FMD values are presented, which are statistically adjusted for changes in diameter and shear rate. *Post hoc pairwise comparison analysis represents a significant decrease in FMD between pre- and post-5-km TT in the sham trial (P = 0.02), whereas FMD was unchanged after RIPC between pre- and post-5-km TT (P = 0.60). Bailey, T. G., Birk, G. K., Cable, N. T., Atkinson, G., Green, D. J., Jones, H., et al. (2012). “Remote ischemic preconditioning prevents reduction in brachial artery flow-mediated dilation after strenuous exercise.” Image from the American Journal of Physiology - Heart and Circulatory Physiology, © 2012.

Remote Ischemic Preconditioning — Influence on Exercise Due to its ability to increase blood flow in muscle tissue, remote ischemic preconditioning has been studied for its role in exercise performance. Since one of the largest factors in the development of muscle fatigue is perfusion, and RIPC increases blood flow to in skeletal muscles during reperfusion, RIPC should delay the onset of fatigue in muscle tissue (Barbosa, Machado, Braz, Fernandes, Vianna, Nobrega, & Silva, 2015). It is possible that the RIPC protocol, in addition to the shear stress induced by occlusion and resultant metabolite or NO-mediated reactive hyperaemia, could explain the observed benefits in exercise performance shortly after cuff occlusion. The short hyperaemic window observed with occlusion could be exploited to possibly improve oxygen delivery and mechanical output during high intensity exercise (Figure 4) (Cunniffe, Sharma, Cardinale, & Yellon, 2017). The phenomenon of functional sympatholysis during exercise, which refers to blunting of sympathetically mediated vasoconstriction, can also be impacted by ischemic preconditioning (IPC) and remote ischemic preconditioning (RIPC) to improve exercise performance and muscle oxygenation (Horiuchi, Endo, & Thijssen, 2015). The use and understanding of a RIPC protocol is important because it reveals a noninvasive and easy application in humans to prevent the reduction in endothelial function after strenuous exercise. For example, RIPC of the lower limbs has been shown to maintain post-exercise brachial artery endothelium-dependent function at pre-exercise level (Bailey, Birk, Cable, Atkinson, Green, Jones, & Thijssen, 2012). Additionally, there is debate as to the benefits of ischemic preconditioning as opposed to remote ischemic preconditioning. There is no debate that local IPC increases blood flow during exercise and enhances post-exercise vascular function, however it is still under scrutiny whether RIPC provides the same level of protection (Cocking, Cable, Wilson, Green, Thijssen, & Jones, 2018).

Remote Ischemic Preconditioning — Influence on Cognitive Function To date, there has only been one study done that has examined the effects of remote ischemic preconditioning on cognitive function. In a Proceedings 2018

HEALTH AND SPORT SCIENCE pilot study done by Joung, Rhim, Chin, Kim, Choice, Lee, et al., (2013), the team examined the effects of RIPC on cognitive function after off-pump coronary artery bypass grafts. After this particular procedure, there is an increased occurrence of patients with postoperative cognitive dysfunction. The researchers proposed that a RIPC procedure before coronary artery anastomosis would provide protection against cognitive decline; however, the results of the study indicated that remote ischemic preconditioning did not reduce the incidence of postoperative cognitive dysfunction during the immediate postoperative period. Researchers indicated that there were limitations to this study and, as it is the only one if its kind, more testing is needed to procure a definitive conclusion as to the effects of RIPC on cognitive function.

in a post-operative condition where other factors may have influenced cognitive functioning and did not examine the effects of RIPC on cognitive function in a hypoxic environment.

Acknowledgements I would like to thank the University of Dayton Honors Program and the Berry Family for funding my research this summer. I would also like to thank my advisor, Dr. Anne Crecelius, for her support and guidance throughout the research process. Finally, I would like to thank the entire IHPL lab for their support and assistance with my research.

References Areas for Study

Andersen, P., & Saltin, B. (1985). Maximal perfusion of skeletal muscle in man. J Physiol, 366, 233-249.

There is a need to determine whether RIPC can attenuate the hypoxia-induced decline in cognitive function and exercise performance in young, healthy adults. Without adequate oxygenation to muscle and neural tissue, functions can decline and a sustained lack of oxygen can lead to cell death. There are significant populations that live and work at high altitudes or have exposure to hypoxia (e.g. people with sleep apnea) that are at risk for hypoxia-induced diseases and loss of normal functioning.

Anrep, G. V., & von Saalfeld, E. (1935). The blood flow through the skeletal muscle in relation to its contraction. J Physiol, 85(3), 375-399.

Inadequate research has been done, with the only other study on the subject being conducted by Rieger, Hoiland, Tremblay, Stembridge, Bain, Flück, et al., 2017. Their results indicated an increase in blood flow in the internal carotid arteries and an increase in blood velocity in the middle cerebral artery in both the RIPC and control procedures. This study concluded that no benefits could be seen from treatment with RIPC in terms of pulmonary vascular protection or preservation of peripheral endothelial functioning during acute and chronic hypoxia, but continued exploration of potential protective benefits would be useful particularly with respect to hemodynamics during exercise. To the best of our knowledge, the study done by Joung, Rhim, Chin, Kim, Choi, Lee, et al., (2013), is the only study to have explored the potential protective effect of RIPC on cognitive functioning. However, this study was a pilot study that only examined protection Proceedings 2018

Bailey, T. G., Birk, G. K., Cable, N. T., Atkinson, G., Green, D. J., Jones, H., et al. (2012). Remote ischemic preconditioning prevents reduction in brachial artery flow-mediated dilation after strenuous exercise. Am J Physiol Heart Circ Physiol, 303(5), H533-538. Banzett, R. B., Garcia, R. T., & Moosavi, S. H. (2000). Simple contrivance "clamps" end-tidal PCO(2) and PO(2) despite rapid changes in ventilation. J Appl Physiol (1985), 88(5), 1597-1600. Barbosa, T. C., Machado, A. C., Braz, I. D., Fernandes, I. A., Vianna, L. C., Nobrega, A. C., et al. (2015). Remote ischemic preconditioning delays fatigue development during handgrip exercise. Scand J Med Sci Sports, 25(3), 356-364. Berger, M. M., Macholz, F., Mairbäurl, H., & Bärtsch, P. (2015). Remote ischemic preconditioning for prevention of high-altitude diseases: Fact or fiction? J Appl Physiol (1985), 119(10), 1143-1151. Buck, T. M., Sieck, D. C., & Halliwill, J. R. (2014). Thin-beam ultrasound overestimation of blood flow: How wide is your beam? J Appl Physiol (1985), 116(8), 1096-1104. Casey, D. P., & Joyner, M. J. (2011). Local control of skeletal muscle blood flow during exercise: Influence of available oxygen. J Appl Physiol (1985), 111(6), 1527-1538. Cocking, S., Cable, N. T., Wilson, M. G., Green, D. J., Thijssen, D. H. J., & Jones, H. (2018). Conduit artery diameter during exercise is enhanced after local, but not remote, ischemic preconditioning. Front Physiol, 9, 435. Collie, A., Maruff, P., Darby, D. G., & McStephen, M. (2003). The effects of practice on the cognitive test performance of neurologically normal individuals assessed at brief testretest intervals. J Int Neuropsychol Soc, 9(3), 419-428. Crecelius, A. R., Kirby, B. S., & Dinenno, F. A. (2015). Intravascular ATP and the regulation of blood flow and oxygen delivery in humans. Exerc Sport Sci Rev, 43(1), 5-13.

HEALTH AND SPORT SCIENCE Crecelius, A. R., Kirby, B. S., Hearon, C. M., Luckasen, G. J., Larson, D. G., & Dinenno, F. A. (2015). Contracting human skeletal muscle maintains the ability to blunt Îą1 -adrenergic vasoconstriction during KIR channel and Na(+) /K(+) -ATPase inhibition. J Physiol, 593(12), 2735-2751. Crecelius, A. R., Kirby, B. S., Voyles, W. F., & Dinenno, F. A. (2011). Augmented skeletal muscle hyperaemia during hypoxic exercise in humans is blunted by combined inhibition of nitric oxide and vasodilating prostaglandins. J Physiol, 589(Pt 14), 3671-3683.

Turner, C. E., Barker-Collo, S. L., Connell, C. J., & Gant, N. (2015). Acute hypoxic gas breathing severely impairs cognition and task learning in humans. Physiol Behav, 142, 104-110. Wilkins, B. W., Pike, T. L., Martin, E. A., Curry, T. B., Ceridon, M. L., & Joyner, M. J. (2008). Exercise intensity-dependent contribution of beta-adrenergic receptor-mediated vasodilatation in hypoxic humans. J Physiol, 586(4), 1195-1205. doi:10.1113/jphysiol.2007.144113.

Crecelius, A. R., Luckasen, G. J., Larson, D. G., & Dinenno, F. A. (2014). KIR channel activation contributes to onset and steady-state exercise hyperemia in humans. Am J Physiol Heart Circ Physiol, 307(5), H782-791. Crecelius, A. R., Richards, J. C., Luckasen, G. J., Larson, D. G., & Dinenno, F. A. (2013). Reactive hyperemia occurs via activation of inwardly rectifying potassium channels and Na+/K+-ATPase in humans. Circ Res, 113(8), 1023-1032. Crum, E. M., O'Connor, W. J., Van Loo, L., Valckx, M., & Stannard, S. R. (2017). Validity and reliability of the Moxy oxygen monitor during incremental cycling exercise. Eur J Sport Sci, 17(8), 1037-1043. Cunniffe, B., Sharma, V., Cardinale, M., & Yellon, D. (2017). Characterization of muscle oxygenation response to vascular occlusion: implications for remote ischaemic preconditioning and physical Performance. Clin Physiol Funct Imaging, 37(6), 785-793. Griva, K., Stygall, J., Wilson, M. H., Martin, D., Levett, D., Mitchell, K., et al. (2017). Caudwell Xtreme Everest: A prospective study of the effects of environmental hypoxia on cognitive functioning. PLoS One, 12(3), e0174277. Gualtieri, C. T., & Johnson, L. G. (2006). Reliability and validity of a computerized neurocognitive test battery, CNS Vital Signs. Arch Clin Neuropsychol, 21(7), 623-643. Heistad, D. D., & Abboud, F. M. (1980). Dickinson W. Richards Lecture: Circulatory adjustments to hypoxia. Circulation, 61(3), 463-470. Horiuchi, M., Endo, J., & Thijssen, D. H. (2015). Impact of ischemic preconditioning on functional sympatholysis during handgrip exercise in humans. Physiol Rep, 3(2). Joung, K. W., Rhim, J. H., Chin, J. H., Kim, W. J., Choi, D. K., Lee, E. H., et al. (2013). Effect of remote ischemic preconditioning on cognitive function after off-pump coronary artery bypass graft: a pilot study. Korean J Anesthesiol, 65(5), 418-424. Komiyama, T., Katayama, K., Sudo, M., Ishida, K., Higaki, Y., & Ando, S. (2017). Cognitive function during exercise under severe hypoxia. Sci Rep, 7(1), 10000. McMorris, T., Hale, B. J., Barwood, M., Costello, J., & Corbett, J. (2017). Effect of acute hypoxia on cognition: A systematic review and meta-regression analysis. Neurosci Biobehav Rev, 74(Pt A), 225-232. Rieger, M. G., Hoiland, R. L., Tremblay, J. C., Stembridge, M., Bain, A. R., FlĂźck, D., et al. (2017). One session of remote ischemic preconditioning does not improve vascular function in acute normobaric and chronic hypobaric hypoxia. Exp Physiol, 102(9), 1143-1157. Thijssen, D. H., Maxwell, J., Green, D. J., Cable, N. T., & Jones, H. (2016). Repeated ischaemic preconditioning: A novel therapeutic intervention and potential underlying mechanisms. Exp Physiol, 101(6), 677-692.

Proceedings 2018

HEALTH AND SPORT SCIENCE

Youth Sport Concussion Management: A Review Cordell J. Stover1,2,3 University of Dayton, 300 College Park, Dayton, OH, 45469 1. Department of Health and Sport Science 2. Berry Summer Thesis Institute 3. University Honors Program

Mentor: Corinne M. Daprano, Ph.D. Department of Health and Sport Science Corresponding Author: Corinne M. Daprano, Ph.D. Email: cdaprano1@udayton.edu

Abstract Concussions have become a popular subject recently in the sport world. This review will describe what has been learned through concussion research within the last 50 years. It will entail recent studies and the information that has been discovered concerning youth sport coaches and parent’s knowledge of sportrelated concussions. It will then explain what the knowledge gaps are and potential future studies. The goal is to find what concussion topics youth coaches and parents need more information on and how to most effectively convey that information. This will help to make sports at all levels safer for athletes.

History of Concussions Sport-related concussions have become a growing public health concern over the past decade and affects all levels of sport. A recent study done by the Ohio Department of Health found that “visits for sport-related TBIs… increased by 110 percent from 2002 to 2010” (2016). This increase in the number of sport-related concussions has resulted in an increase of knowledge and education. Concussions in sport have always been present but an understanding of the consequences of these injuries have only recently been discovered. According to Cardenas (2017) the definition of a sport-related concussion was, “a loss of consciousness due to a blow to the head.” However, this definition lacked details regarding the neurological effects of sport-related concussions. Proceedings 2018

Not much was known about the effects of concussions in earlier years, so there was little importance given to monitoring athletes after a blow to the head. For example, one Penn football player described his experience “The only serious injury I received was in the game with Harvard in 1883, when in a scrimmage behind the goal I was knocked insensible, but recovered in about fifteen minutes” (Harrison, 2014). At the turn of the century and up until recently, if a player felt better after being knocked out, they could go back into the game. One possible reason for the increase in documented sport-related concussions is the growing understanding of the signs and symptoms of concussions. Through research and education, new laws and regulations have been put in place. For example, the 5th International Conference of Concussion in Sport Group held in Berlin in 2016 defined sport-related concussions as, “a traumatic brain injury induced by biomechanical forces. Several common features that may be utilized in clinically defining the nature of a concussive head injury include:…” a direct blow to the head, or body with an impulsive force transmitted to the head, short-lived impairment of neurological function, neuropathological changes, or a range of clinical signs and symptoms that may or may not involve loss of consciousness (McCrory, et al., 2017). This is an example of how complicated it is to define all the aspects of a sport-related concussion, as there are several different ways to receive a concussion and then several different neurological responses to each type of concussion. 45

HEALTH AND SPORT SCIENCE Even though much has been discovered in the past decade, more knowledge is still needed to understand the full effects of sport-related concussions, which will allow for better understanding of exact neurological effects.

What We Know As knowledge about sport-related concussions increases, the importance of teaching parents and students about the signs, symptoms and consequences of concussions has also increased. The better educated the public is the safer athletes can be. More specifically this knowledge needs to be taught to parents and coaches of athletes at all levels. The more that parents and coaches are taught the better they will be able to manage their athletes. One study conducted asked coaches in a post Heads-up training survey if they were better able to identify concussion signs and symptoms in their athletes after having gone through the Heads-up material. They found that 77 percent of youth coaches agreed that they were better prepared to identify concussion signs and symptoms after the training (Covassin, Elbin, & Sarmiento, 2012). This shows that it is important to not only train more coaches but also make the training more effective so that all coaches are able to identify concussion signs and symptoms. Another reason why it is very important to train coaches is that many athletes do not tell their coaches they have a concussion or recognize the signs and symptoms of a concussion. For example, one survey asked high school athletes each week throughout their season if they had a concussion. If they did then they asked if the athletes told their coaches. They found that 40 percent of high school players did not tell their coach that they had a concussion (Rivara, Schiff, Chrisman, Chung, Ellenbogen, & Herring, 2014). This reinforces the need for coaches to be able to identify the signs and symptoms of concussions in their players and not to rely on the athletes for this information. Another study used a focus group of high school athletes and asked them if they would leave a game if they experienced concussion symptoms or knew they had a concussion. Many of them said they would not leave. Some of the reasons included ensuring that it was a concussion rather than sickness or dehydration, not wasting all their training to miss the season, being scared 46

of telling their coaches and not letting their team down by leaving a game (Chrisman, Quitiquit, & Rivara, 2013). Players do not always look at the long-term effects that can happen by not taking care of their concussion symptoms, which is why coaches need to be trained to be able to help the athletes understand the importance of leaving the game and being treated so they are ready to return to play. Many coaches understand and can identify most of the signs and symptoms of a concussion but there is a knowledge gap between knowing how to manage an athlete with a concussion and determining when it is safe for that athlete to return to play. A Canadian survey found that coaches had a good recognition of physical and cognitive symptoms of concussions but could not recognize mental health symptoms. Some of the physical symptoms included headaches, dizziness, and blurred vision. The cognitive symptoms were confusion, problems concentrating and problems remembering; mentalhealth symptoms included irritability, sadness or depression and nervousness or anxiety (Topolovec-Vranic, et al., 2015). Studies like this one demonstrate that coaches can typically identify early symptoms, but they cannot tell when a player is safe to return to play. Another survey found that 90 percent of coaches could correctly identify at least 5 of 8 concussion symptoms but less than 50 percent understood the risk of a second concussion. Similarly, less than 25 percent knew that young players typically take longer to recover from a concussion compared to adults (White, et al., 2014). Coaches need to be better educated on long-term effects of concussions for athletes of all ages. Most research on coach and parent knowledge about concussions has been done at the high school and college levels. Very little research has examined concussion knowledge among parents and coaches of youth sport participants. The research that is being done with younger athletes found they have different responses to concussions and they take longer to recover from those concussions. It is important that youth sport coaches are fully educated so they can identify the signs and symptoms in a youth athlete and be able to manage those symptoms. One study that demonstrated this lack of youth coach and parent knowledge regarding concussions used a survey with patients aged 5 to 18 Proceedings 2018

HEALTH AND SPORT SCIENCE who came into the emergency department of a hospital with a concussion. The survey was part oral, during the time of the visit, and then a phone call later. Researchers found that 19 percent of those concussed patients did not immediately leave play, 29 percent continued play on the same day, 42 percent were not managed using proper guidelines and 27 percent were not assessed by qualified personnel (Haran, Bressan, Oakley, Davis, Anderson, & Babl, 2016). This study illustrates the need for more concussion knowledge and education for youth sport coaches and parents. The more that is known about concussions the more people will understand the dangers and how to protect an athlete with a diagnosed concussion from serious long-term damage.

Future Studies Future studies should focus on what youth sport coaches and parents know about the management of sport-related concussions. This will help find a better way to educate youth sport coaches and parents, which will decrease the knowledge gap. Other studies should examine what tools used in concussion trainings coaches found most effective in helping them learn about sportrelated concussions. This will enable researchers to determine the most effective ways to educate coaches and inform parents about what they need to know about concussions. All of this will increase the safety of athletes at all levels.

Proceedings 2018

References Cardenas, J., M.D. (2017, October 26). Concussion in sports past, present and future. Retrieved from https://www.nfhs. org/articles/concussion-in-sports-past-present-and-future/. Chrisman, S. P., Quitiquit, C., & Rivara, F. P. (2013). Qualitative study of barriers to concussive symptom reporting in high school athletics. Journal of Adolescent Health, 52(3), 330-335. Covassin, T., Elbin, R. J., & Sarmiento, K. (2012). Educating coaches about concussion in sports: Evaluation of the CDC’s “Heads Up: Concussion in Youth Sports” Initiative. Journal of School Health, 82(5), 233–238. Haran, H. P., Bressan, S., Oakley, E., Davis, G. A., Anderson, V., & Babl, F. E. (2016). On-field management and return-toplay in sports-related concussion in children: Are children managed appropriately? Journal of Science and Medicine in Sport, 19(3), 194–199. Harrison, E. A. (2014). The first concussion crisis: Head injury and evidence in early American football. American Journal of Public Health, 104(5), 822–833. Macdonald, I. & Hauber, R. (2016). Educating parents on sports-related concussions. Journal of Neuroscience Nursing, 48(6), 297-302. McCrory, P., Meeuwisse, W., Johnston, K., Dvorak, J., Aubry, M., Molloy, M., & Cantu, R. (2009). Consensus statement on concussion in sport—the 3rd international conference on concussion in sport held in Zurich, November 2008. PM&R, 1(5), 406–420. Rivara, F. P., Schiff, M. A., Chrisman, S. P., Chung, S. K., Ellenbogen, R. G., & Herring, S. A. (2014). The effect of coach education on reporting of concussions among high school athletes after passage of a concussion law. The American Journal of Sports Medicine, 42(5), 1197–1203. Topolovec-Vranic, J., Zhang, S., Wong, H., Lam, E., Jing, R., Russell, K., & Cusimano, M. D. (2015). Recognizing the symptoms of mental illness following concussions in the sports community: A need for improvement. PLoS ONE, 10(11). https://doi.org/10.1371/journal.pone.0141699. White, P. E., Newton, J. D., Makdissi, M., Sullivan, S. J., Davis, G., McCrory, P., … Finch, C. F. (2014). Knowledge about sports-related concussion: Is the message getting through to coaches and trainers? British Journal of Sports Medicine, 48(2), 119–124.

LIFE AND PHYSICAL SCIENCES

The Human Gut Microbiome, Microbial Metabolites and the Development of Analytical Techniques of SCFA Analysis Ashton N. Dix1,2,3 University of Dayton, 300 College Park, Dayton, OH, 45469 1. Department of Biology 2. Berry Summer Thesis Institute 3. University Honors Program

Mentors: Yvonne Sun, Ph.D.,Department of Biology Erick Vasquez, Ph.D., Department of Chemical and Materials Engineering Corresponding Author: Yvonne Sun, Ph.D. Email: ysun02@udayton.edu

Abstract The gut microbiota and its impacts on human health has recently become an up and rising area of interest. In the following article, I will review established research on the interplay between the gut microbiota and different health issues, the mechanisms behind how microbial metabolites impact these health issues, the metabolite (SCFA) that my research is focused on, how SCFAs can be analyzed using a gas chromatograph and the foodborne pathogen Listeria monocytogenes. I will also give a brief description of the background and purpose of my projectâ&#x20AC;&#x201D;development of an analytical method for SCFA analysis.

Literature Review The Significance of the Human Gut Microbiome on Human Health The human gut microbiome has a much larger impact on human health than previously known. One example of this involves obesity, which plagues much of our world today. According to the CDC, in 2016 every state in the U.S. had 20 percent or more of its adult population classified as obese. In some states, such as Alabama, the percentage jumped to 35 percent or more.1 Obesity is connected with decreased variety of gut bacteria, which in turn is associated with higher levels of systematic inflammation.2 48

Because this inflammation plays a role in the development of some cancers, it is likely that the gut microbiota have a connection in the affiliation that adiposity has with gastrointestinal cancers.3 The impact that the gut microbiome has on health is further established in the discussion on the gut-brain axis. The gut-brain axis is the signaling pathway between the gastrointestinal tract (gut) and central nervous system. However, despite the recognition and awareness of the gut microbiota as a key factor in human health, whether the relationship is correlative or causal is difficult to establish. Using rodents as experimental hosts, the gut microbiome was shown to play a role in modifying emotional behavior. Bifidobacterium and Lactobacillus have both been shown to exert positive effects on anxious and depression-like behavior.4 In human studies, children with autism spectrum disorder (ASD) have reduced protein digestion, which results in higher amounts of peptides going into circulation and the distal gastrointestinal tract.5 This could cause dysbiosis, which is a lack of harmony between the good and harmful species in the gut.5 Dysbiosis could cause changed immune reactions and elevated risk of diseases.6 It has also been suggested that gastrointestinal issues, which could be linked to dysbiosis, may have a causal relationship with ASD because of the frequency that children with ASD have had these issues.7

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LIFE AND PHYSICAL SCIENCES Alterations in diet, which consequentially alter the gut microbiota, are known to play a role in autoimmune diseases (like multiple sclerosis).8 For example, high consumption of salt could be a factor in autoimmune disease development because of certain pathogenic cell induction.9 The human gut microbiota plays a key role in priming the immune system in multiple ways, such as providing immune-stimulatory signals which may trigger immune responses.8 Not only does the gut microbiota influence the immune system, but the immune system also controls the composition of the microbiota.10

Mechanisms of Microbiome Effect on Human Health Trimethylamine N-oxide (TMAO) is an example of an intestinal metabolite that impacts human cardiovascular health. TMAO is derived from choline and phosphatidylcholine in oneâ&#x20AC;&#x2122;s diet.11 Choline is first metabolized to trimethylamine (TMA) by the gut microbiota, and TMA is in turn converted to TMAO by FMO3, which is an enzyme in the liver.12 Interestingly, higher amounts of TMAO have been associated with higher risk of adverse cardiovascular events13 because this TMA/FMO3/TMAO pathway regulates inflammation and lipid metabolism in many different ways11 that result in the deposit of fatty materials in the artery linings, or atherosclerosis. TMAO promotes expression of scavenger receptors in macrophages to increase their proinflammatory responses. TMAO also significantly alters cholesterol and sterol metabolism by altering bile-acid synthesis and cholesterol transport.14 Moreover, because phosphocholines are enriched in red meat, subjects that had a combined meat-plantbased diet produced more TMAO than those with only a plant-based diet following the same ingestion.14 As a result of this research field, the

gut microbiota has demonstrated a direct role in contributing to cardiovascular disease. Other examples of metabolites that impact health are long-chain fatty acids (LCFAs) and short-chain fatty acids (SCFAs). The fascinating thing about these molecules is a minor difference in chain length can have a large impact on their health consequences in the human body. LCFAs, which are microbial metabolites found in large quantities in the modern Western diet, may be an underlying cause of diseases because they promote differentiation and proliferation of T helper 1 (Th1) and/or T helper 17 (Th17) cells and diminish sequestration of Th1 and Th17 cells in the intestines. SCFAs are metabolites produced by microbial fermentation in the intestines and play multiple roles in benefiting our health.15 They are able to play a role in the human metabolic regulation, immune homeostasis and the performance of the nervous system through the activation of G-protein-coupled receptors (GPCRs).16 SCFAs are the ligands of the GPCRs free fatty-acid receptors 2 and 3 (FFA2 and FFA3). FFA2 has been found to alter the supply of gut immune cells and inflammatory responses that manage immune homeostasis and protect the epithelial wall of the intestine against tumor development and pathogens, and both FFA2 and FFA3 regulate glucose homeostasis.17

What SCFAs Are and How They Are Synthesized SCFAs are fatty acids consisting of only two to six carbons, making them highly difficult to analyze in complex biological samples. They are metabolites produced by microbial fermentation of polysaccharides, starches and endogenous glycans.16 Only specific species of bacteria can carry out this fermentation to produce SCFAs, such as Firmicutes and Bacteroidetes.16 The

Figure 1. Ball-and-stick models of the three most abundant SCFAs produced in the human gut, (from left to right) acetic acid, propionic acid, and butyric acid. Courtesy of molview.org/.

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LIFE AND PHYSICAL SCIENCES most abundant SCFAs produced are acetic acid (C2), propionic acid (C3) and butyric acid (C4), with each usually being produced by a different pathway. The most effective pathway for the production of acetate is the Wood-Ljungdahl pathway. In this pathway, two molecules of carbon dioxide are reduced to form a single molecule of acetate.18 Propionate is usually produced by a carbon dioxide fixation pathway, while butyrate is typically formed by acetyl-S coenzyme A condensation.19

Methods to Measure SCFAs (Sample Prep and GC Instrumentation) Analysis of SCFAs often relies on gas chromatography (GC). However, to detect SCFAs in complex biological samples, sample pretreatment process is required before injection into the GC for cleaner peaks. Three common cleanup treatments include extraction,20 ultrafiltration21,22 and steam distillation.23 Extraction was performed by suspending fecal samples in water and 50 percent sulfuric acid. The sample was then homogenized and centrifuged, creating a supernatant. Using the supernatant and ethyl ether, an extraction was performed by shaking and venting. The suspension was then collected and the chosen internal standard (IS) was added before analysis.20 Ultrafiltration was performed by homogenizing the fecal sample in sulfuric acid and water. The homogenized sample was then centrifuged. The supernatant was filtered through a microconcentrator and then the sample was acidified and distilled using steam distillation. Acidification and steam distillation steps are used to help with the detection of the wanted compounds.21 Other literature discussed the use of an ultrafiltration unit.22 Steam distillation was performed by using a microKjeldahl apparatus.23 Successful experimental conditions of the GC are vital for successful separation and detection of the wanted compounds. Many various methodologies have been reported and share several common steps. For example, an IS, such as 2-ethyl butyric acid,24,25 is used for calibration and to adjust for the loss of analyte during analysis in order to obtain accurate results. In other cases, another 6 carbon chemical is used as the IS.26 A flame ionization detector (FID) is a common detector used for analyses of SCFAs24,25 because of its sensitivity, uniform response and linear range.27 The column used in reported 50

studies vary; some used a fused-silica capillary column,24,26 and others used a high polarity capillary column.25 Some columns had a free fatty acid phase (FFAP)24,25 in order to better analyze SCFAs. The sizes of the columns also varied in internal diameters – 30m x 0.53mm24,26 and 30m x 0.25mm.25 The film coating thickness varied from 0.5µm,24 0.25µm,25 and 1µm.26 Carrier gases are used in GC as the mobile phase, and those used include helium24,26 and nitrogen.25 The flow rates of these carrier gases varied greatly— from 1 mL/min,25 14.4 mL/min,24 to 24 mL/min.26 The detector temperatures were maintained at 240°C24,25 or 300°C26 to vaporize the analytes. The injection port temperatures were 200°C24,26 and 220°C.25 In each study, the sample volume used was 1µL.24–26 Flow rates of hydrogen, air and nitrogen were 20-30mL/min, 300mL/min and 20-30mL/min, respectively.24,25

Listeria Monocytogenes Listeria monocytogenes is a rod-shaped, Gram-positive, facultative anaerobic bacterium28 and a foodborne pathogen that causes listeriosis. Listeriosis cases are generally infrequent in humans, but the mortality rate for those infected is about 20 percent, making it an extremely dangerous disease. Listeria is capable of surviving through food treatment and packaging because it can still propagate in harsh conditions, such as in temperatures of 1.7-45.0°C,29 in a pH range of 4.7-9.230 and hypoxic or anaerobic environments. Listeria, as an enteric pathogen, transits through anaerobic intestinal lumen rich with SCFAs. However, we knew very little about how the bacteria respond to SCFAs or how SCFAs affect Listeria pathogenesis. Better understanding of the relationship between SCFAs and Listeria may identify novel preventative or therapeutic strategies.

My Project Overall Project Goals The main goal of this project is to establish an analytical method for SCFA analysis. Although various methods have been established in literature, including GC, MS, GCMS and HPLC, there are no established methods on the University of Dayton’s campus. Gas chromatography is the most readily available option in Dr. Erick Vasquez’s laboratory and was chosen for this project. Proceedings 2018

LIFE AND PHYSICAL SCIENCES The two main research objectives include (1) Identify the SCFA production by Listeria and (2) Quantify the SCFA production in mouse fecal pellets. More specifically, the project is aimed to identify which SCFAs and at what levels they are produced, and also what environmental factors, such as oxygen availability, may alter SCFA production by Listeria. Quantification of SCFA in fecal pellets will also provide an insight into how animal age or alcohol consumption may alter SCFA productions.

Bibliography

Future Directions The gut microbiome has a large impact on human health, as proven by the vast amount of literature published in recent years. However, there are practically endless avenues that can be taken when it comes to other health implications of gut metabolites such as SCFAs. Not only do we need to be aware of these health implications but also concrete ways to combat them. In terms of listeriosis, further understanding of environmental effects on proliferation is necessary to develop treatments. With my project, I would first like to establish a better understanding of the previous studies that created the foundation of research on gut microbiota, and I will do so by reviewing more literature. I also plan on continuing this project until a reliable SCFA analysis method using a GC has been established. For future experimental goals, the viability of a GCMS in SCFA analysis could be tested.

Acknowledgements I would like to send thanks to Dr. Yvonne Sun for her mentorship and giving me the opportunity to do research in her lab, along with Dr. Vasquez for his co-mentorship and the opportunity to do interdisciplinary research. I would also like to thank the University of Dayton Honors Program and the Berry family for the opportunity to do research over the summer in the Berry Sumer Thesis Institute.

CDC (2017). New adult obesity maps. Centers for Disease Control and Prevention. Available at: https://www.cdc. gov/obesity/data/prevalence-maps.html. (Accessed: 17 August 2018)

Turnbaugh, P. J., et al. (2009). A core gut microbiome in obese and lean twins. Nature 457, 480–484.

Murphy, N., Jenab, M., & Gunter, M. J. (2018)/ Adiposity and gastrointestinal cancers: Epidemiology, mechanisms and future directions. Nat. Rev. Gastroenterol. Hepatol. doi:10.1038/s41575-018-0038-1.

Mayer, E. A., Knight, R., Mazmanian, S. K., Cryan, J. F., & Tillisch, K. (2014). Gut microbes and the brain: Paradigm shift in neuroscience. J. Neurosci. 34, 15490–15496.

Sanctuary, M. R., Kain, J. N., Angkustsiri, K., & German, J. B. (2018). Dietary considerations in autism spectrum disorders: The potential role of protein digestion and microbial putrefaction in the gut-brain axis. Front. Nutr. 5, 40.

Brown, K., DeCoffe, D., Molcan, E., & Gibson, D. L. (2012) Diet-induced dysbiosis of the intestinal microbiota and the effects on immunity and disease. Nutrients 4, 1095–1119.

de Theije, C. G. M., et al. (2011). Pathways underlying the gut-to-brain connection in autism spectrum disorders as future targets for disease management. Eur. J. Pharmacol. 668 Suppl 1, S70-80.

Haase, S., Haghikia, A., Wilck, N., Müller, D. N., & Linker, R. A. (2018). Impacts of microbiome metabolites on immune regulation and autoimmunity. Immunology 154, 230–238.

Kleinewietfeld, M., et al. (2013). Sodium chloride drives autoimmune disease by the induction of pathogenic TH17 cells. Nature 496, 518–522.

10. Zhang, H., Sparks, J. B., Karyala, S. V., Settlage, R., & Luo, X. M. (2015). Host adaptive immunity alters gut microbiota. ISME J. 9, 770–781. 11. Bu, J. & Wang, Z. (2018). Cross-talk between gut microbiota and heart via the routes of metabolite and immunity. Gastroenterol. Res. Pract. 2018, 6458094. 12. Bennett, B. J., et al. (2013). Trimethylamine-N-oxide, a metabolite associated with atherosclerosis, exhibits complex genetic and dietary regulation. Cell Metab. 17, 49–60. 13. Tang, W. H. W., et al. (2013). Intestinal microbial metabolism of phosphatidylcholine and cardiovascular risk. N. Engl. J. Med. 368, 1575–1584. 14. Koeth, R. A., et al. (2013). Intestinal microbiota metabolism of L-carnitine, a nutrient in red meat, promotes atherosclerosis. Nat. Med. 19, 576–585. 15. Haghikia, A., et al. (2015). Dietary fatty acids directly impact central nervous system autoimmunity via the small intestine. Immunity 43, 817–829. 16. Fiori, J., Turroni, S., Candela, M., Brigidi, P., & Gotti, R. (2018). Simultaneous HS-SPME GC-MS determination of shortchain fatty acids, trimethylamine and trimethylamine N-oxide for gut microbiota metabolic profile. Talanta 189, 573–578. 17. Priyadarshini, M., Kotlo, K. U., Dudeja, P. K., & Layden, B. T. (2018). Role of short chain fatty acid receptors in intestinal physiology and pathophysiology. Compr. Physiol. 8, 1091–1115.

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LIFE AND PHYSICAL SCIENCES 18. Fast, A. G. & Papoutsakis, E. T. (2012). Stoichiometric and energetic analyses of non-photosynthetic CO2-fixation pathways to support synthetic biology strategies for production of fuels and chemicals. Curr. Opin. Chem. Eng. 1, 380–395. 19. Miller, T. L. & Wolin, M. J. (1996). Pathways of acetate, propionate, and butyrate formation by the human fecal microbial flora. Appl. Environ. Microbiol. 62, 1589–1592. 20. Tao, J., et al. (2016). Simultaneous determination of six short-chain fatty acids in colonic contents of colitis mice after oral administration of polysaccharides from Chrysanthemum morifolium Ramat by gas chromatography with flame ionization detector. J. Chromatogr. B 1029–1030, 88–94. 21. Chen, H. M. & Lifschitz, C. H. (1989). Preparation of fecal samples for assay of volatile fatty acids by gas-liquid chromatography and high-performance liquid chromatography. Clin. Chem. 35, 74–76. 22. Cummings, K. C. & Jatlow, P. I. (1982). Sample preparation by ultrafiltration for direct gas chromatographic analysis of ethylene glycol in plasma. J. Anal. Toxicol. 6, 324–326. 23. Fleming, S. E. & Rodriguez, M. A. (1983). Influence of dietary fiber on fecal excretion of volatile fatty acids by human adults. J. Nutr. 113, 1613–1625. 24. Zhao, G., Nyman, M., & Jönsson, J. Å. Rapid determination of short-chain fatty acids in colonic contents and faeces of humans and rats by acidified water-extraction and direct-injection gas chromatography. Biomed. Chromatogr. 20, 674–682. 25. Wang, L.-L., et al. (2017). Comprehensive evaluation of SCFA production in the intestinal bacteria regulated by berberine using gas-chromatography combined with polymerase chain reaction. J. Chromatogr. B 1057, 70–80. 26. Schäfer, K. (1995). Analysis of short chain fatty acids from different intestinal samples by capillary gas chromatography. Chromatographia 40, 550–556. 27. Jorgensen, A. D., Picel, K. C., & Stamoudis, V. C. (1990). Prediction of gas chromatography flame ionization detector response factors from molecular structures. Anal. Chem. 62, 683–689. 28. Müller-Herbst, S., et al. (2014). Identification of genes essential for anaerobic growth of Listeria monocytogenes. Microbiology 160, 752–765. 29. Junttila, J. R., Niemelä, S. I., & Hirn, J. (1988). Minimum growth temperatures of Listeria monocytogenes and non-haemolytic Listeria. J. Appl. Bacteriol. 65, 321–327. 30. Petran, R. L. & Zottola, E. A. (1989). A Study of factors affecting growth and recovery of Listeria monocytogenes Scott A J. Food Sci. 54, 458–460.

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Finding the Switches That Activate Animal Genes Through a Combined in Silico and in Vivo Approach Chad M. Jaenke1,2,3 University of Dayton, 300 College Park, Dayton, OH, 45469 1. Department of Biology 2. Berry Summer Thesis Institute 3. University Honors Program

Mentor: Thomas M. Williams, Ph.D. Department of Biology Corresponding Author: Chad M. Jaenke Email: jaenkec1@udayton.edu

Abstract The DNA sequences of genomes encode the recipes for making functional cellular products, notably proteins and switches that regulate when these products are made. While the genetic code for proteins has been known for decades, a similar code for the regulative switches is lacking. This presents a major challenge to understanding the genetic basis of life, as these switches (called cis-regulatory elements or CREs) may outnumber protein-coding genes by 20-50 fold. Both in vivo and in silico approaches exist to study CREs, but the former approaches are generally low throughput and not up to the scale of vast genomes, and the latter lack validation of predictions. We are merging in silico and in vivo approaches to identify the CREs controlling genes responsible for a fruit fly pigmentation trait. Here, we are leveraging the knowledge of six CREs that switch on the transcription of five different genes from a fruit fly tergite pigmentation gene regulatory network (GRN). We are using the SCRMshaw bioinformatic tool to identify novel predicted CREs controlling genes within this GRN based on underlying similarities in the DNA sequences of the known CREs. From this novel list, we will test 24 for CRE activity in in vivo reporter transgene assays. The results from these tests will reveal to what extent the in silico method succeeded. Novel validated CREs will be compared with the known six to reveal what the molecular functions are for the common DNA motifs as the next stage of this research project. The encoding of information Proceedings 2018

in CREs is a universal feature of life, so future research investigating this topic bears upon life at every level, including the betterment of the human condition.

The Challenge of Annotating in the Genome Era At the heart of biological research is the desire to understand the living worldâ&#x20AC;&#x201D;a world where much of the information as to how organisms are made (development), how they came to be (evolution), how they interact with their environment (ecology) and how function goes awry (disease) is encoded as information in their DNA (Carroll, 2008; Sethupathy & Collins, 2008; Visel, Rubin, & Pennacchio, 2009; Abouheif, et al., 2014). Thus, the path was set decades ago to sequence the entire collections of DNA (so-called genomes) for various species, including our own (Lander, et al., 2001), to obtain inventories of genes. Sequencing the first human genome took 13 years and cost 3 billion dollars (National Human Genome Research Institute, 2010). In spite of this triumph, 15 years after completing the human genome project, the genetics community remains largely mystified as to how this inventory is used (Check Hayden, 2010). During this time, the speed of DNA sequencing has been doubling each year while the costs declined. Sequencing a human genome today takes less than a day and costs less than $1,000 (Burke, 2012). Genome sequencing projects 53

X LIFE AND PHYSICAL SCIENCES have been finished for numerous animal species and more continue to emerge at a quickening pace. The major challenge now is to hasten the timeline for revealing how these gene inventories are used. To appreciate why it is so difficult to discover how tens of thousands of genes are used in the lifespan of an animal, one must appreciate the complexity of animals and the architecture of genes. Adults of species like D. melanogaster and humans are comprised of ~100 and ~200 cell types respectively, and these cells emerge from the primordial first cell of each organism that is known as the zygote (“The Cells in Your Body,” n.d.). The resultant cell types and the traits they produce emerge via differences in which genes are being activated (expression) in these cells. Hence, all features of an organism depend upon the regulated expression of genes. For genes, use comes by expression in which DNA sequence information gets transcribed into messenger RNA (mRNA) molecules which subsequently are translated by cellular ribosomes into proteins. Gene expression is controlled by DNA sequences known as cis-regulatory elements (CREs) which in multicellular animals are responsible for making gene expression occur at desired levels (quantities of mRNAs), at particular life times, in particular cell types, in a specific sex and/or in response to certain environmental triggers (Figure 2) (Davidson,

Figure 1. (A) Genes are pleiotropic and thus generally possess multiple CREs that each control an aspect of the overall (B) temporal and (C) spatial expression pattern. Courtesy of C.M. Jaenke and T.M. Williams, 2018.

2006). It is generally appreciated that the specific expression pattern activated by any particular CRE is determined by its DNA sequence of A, C, T and G letters being arranged into multiple small motifs (each 5-12 base pairs, or bp, in length) that each function as a binding site for a type of protein known as a transcription factor (Arnone & Davidson, 1997). Different transcription factors bind to different DNA motifs, and fruit flies and humans possess around 753 and 1,391 transcription factor genes respectively (Adryan & Teichmann, 2006; Vaquerizas, Kummerfeld, Teichmann, & Luscombe, 2009; Pfreundt, et al., 2010). The combination of transcription factors binding to CREs apparently determines how the regulated gene or genes are expressed, a principle known as combinatorial control (Figure 1) (Reményi, Schöler, & Wilmanns, 2004). Most, but not all, genes encode the information for a string of amino acids, referred to as a protein, which will perform some sort of molecular function. Protein information is encoded in DNA sequence by a set of universal rules known as the genetic code (Griffiths, Wessler, Carroll, & Doebley, 2011; Koonin & Novozhilov, 2009). These rules can be used by computer programs to scan genome sequence to find stretches of DNA that are parts of protein-encoding genes. Proteincoding genes can also be identified in newly sequenced genomes by computer programs searching for similar sequences to genes known to exist in completed genomes (Zhang, 2002).

Figure 2. CREs as transcription factor binding sites for combinatorial control. (A) The fundamental unit of information in CREs are binding sites for a transcription factor protein. (B) Specific CRE activities are due to collaboration between two or more transcription factors. Courtesy of C.M. Jaenke and T.M. Williams 2018.

While binding sites appear to be a central feature of CREs, the preferred binding motif for many transcription factors remains uncharacterized (Mathelier, et al., 2014), and little is known about which of the enormous array of transcription factor binding site combinations actually Proceedings 2018

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Figure 3. The understanding and challenges for protein-coding and CRE sequences. CREs activate gene transcription (arrow), though how the particular activity is encoded in CREs remains elusive, making these sequences more difficult to identify. Protein-coding sequences encode the identity of amino acids that, following transcription, will be inserted into proteins during translation by the ribosomes (double oval). Courtesy of C.M. Jaenke and T.M. Williams, 2018.

function in a combinatorial manner. Put plainly, we lack a CRE code comparable to the genetic code for protein-coding sequences (Figure 3).

The Importance of Understanding CREs

CREs are a general feature of all living organisms, and for humans, CREs are of central importance to understanding and improving the human condition. Genetic studies have implicated CREs as the primary location where genetic differences are found that influence phenotypic diversity, disease and disease risk (Figure 4) (Visel, Akiyama, et al., 2009; Musunuru, et al., 2010; Claussnitzer, et al., 2015). The inability of transcription factors to bind to their respective sites by CRE mutations has been linked to an increased risk for diabetes mellites, cardiovascular disease, developmental disorders, and cancer (Horn, et al., 2013; Lee & Young, 2013). In spite of this importance, the genetic community continues to struggle to understand how information is encoded in these sequences and how certain genetic differences in Figure 4. CREs control gene expression patterns, and mutations in CREs are: (A) suspected to CREs alter their function. be a driver of morphological variation between diverse animals, (B) the cause of human lactose tolerance/intolerance, (C) a genetic cause of obesity and (D) responsible for some cases of polydactyly, amongst other variations. Courtesy of C.M. Jaenke and T.M. Williams, 2018.

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Figure 5. Reporter transgene assays: the “gold standard” approach for validating putative CREs. (A) yellow gene with predicted CRE upstream of the transcription start site which controls (B) yellow expression in the A5 and A6 segments in D. melanogaster. (C) placement of Green Fluorescent Protein (GFP) adjacent to a promoter and a CRE, leading to (D) observable expression in the A5 and A6 segments. Courtesy of C.M. Jaenke and T.M. Williams, 2018.

Current Approaches to Identifying CREs For non-human species, in vivo approaches are available to test DNA sequences for CRE activity using what are known as reporter transgenes (Rebeiz & Williams, 2011). Here, a piece of DNA, typically 500-1,000 bp, to be tested for CRE activity is placed next to a DNA sequence for a gene that makes an easy to visualize product when expressed, such as Green Fluorescent Protein (GFP). The CRE activity can then be observed based on the pattern of GFP expression (Figure 5). However, even for a relatively cheap to work with species like D. melanogaster, it is prohibitively expensive to screen all DNA sequences in a genome for CRE activity. Even if such a screen could be afforded, one would need to spend an unrealistic amount of time looking for GFP expression in all cell types, life time points and in response to a wealth of environmental perturbations to reveal what, if any, type of CRE activity each sequence encodes. Thus, in vivo and in vitro approaches in isolation offer little hope to find and understand the biology of CREs. Recently, a computational approach known as “supervised Cis-Regulatory Module prediction” (or “SCRMshaw”) was devised that can evaluate a small set of unique CREs that are known a priori to drive a similar pattern of gene expression in vivo (Figure 6) (Kantorovitz, et al., 2009). These 56

CRE sequences are used as “training data” for an algorithm to build a statistical model of short DNA motif count and distribution common in the training data. The model is then used to score a target genome(s) in overlapping 500 bp windows to find 500 bp sequences that are predicted to be CREs with functions similar to those of the training set. A key aspect of this SCRMshaw is that it is not a prerequisite to know what interacts with the common motifs. A few studies validated several of the SCRMshawidentified sequences for CRE activity in reporter transgene assays (Kazemian, Zhu, Halfon, & Sinha, 2011; Suryamohan & Halfon, 2015). However, additional test cases are needed in order to support the general suitability of this approach to find CREs or whether optimizations are needed. Moreover, it is important to then progress from finding CREs with a similar function to determining the combination of transcription factor-binding motifs to drive the pattern of gene expression.

Fruit Fly Pigmentation as a Model for CRE discovery Morphological traits are constructed during development by the collective actions of scores to hundreds of genes, many whose identities are not known. The collaborations of binding

Figure 6. SCRMshaw’s machine-learning approach used to evaluate statistics of short word counts of known CREs versus a background control set of sequences. Based on Suryamohan and Halfon, 2015. Courtesy of C.M. Jaenke and T.M. Williams, 2018.

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Figure 7. The abdomens of male fruit flies evolved diverse patterns of pigmentation. Green background indicates the Sophophora subgenus. In Sophophora ancestry, male-specific tergite pigmentation was initially (1) gained, and this pattern (4 and 5) expanded, (2 and 3) contracted and was (6 and 7) repeatedly lost. Courtesy of C.M. Jaenke and T.M. Williams, 2018.

sites for most CREs remain unknown. Thus, a major challenge for evolutionary developmental biology, or evo-devo, research is to find the genes and their regulative CREs that comprise GRNs in order to facilitate investigations into how trait diversity evolved. The lab of Dr. Thomas Williams (UD Biology) has established the diversity of abdominal pigmentation patterns on fruit fly abdomens as a model for elucidating how a trait develops and evolves (Figure 7) (Rebeiz & Williams, 2017). They have identified six CREs that each activate a different gene involved in making the D. melanogaster

pigmentation pattern (Williams, et al., 2008; Rebeiz, Pool, Kassner, Aquadro, & Carroll, 2009; Camino, et al., 2015). These six CREs activate gene expression in the dorsal abdominal epidermis of D. melanogaster, though they differ in the time point of their activity onset and which sex or sexes they activate expression in (Figure 8). A major impediment to the progression of this model is finding the other genes and CREs that comprise the full GRN at a time when we still do not understand the transcription factor binding site encodings of the first six CREs.

A Thesis Involving an in vivo and in silico Approach to CRE Identification

Figure 8. Twenty years of research has revealed a rudimentary GRN for D. melanogaster tergite pigmentation, which includes pigment metabolism genes and several expression-regulating transcription factor genes. Six CREs were identified that can drive patterned expression of the Green Fluorescent Protein (GFP) reporter gene. Courtesy of C.M. Jaenke and T.M. Williams 2018.

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In collaboration with Dr. Marc Halfon (University at Buffalo-SUNY), we will use an initial training set of CREs to generate and empirically validate a set of predicted abdominal epidermis CREs within the D. melanogaster genome. Using the SCRMshaw approach, our research will involve validating 24 predicted abdominal epidermis CREs through the use of GFP reporter transgenes. These transgenes will be integrated into the germline of D. melanogaster fruit flies using standard transgenesis methods (Groth, Fish, Nusse, & Calos, 2004). We will then assay whether GFP expression occurs in the 57

X LIFE AND PHYSICAL SCIENCES abdomen epidermis which will validate or refute the prediction that these DNA sequences are abdominal epidermis CREs. Additional thesis research would involve refining the SCRMshaw technique by using identified abdominal epidermis CREs from the 24 tested and making them a part of an expanded test data set. The identification of just a single novel abdominal epidermis CRE would provide the Williams lab with opportunities to expand their genetic model for trait development and evolution to include an additional gene. In addition, we hypothesize that this test set of CREs and those that I identify from the BSTI research share a combinatorial code of transcription factor binding sites. Future research will investigate these CREs for similar motifs in order to implicate which of the 753 D. melanogaster transcription factors bind to these motifs (Pfreundt, et al., 2010). This hypothesis can be approached by combining in silico searches for which a transcription factor binds to such motifs with RNA-interference screens where we turn down the expression of a transcription factor and observe whether CRE activities are abnormal in reporter transgene assays (Mathelier, et al., 2014; Rogers, et al., 2014). The Williams lab also has information as to which transcription factors bound two of the test data CREs from an in vitro yeast one-hybrid assay (Deplancke, Dupuy, Vidal, & Walhout, 2004). These factors would be top candidates to investigate by RNA-interference.

Acknowledgements The authors would like to thank the Berry Family and the Berry Summer Thesis Institute. Thanks are owed to Dr. Marc Halfon as a collaborator for this project and for the development of the SCRMshaw approach. Additional thanks are owed to the members of the Williams lab for their support of this thesis research. C.M. Jaenke was supported by the University of Dayton Honors Program and the Berry Summer Thesis Institute.

Bibliography Abouheif, E., Fave, M.J., Ibarraran-Viniegra, A. S., Lesoway, M. P., Rafiqi, A. M., & Rajakumar, R. (2014). Eco-evo-devo: The time has come. In C. R. Landry and N. Aubin-Horth (Eds.), Advances in Experimental Medicine and Biology (pp. 107–126). Springer. Adryan, B. & Teichmann, S. A. (2006). FlyTF: a systematic review of site-specific transcription factors in the fruit fly Drosophila melanogaster. Bioinformatics (Oxford, England), 22(12), 1532–3. https://doi.org/10.1093/bioinformatics/btl143. American Association for the Advancement of Science. The cells in your body. (n.d.). Science Net Links. Retrieved from http://sciencenetlinks.com/student-teacher-sheets/cellsyour-body/. Arnone, M. I. & Davidson, E. H. (1997). The hardwiring of development: Organization and function of genomic regulatory systems. Development (Cambridge, England), 124(10), 1851–64. Burke, A. (2012, January 12). DNA Sequencing Is Now Improving Faster Than Moore’s Law! Forbes. Retrieved from https://www.forbes.com/sites/techonomy/2012/01/12/ dna-sequencing-is-now-improving-faster-than-mooreslaw/#12d2537b5e4f. Camino, E. M., Butts, J. C., Ordway, A., Vellky, J. E., Rebeiz, M., & Williams, T. M. (2015). The evolutionary origination and diversification of a dimorphic gene regulatory network through parallel innovations in cis and trans. PLOS Genetics, 11(4), e1005136. https://doi.org/10.1371/journal.pgen.1005136. Carroll, S. B. (2008). Evo-devo and an expanding evolutionary synthesis: A genetic theory of morphological evolution. Cell, 134(1), 25–36. https://doi.org/10.1016/j.cell.2008.06.030. Check Hayden, E. (2010). Life is complicated. Nature, 464. https://doi.org/10.1038/464664a. Claussnitzer, M., Dankel, S. N., Kim, K.-H., Quon, G., Meuleman, W., Haugen, C., … Kellis, M. (2015). FTO obesity variant circuitry and adipocyte browning in humans. New England Journal of Medicine, 150819140043007. https://doi. org/10.1056/NEJMoa1502214. Davidson, E. H. (2006). The Regulatory Genome: Gene Regulatory Networks in Development and Evolution. Amsterdam: Elsevier Academic Press. Deplancke, B., Dupuy, D., Vidal, M., & Walhout, A. J. M. (2004). A Gateway-compatible yeast one-hybrid system. Genome Research, 14(10b), 2093–2101. http://doi.org/10.1101/ gr.2445504. Griffiths, A., Wessler, S., Carroll, S. B., & Doebley, J. (2011). Introduction to Genetic Analysis (10th ed.). New York, NY: W. H. Freeman and Company. Groth, A. C., Fish, M., Nusse, R., & Calos, M. P. (2004). Construction of Transgenic Drosophila by using the sitespecific integrase from phage phiC31. Genetics, 166(April), 1775–1782. Horn, S., Figl, A., Rachakonda, P. S., Fischer, C., Sucker, A., Gast, A., … Kumar, R. (2013). TERT promoter mutations in familial and sporadic melanoma. Science, 339(6122), 959–961. https://doi.org/10.1126/science.1230062. Kantorovitz, M. R., Kazemian, M., Kinston, S., MirandaSaavedra, D., Zhu, Q., Robinson, G. E., … Sinha, S. (2009). Motif-blind, genome-wide discovery of cis-regulatory nodules in Drosophila and mouse. Developmental Cell, 17(4), 568–579. https://doi.org/10.1016/j.devcel.2009.09.002.

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LIFE AND PHYSICAL SCIENCES X Kazemian, M., Zhu, Q., Halfon, M. S., & Sinha, S. (2011). Improved accuracy of supervised CRM discovery with interpolated Markov models and cross-species comparison. Nucleic Acids Research, 39(22), 9463–9472. https://doi. org/10.1093/nar/gkr621.

Suryamohan, K. & Halfon, M. S. (2015). Identifying transcriptional cis-regulatory modules in animal genomes: Identifying transcriptional cis-regulatory modules. Wiley Interdisciplinary Reviews: Developmental Biology, 4(2), 59–84. https://doi.org/10.1002/wdev.168.

Koonin, E. and Novozhilov, A. (2009). Origin and evolution of the genetic code: the universal enigma. International Union of Biochemistry and Molecular Biology Life, 61, 99–111.

Vaquerizas, Juan M., Kummerfeld, Sarah K., Teichmann, Sarah A., & Luscombe, N. M. (2009). A census of human transcription factors: Function, expression and evolution. Nature Reviews Genetics, 10, 252–263.

Lander, E. S., Heaford, A., Sheridan, A., Linton, L. M., Birren, B., Subramanian, A., … Szustakowki, J. (2001). Initial sequencing and analysis of the human genome. Nature, 409(6822), 860–921. Lee, T. I. & Young, R. A. (2013). Transcriptional regulation and its misregulation in disease. Cell, 152(6), 1237–1251. https://doi.org/10.1016/j.cell.2013.02.014. Mathelier, A., Zhao, X., Zhang, A. W., Parcy, F., Worsley-Hunt, R., Arenillas, D. J., … Wasserman, W. W. (2014). JASPAR 2014: An extensively expanded and updated open-access database of transcription factor binding profiles. Nucleic Acids Research, 42(Database issue), D142–7. https://doi. org/10.1093/nar/gkt997. Musunuru, K., Strong, A., Frank-Kamenetsky, M., Lee, N. E., Ahfeldt, T., Sachs, K. V., … Rader, D. J. (2010). From noncoding variant to phenotype via SORT1 at the 1p13 cholesterol locus. Nature, 466(7307), 714–9. https://doi.org/10.1038/nature09266.

Visel, A., Akiyama, J. A., Shoukry, M., Afzal, V., Rubin, E. M., & Pennacchio, L. A. (2009). Functional autonomy of distantacting human enhancers. Genomics, 93(6), 509–13. https://doi. org/10.1016/j.ygeno.2009.02.002. Visel, A., Rubin, E. M., & Pennacchio, L. A. (2009). Genomic views of distant-acting enhancers. Nature, 461(7261), 199–205. https://doi.org/10.1038/nature08451. Williams, T. M., Selegue, J. E., Werner, T., Gompel, N., Kopp, A., & Carroll, S. B. (2008). The regulation and evolution of a genetic switch controlling sexually dimorphic traits in Drosophila. Cell, 134(4), 610–23. https://doi.org/10.1016/j. cell.2008.06.052. Zhang, M. Q. (2002). Computational prediction of eukaryotic protein-coding genes. Nature Reviews Genetics, 3(9), 698–709. https://doi.org/10.1038/nrg890.

National Human Genome Research Institute (2010). The human genome project completion: Frequently asked questions. Retrieved from https://www.genome.gov/11006943/ human-genome-project-completion-frequently-askedquestions/. Pfreundt, U., James, D. P., Tweedie, S., Wilson, D., Teichmann, S. A., & Adryan, B. (2010). FlyTF: improved annotation and enhanced functionality of the Drosophila transcription factor database. Nucleic Acids Research, 38(Database issue), D443–7. https://doi.org/10.1093/nar/gkp910. Rebeiz, M., Pool, J. E., Kassner, V. A., Aquadro, C. F., & Carroll, S. B. (2009). Stepwise modification of a modular enhancer underlies adaptation in a Drosophila population. Science (New York, N.Y.), 326(5960), 1663–7. https://doi.org/10.1126/ science.1178357. Rebeiz, M. and Williams, T. M. (2011). Experimental Approaches to evaluate the contributions of candidate cis-regulatory mutations to phenotypic evolution. In V. Orgogozo and M. V. Rockman (Eds.), Methods in Molecular Biology (Vol. 772, pp. 351–375). Totowa: Humana Press. https://doi.org/10.1007/978-1-61779-228-1. Rebeiz, M. & Williams, T. M. (2017). Using Drosophila pigmentation traits to study the mechanisms of cis-regulatory evolution. Current Opinion in Insect Science, 19, 1–7. https:// doi.org/10.1016/j.cois.2016.10.002. Reményi, A., Schöler, H. R., & Wilmanns, M. (2004). Combinatorial control of gene expression. Nature Structural and Molecular Biology, 11(9), 812–815. https://doi.org/10.1038/ nsmb820. Rogers, W. A., Grover, S., Stringer, S. J., Parks, J., Rebeiz, M., & Williams, T. M. (2014). A survey of the trans-regulatory landscape for Drosophila melanogaster abdominal pigmentation. Developmental Biology, 385(2), 417–432. https://doi. org/10.1016/j.ydbio.2013.11.013. Sethupathy, P. & Collins, F. S. (2008). MicroRNA target site polymorphisms and human disease. Trends in Genetics : TIG, 24(10), 489–97. https://doi.org/10.1016/j.tig.2008.07.004.

Proceedings 2018

LIFE AND PHYSICAL SCIENCES

Effect of Bacterial Membrane Composition on Antibiotic Susceptibility Samantha L. Neanover1,2,3,4 University of Dayton, 300 College Park, Dayton, OH, 45469 1. Department of Biology 2. The Anaerobic Microbiology Lab 3. Berry Summer Thesis Institute 4. University Honors Program

Mentor: Yvonne Sun, Ph.D. Department of Biology Corresponding Author: Yvonne Sun, Ph.D. Email: ysun02@udayton.edu

Introduction

Bacterial Membrane

Antibiotic resistance has become a global crisis as once easily treated bacterial infections are now deadly. Current studies estimate that by 2050, 10,000,000 lives will be lost, exceeding the 700,000 lives that are currently lost to antibiotic resistant infections.1 The crisis has been perpetuated by the over-prescription and misuse of antibiotics in health care and agriculture. Moreover, current methods to combat antibiotic resistance have not been adequate to keep up with the rapid and continuous evolution of bacteria to become resistant. In order to prevent this cycle of resistance, mechanisms behind resistance in bacteria must be better understood. Crossing the bacterial membrane is a major requirement for most antibiotics to work. As a result, bacteria often develop membrane-based resistance mechanisms. One way to understand the efficacy of these mechanisms is to study whether alterations in the membrane composition can affect antibiotic susceptibility.2 Exposing bacteria to short-chain fatty acids can potentially alter the bacterial membrane, but little is known on how this affects antibiotic susceptibility. If alterations of the membrane fatty acid composition affect the passage of antibiotics across the bacterial membrane, then bacteria with shortchain fatty acids may exhibit different levels of antibiotic resistance. Better understanding in the role of membrane composition in bacterial antibiotic resistance will contribute to solving the impending crisis.

Composition

Bacteria contain both cell walls and membranes that determine morphology and transportation of molecules. The cell wall is mainly composed of peptidoglycan to maintain its structure but varies in thickness between Gram-positive and negative bacteria. Gram-positive bacteria contain a thicker peptidoglycan layer that absorbs dye well to stain the cell purple in images. On the other hand, Gram-negative bacteria contain a much thinner peptidoglycan layer that does not absorb the dye well. Therefore, Gram-negative bacteria are characteristically pink in color after the standard Gram staining procedure. The cell membrane is composed of a phospholipid bilayer and proteins. The phospholipid bilayer is amphipathic by having both hydrophilic and hydrophobic characteristics. The phosphate groups that face outward are hydrophilic, but the fatty acids inside the bilayer are hydrophobic (Figure 1). The fatty acids within the bilayer differ in length, anywhere from 14 to 24 carbons, and affect the fluidity of the membrane due to the level of saturation.3

Short-chain Fatty Acids While the fatty acids in the bacterial membrane reach 18 carbons in length, short-chain fatty acids do not exceed 6. They vary from 1 to 6 carbons in length and contain a carboxylic acid functional group. Short-chain fatty acids Proceedings 2018

LIFE AND PHYSICAL SCIENCES between a high fiber diet and a reduced risk of cancer. After studies adjusted for age, dietary intake and other risk factors, there was not a significant difference between groups with and without a high-fiber diet.7

Figure 1. Phospholipid bilayer from cell membrane containing fatty acids. Courtesy of OpenStax Anatomy and Physiology. May 18, 2016.

are produced in the human digestive system by microbial fermentation. This produces three main short-chain fatty acids: butyrate, propionate and acetate. They contain 2, 3 and 4 carbons, respectively (Figure 2). These acids account for 90 to 95 percent of a short-chain fatty acids found in the gut and have the most influential effects on maintaining the digestive environment by influencing the physiology of the colon, directing host-signaling mechanisms, and influencing health outcomes.4 Specifically, the role of butyrate has been investigated for its ability to prevent tumor growth and destroy malignant cells in the colon.5 High-fiber diets have also been associated with a reduced risk of colon cancer. When mice were fed a highfiber diet, there were 75 percent fewer tumors in guts that produced butyrate. However, only the presence of both microbiota and butyrate in the digestive system were correlated with tumor suppression. The presence of just either microbiota or butyrate were not enough to have tumor suppression effects.6 Yet, this correlation has not been supported among similar studies. Others have found that there is no correlation

Butyrate, propionate and acetate are known for their anti-inflammatory properties to relieve inflammation in the digestive system caused by disorders such as, diarrhea, Chronâ&#x20AC;&#x2122;s disease and ulcerative colitis. Patients who take short-chain fatty acid supplements such as butyrate and acetate saw improvement in the management of these disorders. Also, patients with lower levels of these acids reported worsened ulcerative colitis.8 Overall, short-chain fatty acids are unique molecules for their multiple functions in the human body.

Bacterial Fitness Antibiotic Resistant Genes Bacterial fitness is a measurement of bacterial growth and survival that is used to define how well bacteria adapt to an environment under pressure. Therefore, it is heavily influenced by environmental factors such as exposure to antibiotics. Prolonged and frequent exposure to antibiotics has led to the development and dissemination of antibiotic resistance in the environment that improve the fitness of bacteria. Antibiotic resistance has become a growing concern as bacterial infections are more challenging to treat. Resistance to antibiotics has increased by over prescriptions in medical settings, inappropriate use in agriculture and irresponsible disposal of these drugs. Before antibiotics were fully understood for their specificity, they were prescribed to treat a variety of infections and diseases, both viral and bacterial. However, antibiotics are only effective against bacterial infections and play no role in relieving

Figure 2. Structure of three common short chain fatty acids. Courtesy of Ben Mills. July 29, 2007.

Proceedings 2018

LIFE AND PHYSICAL SCIENCES viral infections. For example, approximately 30 percent of antibiotics prescribed in outpatient settings are unnecessary.9 In agricultural settings, antibiotics are commonly deposited into animal feed to promote growth and meat production. According to the Food and Drug Administration, roughly 70 percent of antibiotics produced are sold for use in animals. These antibiotics enter the environment through manure and are left to decompose in soil and waterways. In addition, consumed meat products cause second hand exposure to these antibiotics. Finally, the method of disposal after a course of antibiotic treatment is inconsistent. When a patient does not follow medical recommendations, the remaining antibiotics enter the environment after being flushed or thrown away. All these activities create a selective pressure for antibiotic resistant bacteria that can outcompete those susceptible to antibiotics. Antibiotic resistance can be conferred by resistant genes that occur naturally through random variation within the bacterial genome by mutation or horizontal gene transfer. Overall, bacteria populations tend to have low mutations rates to avoid lethal effects. Yet research suggests that there are hypermutant populations of bacteria that are more prone to mutating than normal populations.10 This tendency to mutate suggests a damaged DNA repair mechanism often induced by stressful environments like infections, bacteriophages or antibiotic exposure. Therefore, bacteria exposed to more stressful environments have higher rates of mutation to improve bacterial fitness.

Environmental Factor: Presence of Oxygen Although the mechanisms of actions for the major classes of antibiotics have been elucidated, recent studies have revealed a controversial concept that the reactive oxygen species (ROS) generated during antibiotic exposure, in addition to the inhibition of biochemical pathways, contribute to the antibiotic effectiveness. The presence of oxygen in cell growth has been studied for antibiotic-induced apoptosis. There is currently some uncertainty in the role between apoptosis and reactive oxygen species, but research has produced data both supporting and refuting the theory. Apoptosis, or programmed cell death, has been correlated to the production of reactive oxygen species by bactericidal 62

antibiotics. These antibiotics stimulate the production of ROS through Fenton reaction. The Fenton reaction produces these reactive oxygen radicals by reducing hydrogen peroxide to hydroxyl.11 The production of these radicals, measured by hydroxyphenyl fluorescein (HPF), an ROS marker, was positively correlated with an increase of apoptosis with the use of bactericidal antibiotics. Contrary to the previous study, there is also evidence that ROS do not cause apoptosis with the use of bactericidal antibiotics. When the experiment described above was ran in anaerobic conditions, no such correlation was observed. The anaerobic conditions prevented the production of ROS without the presence of oxygen in the system. It was found that the apoptosis rate for anaerobic and aerobic conditions were not significantly different, suggesting that ROS do not play a role in increasing apoptosis. Additionally, HPF markers for ROS were also found in anaerobic conditions. This suggests that HPF may not only be measuring ROS, it could also measure cell death.12 Another study also refuted the correlation between ROS and apoptosis by increasing the concentration of antibiotics. When the concentration increased, the ROS production decreased, unlike what was expected.13 Increasing the concentration of antibiotics then leads to greater apoptosis, so the ROS production should have increased as well if this relationship was valid. If ROS do increase apoptosis, then bacterial fitness should decrease in the presence of oxygen. However, there is equal evidence that this relationship has no effect on bacterial fitness, so this topic must be further studied.

Antibiotic Susceptibility in Model Organism Model Organism: Listeria monocytogenes Listeria monocytogenes is a Gram-positive bacilli and foodborne pathogen responsible for causing a bacterial infection known as listeriosis. Listeriosis commonly afflicts immunocompromised individuals, such as the elderly, infants and pregnant women. These susceptible populations account for approximately 1,600 infections in the United States per year, with 20 percent resulting in death despite treatment with antibiotics.1 Listeria is tolerant to many Proceedings 2018

LIFE AND PHYSICAL SCIENCES antibiotics, including ampicillin and penicillin, but still exhibits susceptibility in most conditions. It is a model organism because it can survive extreme environmental conditions such as a large pH range, high salt concentrations, and refrigeration temperatures. Genetically altering Listeria has shown to affect antibiotic susceptibility. When a gene known as lmo1941 is removed, the resulting mutant becomes more susceptible to cephalosporins, an antibiotic that does not usually have activity against Listeria. Yet this did not lower the susceptibility to beta-lactams such as ampicillin and penicillin. Further investigation into the bacterial membrane proved that the deletion of lmo1941 altered peptidoglycan and the cell wall structure.14 This genetic deletion allowed an inactive antibiotic against Listeria to show activity, suggesting that alterations such as these could be used to combat resistance and tolerance to antibiotics. Susceptibility to cephalosporins would be a positive advancement in the treatment of listeriosis, due to its high affinity for cell membrane passage and bactericidal effects. Many antibiotics used now only cause bacteriostatic effects against Listeria. Tolerance and resistance has also been revealed by exposing Listeria to low concentrations of antibiotics over time. When Listeria was exposed to sublethal concentrations of antibiotics, there was a shift from aerobic to anaerobic mechanisms to prevent what is believed to be the production of reactive oxygen species. This prevention is suspected to be an attempt to prevent apoptosis. In addition to this shift, phenotypes linked to antibiotic tolerance were also observed.15 These conclusions further show that Listeria is an adaptive bacterium, with the ability to introduce metabolic and physiological changes for survival.

Figure 3. Beta-lactam ring in ampicillin. Courtesy of Wikimedia Commons. 2006.

beta-lactam resistance by preventing binding to penicillin-binding proteins, reducing access to the binding proteins, or producing beta-lactamase.16 Ampicillin is commonly used to treat enteric fever after Salmonella Typhi gained resistance to chloramphenicol, the first line of antibiotics used to treat the infection. Ampicillin was used in combination with co-trimoxazole to provide effective treatment to both carriers and hosts of the disease among all age groups.17 As ampicillin became widely used across the globe, multidrug resistant strains of Salmonella surfaced, causing ampicillin to lose its efficacy. Once the use of ampicillin declined, the Salmonella resistance against the antibiotic declined as well. Currently, there is speculation that the selective pressure against ampicillin was relieved when bacteria were no longer exposed. As ampicillin resistance is plasmid mediated, Salmonella may have lost this mechanism and reestablished sensitivity to ampicillin.16 This study shows that antibiotic susceptibility is routinely changing to match environmental pressures.

Conclusion Ampicillin Ampicillin, like penicillin, is commonly prescribed to treat listeriosis in patients. It is a broad-spectrum, beta-lactam antibiotic that typically causes bactericidal activity by inactivating penicillin-binding proteins in the inner membrane of bacteria. The beta-lactam ring is defined as a four-membered lactam, containing a nitrogen atom adjacent to a Î˛-carbon (Figure 3). Ampicillin was derived from penicillin after Staphylococcus aureus became resistant by producing penicillinase, a penicillin-hydrolyzing enzyme. Also, some bacteria have acquired Proceedings 2018

As the bacterial membrane properties are highly dependent on its fatty acid composition, alterations in composition can further be explored to address concerns over antibiotic resistance. The addition of short-chain fatty acids to induce membrane alterations provide an opportunity to test antibiotic susceptibility in both anaerobic and aerobic settings to explore the role of ROS. How additional factors, such as antibiotic exposure length, frequency and concentration can affect bacterial response and overall antibiotic susceptibility will help us understand the development of antibiotic resistance. By addressing these 63

LIFE AND PHYSICAL SCIENCES fundamental physiological questions, we will better understand and help curtail antibiotic resistance.

Acknowledgements I would like to thank the Berry Summer Thesis Institute and the Honors Program for the opportunity to conduct research by providing the necessary tools and accommodations. I would also like to thank my mentor, Dr. Yvonne Sun, for her support and guidance through the research process. Finally, I am thankful for my colleagues in the anaerobic microbiology lab for their helpful insight into my project.

Works Cited 1.

Jansen, K. U., Knirsch, C., & Anderson, A. S. (2018). The role of vaccines in preventing bacterial antimicrobial resistance. Nature Medicine, 24, 10-19.

Rinehart, E., Newton, E., Marasco, M. A., Beemiller, K., Zani, A., Muratore, M. K., … Sun, Y. (2018). Listeria monocytogenes response to propionate is differentially modulated by anaerobicity. Pathogens (Basel, Switzerland), 7(3).

Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., & Walter, P. (2002). The lipid bilayer. Molecular Biology of the Cell. 4th Edition.

Ríos-Covián, D., Ruas-Madiedo, P., Margolles, A., Gueimonde, M., Reyes-Gavilán, D. L., G, C., & Salazar, N. (2016). Intestinal short chain fatty acids and their link with diet and human health. Frontiers in Microbiology, 7.

Tang, Y., Chen, Y., Jiang, H., Robbins, G. T., & Nie, D. (2011). G-protein-coupled receptor for short-chain fatty acids suppresses colon cancer. International Journal of Cancer, 128(4).

Donohoe, D. R., Holley, D., Collins, L. B., Montgomery, S. A., Whitmore, A. C., Hillhouse, A., … Bultman, S. J. (2014). A gnotobiotic mouse model demonstrates that dietary fiber protects against colorectal tumorigenesis in a microbiota- and butyrate–dependent manner. Cancer Discovery, 4(12), 1387–1397.

Fuchs, C. S., Giovannucci, E. L., Colditz, G. A., Hunter, D. J., Stampfer, M. J., Rosner, B., … Willett, W. C. (1999). Dietary fiber and the risk of colorectal cancer and adenoma in women. The New England Journal of Medicine, 340(3), 169–176.

Vieira, E. L. M., Leonel, A. J., Sad, A. P., Beltrão, N. R. M., Costa, T. F., Ferreira, T. M. R., … AlvarezLeite, J. I. (2012). Oral administration of sodium butyrate attenuates inflammation and mucosal lesion in experimental acute ulcerative colitis. The Journal of Nutritional Biochemistry, 23(5), 430–436.

Fleming-Dutra, K. E., Hersh, A. L., Shapiro, D. J., Bartoces, M., Enns, E. A., File, T. M., … Hicks, L. A. (2016). Prevalence of inappropriate antibiotic prescriptions among U.S. ambulatory care visits, 2010-2011. JAMA, 315(17), 1864–1873.

10. Martinez, J. L., Fajardo, A., Garmendia, L., Hernandez, A., Linares, J. F., Martínez-Solano, L., & Sánchez, M. B. (2009). A global view of antibiotic resistance. FEMS Microbiology Reviews, 33(1), 44–65. 11. Kohanski, M. A., Dwyer, D. J., Hayete, B., Lawrence, C. A., & Collins, J. J. (2007). A common mechanism of cellular death induced by bactericidal antibiotics. Cell, 130(5), 797–810. 12. Liu, Y., & Imlay, J. A. (2013). Cell death from antibiotics without the involvement of reactive oxygen species. Science (New York, N.Y.), 339(6124), 1210–1213. 13. Keren, I., Wu, Y., Inocencio, J., Mulcahy, L. R., & Lewis, K. (2013). Killing by bactericidal antibiotics does not depend on reactive oxygen species. Science (New York, N.Y.), 339(6124), 1213–1216. 14. Krawczyk-Balska, A., Korsak, D., & Popowska, M. (2014). The surface protein Lmo1941 with LysM domain influences cell wall structure and susceptibility of Listeria monocytogenes to cephalosporins. FEMS Microbiology Letters, 357(2), 175–183. 15. Knudsen, G. M., Fromberg, A., Ng, Y., & Gram, L. (2016). Sublethal concentrations of antibiotics cause shift to anaerobic metabolism in Listeria monocytogenes and induce phenotypes linked to antibiotic tolerance. Frontiers in Microbiology, 7. 16. Kaushik, D., Mohan, M., Borade, D. M., & Swami, O. C. (2014). Ampicillin: Rise, fall and resurgence. Journal of Clinical and Diagnostic Research: JCDR, 8(5), ME01–ME03. 17. Herzog, C. (1976). Chemotherapy of typhoid fever: A review of literature. Infection, 4(3), 166–173.

Proceedings 2018

LIFE AND PHYSICAL SCIENCES X

Tyrosine Kinase Inhibitors and Their Use as Drugs in Glioblastoma Chemotherapy: A Review Jordan M. Terschluse1,2,3 University of Dayton, 300 College Park, Dayton, OH, 45469 1. Department of Biology 2. Berry Summer Thesis Institute 3. University Honors Program

Mentor: Madhuri Kango-Singh, Ph.D. Department of Biology Corresponding Author: Madhuri Kango-Singh, Ph.D. Email: mkangosingh1@udayton.edu

Abstract Through all the medical advancements made in cancer detection and treatment over the centuries, there still is no cure for most cancer types. Today, there are over 250 drugs being used to treat over 100 different types of cancers prevalent in our society. It is notable that a drug, out of the 250, that helps treat a certain type of cancer could be harmful or ineffective for another type. In fact, certain chemotherapy drugs result in enhancing the tumor (promote its growth) and thus have a negative effect on patient health. A class of chemicals called tyrosine kinase inhibitors seems the most promising drugs, as most cancers show activation of tyrosine kinase dependent oncogenic pathways. Different clinical studies using kinase inhibitors in glioblastoma cells or models have been analyzed, where these drugs have shown a reduction in neoplasia. Some of these studies are now focused on testing different concentrations to determine the most effective dosage in model systems so that clinical trials can be done with human patients. We took a similar approach and tested a chemical library of tyrosine kinase inhibitors on a Drosophila glioma model. Drosophila melanogaster has high percentage of conserved genes that are known to cause diseases in humans. Using these orthologous genes, it is possible to model human diseases like glioma in flies, and the results from flies may be extrapolated to mammalian models. Given the amenability of Drosophila flies to genetic manipulation and large scale production it is advantageous to use Proceedings 2018

Drosophila melanogaster as a model organism to test tyrosine kinase inhibitors on glioblastoma. Results from these studies will be of immense importance to the medical field, specifically dealing with different treatment options for patients.

Introduction Cancer is still one of the leading causes of death worldwide, and although many advances have been made in finding a cure, it is responsible for around 8.2 million deaths yearly (National Cancer Institute, 2018). Gliomas are glia-derived, primary brain tumors with poor prognosis (Modrek, Bayin, & Placantonakis, 2014), and are associated with mutations in developmental pathways (e.g., the Epidermal Growth Factor (EGF), TP53, ATRX, etc). The NCI website states that 250 different types of drugs are being used in the medical field to help cure cancer, and it is these drugs that have proven useful in curing specific types. Previously it was shown that a drug approved for the treatment of one cancer type can work effectively in the treatment of another cancer either in combination with other drugs or as a single therapeutic agent. In this review, we explore how simple model organisms like Drosophila melanogaster can prove useful for initial screening of drugs, followed by a discussion of Tyrosine Kinase inhibitors and their potential in glioblastoma treatment, along with past treatment therapies. The background 65

LIFE AND PHYSICAL SCIENCES information covered in this review is important to the Chemical Drug Screens conducted because it helps give insight to a model organism that effectively expresses glioma, and it also gives insight to specific drugs that have proven to have an effect on cancer, which narrows the search of effective chemical libraries immensely.

Drosophila melanogaster History of Drosophila Drosophila has proven to be a useful model organism in the scientific field since it was first used in 1901 by Thomas Hunt Morgan (Jennings, 2011). Jennings states in her research that Morgan, along with many other scientists, found Drosophila more beneficial than vertebrate models for a number of reasons, including: they are easy and inexpensive to culture in laboratory conditions, have a much shorter life cycle of about 12 days (see, Figure 1), and they produce large numbers of externally laid embryos that can be genetically modified. Morgan used the defining of genes and ability to establish that they were on the chromosomes to further define the theory of inheritance that was proposed by Gregor Mendel (Jennings, 2011). It was this redefining that led Morgan to receive The Nobel Prize of Physiology or Medicine in 1933 for “his discoveries concerning the role played by the chromosome in heredity” (Nobel Prize

Figure 1. Lifecycle of Drosophila Melanogaster: For both male and female flies, they go through 5 different growth stages, starting with the embryo and ending with pupa. Courtesy of Creative Diagnostics, 2009-2018.

and Literature, 2018). This Nobel Prize opened the doors to different research that could be conducted using Drosophila, and it was this continuation of research that went on to win many other awards of their own. This is demonstrated through the findings of Hermann Muller, Christiane Nüsslein-Volhard, and Craig Venter, who all discovered promising breakthroughs using this model organism (Jennings, 2011). Areas of their research concerned “x-ray irradiation”, “the genetic control of early embryonic development”, and also using Drosophila as a “shot-gun approach for sequencing the human genome”. It is this rich history of Drosophila as a research model that allows it to be used to model diseases as well as other physiological differences for a deeper understanding of the mechanisms.

Comparisons between Drosophila and Humans Besides the ability to easily induce desired genetic pathways, Drosophila also makes a good model organism due to it sharing 70 percent of disease genes with humans (Read, et al., 2009). Drosophila has approximately 13,600 genes in its genome, which was sequenced for the first time in 2000 (Mark, Susan, & Holt, 2000). Of these 13,600 genes, there was a separate study that looked at the 929 human disease genes that were associated with at least one mutant allele discovered in Drosophila’s own genome (Reiter, 2001). The OMIM, or Online Mendelian Inheritance in Man, was used as the human comparison group. Reiter, et. al., found that of the 929 OMIM genes, 714 of them contain highly similar (E ≤10−10) cognates in Drosophila (77 percent). The research Reiter et al. conducted, culminated in the generation of the Homophila database that illustrates the connection between Drosophila and human genes, allowed for a variety of successful experiments to be conducted in the future. The 77 percent of shared genes represents the different diseases Drosophila can be a model organism for including cancer, neurological diseases, cardiovascular diseases, malformation syndromes, hematological, immune, endocrine, renal, and metabolic disorders (Fortini, Skupski, Boguski, & Hariharan, 2000). This and other studies (Fortini, et al., 2000; Rubin, et al., 2000) focused on the genetic comparisons between Drosophila and human disease causing genes and laid the groundwork for making Drosophila an ideal system for modelling human diseases (see, Figure 2). Proceedings 2018

LIFE AND PHYSICAL SCIENCES X The P13K Ras Pathway

Figure 2. Illustration of tumor expression in Drosophila. Tumors ranging from Imaginal Disks, Brain, Muscle, and Haemo-lymph tumors can be expressed. These can be further divided into sub-tumors as listed on the right along with the type of pathways used for each. Showcases how Drosophila is a top-tier model organism for expressing the wanted tumors. Courtesy of Villegas, 2017.

Modelling Glioma in Drosophila Glioma (or Glioblastoma) are glia-derived, primary brain tumors with poor prognosis. Given the ease of inducing mutations, and combining several mutations together, it is possible to model glioma that can be replicated repeatedly in Drosophila using the GAL4UAS based misexpression system (our lab and Read, Cavenee, Furnari, & Thomas, 2009; Witte, Jeibmann, Klambt, & Paulus, 2009; Brand and Perrimon, 1993). This strategy is useful in studying the possible causes, as well as changes that can act as inhibitors to the tumor growth. The fly brain is composed of two dorsal hemispheres and the ventral ganglion (see, Figure 3) of which 10 percent of cells are glial in nature. In glioma, the number of glial cells expand significantly making the brain appear overgrown and malformed (see, Figure 3). Other areas that Witte, Jeibmann, etc. cover in their research are the pathways that can be induced in Drosophila to effectively produce glioma. They found promising molecular targets for therapeutic intervention included the tyrosine kinase receptors epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), and vascular endothelial growth factor receptor (VEGFR) and their downstream signaling cascades, the phosphatidylinositol 3-phosphate kinase (PI3K)/ AKT and Ras/Mitogen-Activated Protein kinase pathway (Witte, et al., 2009).

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The three core signaling pathways that are commonly activated in glioma patients are the tumor protein p53 [p53] pathway, the receptor tyrosine kinase/Ras/phosphoinositide 3-kinase signaling pathway and the retinoblastoma pathway (Davis, 2016). Despite these separate pathways, the EGFR-Ras and PI3K pathway continues to be one of the most effective models, as shown in Figure 4. The mutation or amplification of the Epidermal Growth Factor Receptor (EGFR) tyrosine kinase continually shows the most frequent genetic damage in gliomas (Read, et al., 2009). The network EGFR-Ras/ P13K pathway coordinately stimulates oncogenic behaviors, such as cell cycle entry and progression, protein translation and inappropriate cellular growth and migration (Read. et al., 2009). These behaviors were exhibited in the coactivation of these pathways that creates tumor-like growths that mimic human glioma, which is what makes it so useful in a laboratory setting (Read, et al., 2009). When driven by repo-Gal4 in the CNS glia, the P13K-Ras driven pathway induced an accumulation of ~50-fold excess glia causing glioma (Read, et al., 2009). In spite the fact that different pathways initiated the glioma, in the Read, et al., study, they found glial-specific coactivation of EGFR-Ras and PI3K stimulated glial neoplasia, giving rise to CNS enlargement and malformation, neurologic defects, and late larval lethality (Figure 3). Both

Figure 3. Optical projections of whole brain-ventral nerve cord complexes from late 3rd instar larvae approximately 130 hr AED, displayed at the same scale. Dorsal view; anterior upEach brain is composed of 2 symmetrical hemispheres attached to the ventral nerve cord (VNC). In repo>dEGFRÎť;dp110CAAX larvae, both brain hemispheres and the VNC are enlarged and elongated relative to other genotypes. Courtesy of Read, et al., 2009.

X LIFE AND PHYSICAL SCIENCES

Figure 4. Overview of the PI3K/AKT/mTOR pathway and drug targets. Activating nodes (PI3K, AKT, PDK1, mTORC1 and mTORC2) and negative regulators (PTEN, TSC complex) are highlighted. Interaction with RAS and LKB1/AMPK pathways is also displayed. Courtesy of Wikipedia.org signal transduction, 2018.

the PI3K and Ras pathway are known to activate a class of enzymes called the tyrosine kinases to cause changes in proliferation and to induce glioma.

Tyrosine Kinase Model Tyrosine Kinase Function Tyrosine kinases are a family of enzymes, which use ATP to catalyze phosphorylation of select tyrosine residues in target proteins. It is these enzymes, through covalent post-translational modification, that act as a key component of normal cellular communication and maintenance of homeostasis (Paul & Mukhopadhyay, 2004). Some of these cellular functions include: cell proliferation, differentiation, migration, metabolism and programmed cell death. Paul and Mukhopadhyay go on in their research of tyrosine kinases to 68

state that it is the ligand binding to the kinaseâ&#x20AC;&#x2122;s extracellular domain that triggers a response (Figure 5). These ligands are extracellular signal molecules (e.g. EGF, PDGF etc) that induce receptor dimerization, which is a chemical reaction that joins two molecular subunits, forming a dimer (Chemistry LibreTexts, 2017). There are two separate classifications of Tyrosine Kinases, either Receptor Tyrosine

Figure 5. Mechanism of action of tyrosine kinase. 1. Receptor expression at membrane claveola 2. Ligand binding occurs 3. Hetero/homodimerization leads to tyrosine kinase activation and tyrosine transphosphorylation 4. Signal transduction throughout the cell 5. Receptor internalization 6. Receptor activates response of either degradation or re-expression. Courtesy of Wikipedia.org Akt/PKB signaling pathway, 2017.

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LIFE AND PHYSICAL SCIENCES X Kinase (RTK) or non-receptor tyrosine kinase (NRTK). The RTKs are cell surface transmembrane receptors that also possess kinase activity. Paul and Mukhopadhyay found in their research that the RTKs are ligand specific, contain a single pass transmembrane hydrophobic helix and a cytoplasmic portion with a tyrosine kinase domain. NRTKs are cytoplasmic proteins that contain a kinase domain and possess several additional signaling or protein-protein interacting domains (Paul & Mukhopadhyay, 2004). Paul and Mukhopadhyay found that the activation mechanism of NRTKs is more complex than that of RTK, which they hypothesized made the RTKs more frequently activated. Tyrosine kinases activity are tightly regulated in normal cells, but they have the ability to acquire transforming functions due to mutation(s), overexpression and autocrine paracrine stimulation, leading to malignancy (Paul & Mukhopadhyay, 2004). It is these different functions that the kinase takes on that allow it to represent a major portion of the oncoproteins that play a powerful role in the plethora of cancers, which includes glioma.

Tyrosine Kinase Role in Cancer To ensure normal tissue patterning in an organism, there must be tight control of cell proliferation and morphogenesis in conjunction with programmed apoptosis. It is when imbalances occur within these cell signals that oncogenesis can occur (Sangwan & Park, 2006). As stated in earlier research (Mukhopadhyay & Paul, 2004), it is the more frequently activated RTKs that have the potential to cause dimerization to the kinases, and this change in conformation leads to activation of the kinase and transphosphorylation of the receptor on specific tyrosine residues. The phosphorylated tyrosine residues are a critical aspect because they provide docking sites on the receptor for signaling proteins, and it is these signaling proteins that act to relay the signal from the receptor into the cell. When these signaling pathways become altered through mutations or chromosomal translocation, the RTKs can deliver a continuous or enhanced signal, forming it into a potent oncogene (Sangwan & Park, 2006). As of 2001, 58 genes encoding RTKs have been identified in the human genome, 30 of which have been found to be dysregulated in human cancers (Blume-Jensen & Hunter, 2001). Different discovered tyrosine kinases are shown in Figure 6.

Figure 6. These are structures of some of the important kinase receptors discovered so far. Courtesy of S. Grant, 2008.

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X LIFE AND PHYSICAL SCIENCES Together with these behaviors, Sangwan & Park (2006) found that growthfactor stimulation within a cell also aids in producing oncogenic behaviors. It is the non-scheduled expression of these growth factors that may result in a constant stimulation of cell growth in addition to a block in apoptosis. The combination of the mis-regulated RTKs and growth factors plays a critical role in regulating the tumor microenvironment, by enhancing both the proliferation and invasion by tumor cells (Sangwan and Park, 2006). This invasion of tumor cells can form lung, breast and brain cancer, and Sangwan and Park in their research explain how the pathways are initiated, and also possible routes that can be taken to halt the tumor progression in its track.

Figure 7. Activation of Ras/Raf/MEK/ERK signaling pathway. RTK, receptor tyrosine kinase; SOS, mammalian son-of-sevenless; Shc, homology 2 domain-containing protein; MEK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase. Courtesy of Wikipedia.org MTOR-ger.png., 2017.

Tyrosine Kinase Expression in Drosophila Through the sequencing completed by the Human Genome Project, it was found that the human genome contains 90 tyrosine kinases (Robinson, Wu, & Lin, 2000). Of these 90 tyrosine kinases, 58 are the receptor types (RTKs). Of this large amount of RTKs present in the human genome, only 20 RTKs have been identified in the Drosophila genome. Just like what has been found in human kinases, these different receptors share many of the same effectors and their hierarchical organization is retained in biological contexts (Sopko & Perrimon, 2013). While Sopko and Perrimon in their research may have found this to be true for the identified Drosophila tyrosine kinase receptors, very little is still known for approximately half of them. Of the many pathways that RTKs can signal in the Drosophila genome, the one most commonly

linked to glioma is the Epidermal Growth Factor Receptor (EGFR). EGFR plays a multitude of roles inside the genome, such as dorsal/ ventral patterning of the embryonic ectoderm, establishment of neuroectoderm, wing development, photoreceptor differentiation, and the specification of muscle precursors (Sopko & Perrimon, 2013). It is able to signal these specific pathways by predominantly mediating short-range signaling that is restricted to cells producing EGF or to cells positioned 1-2 cells away. A pathway that falls into this category is the Ras/Raf/MEK/ERK pathway, showcased in Figure 7. There are four EGFR ligands in Drosophila: Spitz, Keren, Gurken and Vein, and Sopko and Perrimon found in their study that all four play a critical role in the signaling of the pathway. By monitoring these different ligands and sub-pathways in EGFR, Sopko and Perrimon opened up the research field to possibly triggers or onsets of diseases, such as ones like glioma.

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LIFE AND PHYSICAL SCIENCES X Success of Tyrosine Kinase Inhibitors with Glioma The modes of oncogenic activation can be targeted using different approaches for tyrosine kinase inhibition, such as small molecule inhibitors, monoclonal antibodies, heat shock proteins, immunoconjugates, antisense and peptide drugs (Paul & Mukhopadhyay, 2004). The small-molecule compounds that inhibit the kinase domain have recently changed clinical practice for several cancers. Lapatinib has shown positive effects in HER2-positive metastatic breast cancer (Geyer, Forster, & Lindquist, 2006); Sunitinib positively influences metastatic renal-cell carcinoma (Motzer, Hutson, & Tomczak, 2007); and Sorafenib is beneficial in carcinoma treatment due to it inhibiting the targeted kinase domain (Joseph, Ricci, & Mazzaferro, 2008). Taking this knowledge on the kinase inhibitors that work, De Witt Hammer in his study (2010) systematically reviewed the efficacy, toxicity and tissue analysis of smallmolecule kinase inhibitors in adult patients with glioblastoma as reported in published clinical studies. De Witt Hammer also determined which kinases have been targeted by the inhibitors used in these studies, by using publications from a MEDLINE search. From the search, 60 studies qualified for inclusion, and 2385 glioblastoma patients receiving kinase inhibitors could be evaluated. The extracted data included radiological response, progression-free survival, overall survival, toxicity and biomarker analysis (De Witt Hammer, 2010). This data could be analyzed to determine the overall effects of kinase inhibition from past studies, by looking at the effects it showed on the patients during and after treatment. De Witte Hammer found through analysis that (i) efficacy of small-molecule kinase inhibitors in clinical studies with glioblastoma patients does not yet warrant a change in standard clinical practice and (ii) 6 main kinase targets for inhibitors have been evaluated in these studies: EGFR, mTOR, KDR, FLT1, PKCβ, and PDGFR. Although in this study the promise of kinase inhibitors being effective in cancer treatment was not strong, De Witte Hammer overlooks the fact that there are many limitations to his study. Some of these limitations include, not having a control group, small sample sizes, and many of the 60 studies were not designed to determine the efficacy of therapy. The efficacy of the therapy is especially important, because Proceedings 2018

by not having that the pathobiology of the drug may not be accurately studied in glioblastoma patients, along with the inhibitor may have failed to inactivate the target in glioblastoma cells. Knowing the efficacy would aid to ruling out these errors and producing more accurate studies in the future. Different from De Witte Hammer’s study (2010), Mellinghoff, Schultz, Mischel and Cloughesy, (2012), looked more specifically at how kinase inhibitors perform as glioblastoma drugs, specifically when targeting the PI3K pathway. Throughout their study they look at results from clinical trials, the structure of the human genome, and techniques that can be applied to glioblastoma to halt tumor growth. The clinical study looked specifically at mTOR, which was the first member of the PI3K pathway for which a clinical grade inhibitor became available, and its effect on patients with PTEN-deficient, recurrent glioblastoma (Podsypanina, Lee, & Politis, 2001). After 1-2 weeks, the effect that inhibition of mTOR had on glioblastoma was analyzed, and it was found that although there was reduction of neoplasia, it was not enough to cause effective change on the tumor. Although these results do not seem promising, it must be taken into account that the information was preliminary because tumors with the most informative genotype(s) and strong basal pathway activation were generally underrepresented in the studies and because of difficulties to assemble a sufficient drug-naive “control” tumor samples (Mellinghoff, et al., 2012). To end their study, Mellinghoff, et al., (2012) talks about

Figure 8. Results from clinical trial of patient treated with Radiotherapy plus Temozolomide . A) Initial MRI on February 16, 2005, shows a tumor in the right and left frontal lobe as well as the right thalamus. (B) MRI after surgery, radiation and chemotherapy. The tumor has completely disappeared. Courtesy of Wikipedia.org Archivo, 2008.

X LIFE AND PHYSICAL SCIENCES factors that would increase the therapeutic window of individual kinase inhibitors, which includes different dosing schedules (e.g., intermittent or “pulsatile” dosing) (Shah, et al., 2008) and isoform-specific (e.g., PI3K) or mutantspecific (e.g., BRAF) compounds. The different dosage levels effectiveness can be represented through cytotoxicity, and the results are shown in Figure 8. The different dosage levels is the strongest candidate for positive results, given it was this fact that was also highlighted in De Witte Hammer’s (2010) study of kinase inhibitors with glioblastoma.

History of Glioma Treatment Surgery Standard treatment of glioblastoma includes maximal safe surgical resection, followed by concurrent radiation with Temozolomide (TMZ) (Temodar®), an oral alkylating chemotherapy agent, and then adjuvant chemotherapy with TMZ (National Comprehensive Cancer Network [NCCN], 2015). Although surgically removing the tumor would seem like a promising therapy, extensive and complete surgical resection of glioblastoma is difficult because these tumors are frequently invasive and are often in eloquent areas of the brain, including areas that control speech, motor function and the senses (Davis, 2016). In the study conducted by Kuhnt, Becker, Ganslandt, Bauer, Buchfelder and Nimsky (2011), they focus on how the more total resection for the patients possible, the more beneficial surgery will be in glioblastoma therapy. They came to this conclusion by having 135 glioma patients undergo tumor resection aided by 1.5T intraoperative MRI (iMRI) and integrated multimodal navigation. The media survival was 14 months for patients who underwent an extent of resection ≥98 percent, which is a significant improvement in patient survival. Kuhnt, et al., (2011) in their study came to the conclusion that results like this can be achieved with iMRI and an intraoperative update of navigation data, along when performed on patients <65 years of age. Although, Kuhnt, et al., (2011) study showed promising results, the prognosis for patients with GBM remains poor, with a median survival of 15 months (Thakkar, et al., 2014). In conclusion, both Thakkar, et al., (2014) and Kuhnt, et al., (2011) found that patients with a lower age and higher performance status experience longer survival.

Chemoradiation Chemoradiation as means of glioblastoma treatment has been seen to produce more promising results compared to chemotherapy and radiation alone. Although it is one of the most popular choices of glioblastoma therapy, usually occurring around four weeks after surgery, it holds the potential to cause more consequences than advantages. Shih and Batchelor’s study (2017) analyzed the different techniques of chemoradiation practiced (Adjuvant radiotherapy, treatment target, Intensity-modulated RT, etc.) and stated the similar limitations found in each of them. It was found that the similar consequences of chemoradiation on glioblastoma include radiation-induced gliomas, neurocognitive toxicity, RT-induced leukoencephalopathy, and endocrinopathies (Shih & Batchelor, 2016). Other side effects do occur because of chemoradiation, but they are more specific to the route of chemoradiation chosen. Together with all these side effects, it was found that the survival rate of patients was still low, with at 17 months after treatment 72 percent of patients developed recurrent glioblastoma (Milano, et al., 2010). Comparing Shih and Batchelor’s (2016) findings to present chemoradiation practice, the adequate dose of chemoradiation necessary to cause a high survival rate with low cytotoxicity levels is required to maximize the survival benefit. Furthermore, these studies done in the past on glioblastoma chemoradiation treatment, which included the benefits and limitations of it, showed that further therapy options must be taken either before or after this to ensure an increase in overall patient survival.

Temozolomide Chemoradiation starting showing much higher patient survival rates and less lethal side effects when it contained Temozolomide (TMZ), rather than simple radiation alone, shown in Figure 8. A separate study was conducted to test and analyze the results of TMZ used in clinical practice, and the results from it were promising (Stupp, Hegi, & Mason, 2005). Stupp, et al., (2005) conducted the study so 573 patients randomly received radiotherapy alone or radiotherapy plus continuous daily temozolomide, followed by six cycles of adjuvant temozolomide. At the median follow-up of 28 months, the median survival was 14.6 months with radiotherapy plus temozolomide and 12.1 months with radiotherapy alone (Stupp, Proceedings 2018

LIFE AND PHYSICAL SCIENCES X et al., 2005). The two-year survival rate was 26.5 percent with radiotherapy plus temozolomide and 10.4 percent with radiotherapy alone. These results were clinically beneficial and showed statistically significant survival benefit with minimal additional toxicity. Temozolomide acts through DNA alkylation, and the analysis of this led to another strong predictor of patient-related outcomes: the methylation of the MGMT gene (Stupp, et al., 2009). Methylated (not activated) MGMT exhibit compromised DNA repair, so when MGMT becomes activated it can interfere with the effects of treatment. Radiotherapy and alkylating chemotherapy exert their therapeutic effects by causing DNA damage, cytotoxicity, and triggering apoptosis. Therefore, the expression of methylated MGMT is beneficial for patients undergoing temozolomide chemotherapy and radiation. In this study conducted by Stupp, et al., (2009), the methylation of MGMT was a strong predictor of better outcomes for temozolomide treatment.

Conclusion Out of the 250 drugs being used to treat over 100 different types of cancers prevalent in our society, it is important to note that a certain drug being used to treat a certain kind of cancer could be harmful or ineffective for another type. Scientific research on the topic of cancer is continuously looking into different treatment options and, through this research, tyrosine kinase inhibitors have shown to play a key role in the inhibition of glioblastoma, a type of brain tumor. The literature reviewed in this paper covers the topics of tyrosine kinase and its role in glioblastoma, its success so far in clinical trials and why Drosophila melanogaster is considered one of the most efficient and successful model organisms used in cancer research. The research presented shows that kinase molecules play a significant role in the regulation of gene expression by the RTKs having the potential to cause dimerization to the kinases, and this change in conformation leads to activation of the kinase and transphosphorylation of the receptor on specific tyrosine residues. This downstream activation of these kinases has the potential to cause proliferation in tumor growth, so by treating a tumor with a kinase inhibitor it has shown to have the potential to reduce the growth. Areas for future research involving Proceedings 2018

these kinases are furthering the knowledge on how they specifically go about activating or inhibiting a tumor, and also their effect on tumor growth when combined with another drug such as Temozolomide. Looking into the additive effects of these different drugs on tumor growth would not only be of great practice in the medical community when it comes to treating patients, but it would also further the scientific communities knowledge on the different genetic pathways that can be utilized to decrease the progression or growth of a tumor.

Acknowledgments The author would like to thank the University of Dayton Honors Program, the Berry Family Foundation, and the Brother Joseph W. Stander Symposium for providing the funding necessary to pursue research over the summer. Thanks are owed to Dr. Madhuri Kango-Singh, for making the project possible along with all the time and energy she invested into it. Additional thanks are owed to lab members Kirti Snigdha and Karishma Gangwani, for their efforts in maintaining the project and all their help and support this summer.

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PHOTOGRAPH AND ILLUSTRATION CREDITS Cover Row 1, left to right The First Issue with CMAA Seal of Approval. Illustrations courtesy of Treasure Chest of Fun and Facts, Volume 10, Issue No. 20 (Schaller, 1955), Catholic University of America Digital Collections. Pleiotropic Genes. Illustration courtesy of C.M. Jaenke and T.M. Williams, 2018. Row 2, left to right Ball-and-stick Models of SCFAs. Illustration courtesy of molview.org/. Phospholipid Bilayer from Cell Membrane Containing Fatty Acids. Illustration courtesy of OpenStax Anatomy and Physiology, 2016. Middle Cerebral Artery Mean Velocity During Cognitive Tasks. Illustration courtesy of http:// creativecommons.org/licenses/by/4.0/. Produce from a Lower-Income Area. Photograph courtesy of Katrina Coleman 2018. Row 3, left to right Football Helmet. Courtesy of Clipart Library. Activation of Ras/Raf/MEK/ERK Signaling Pathway. Illustration courtesy of Wikipedia.org MTOR-ger.png., 2017.

Articles Page 5: Largest Advertisement in Lower-Income area. Photograph courtesy of Katrina Coleman 2018. Page 5: Part of the Produce Section at Middle-to-Higher Income Location. Photograph courtesy of Katrina Coleman 2018. Page 5: Produce from a Middle-to-Higher Income Area. Photograph courtesy of Katrina Coleman 2018. Page 5: Produce from a Lower-Income Area. Photograph courtesy of Katrina Coleman 2018. Page 19: The First Issue with CMAA Seal of Approval. Illustrations courtesy of Treasure Chest of Fun and Facts, Volume 10, Issue No. 20 (Schaller, 1955), Catholic University of America Digital Collections. Page 19: The Last Issue with CMAA Seal of Approval. Illustrations courtesy of Treasure Chest of Fun and Facts, Volume 22, Issue No. 20 (Langdon, 1967b), Catholic University of America Digital Collections. Page 21: We Built America. Illustrations courtesy of Treasure Chest of Fun and Facts, Eaglin, 1955a, p. 31, Catholic University of America Digital Collections. Page 21: Civics Club Meeting. Illustrations courtesy of Treasure Chest of Fun and Facts, Ostendorf, 1950, p. 13, Catholic University of America Digital Collections. Page 22: America, The Melting Pot. Illustrations courtesy of Treasure Chest of Fun and Facts, Ostendorf, 1963, p. 18-19, Catholic University of America Digital Collections. Page 23: Only the Best Person Should be Elected. Illustrations courtesy of Treasure Chest of Fun and Facts, Schaller, 1949b, p. 34, Catholic University of America Digital Collections 76

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Page 23: “Where Do We Stand?” Illustrations courtesy of Treasure Chest of Fun and Facts, Ostendorf, 1951b, p. 4, Catholic University of America Digital Collections Page 24: Subversion. Illustrations courtesy of Treasure Chest of Fun and Facts, Ostendorf, 1951a, p. 13, Catholic University of America Digital Collections. Page 24: Prayers and the Spreading of Truth. Illustrations courtesy of Treasure Chest of Fun and Facts, Crandall, 1961a, p. 8, Catholic University of America Digital Collections. Page 25: Marx and Hegel Philosophy. Illustrations courtesy of Treasure Chest of Fun and Facts, Crandall, 1961b, p. 31, Catholic University of America Digital Collections. Page 26: Colonists Celebrate a “Charter of Liberties.” Illustrations courtesy of Treasure Chest of Fun and Facts, Klosterman, 1965a, p. 4, Catholic University of America Digital Collections. Page 26: Dr. Pat Smith Serves in Vietnam. Illustrations courtesy of Treasure Chest of Fun and Facts, Grady, 1967, p. 34, Catholic University of America Digital Collections. Page 28: Good Citizenship. Illustrations courtesy of Treasure Chest of Fun and Facts, Schaller, 1950, p. 28, Catholic University of America Digital Collections. Page 28: Humility Is a Virtue. Illustrations courtesy of Treasure Chest of Fun and Facts, Pflaum, 1946e, p. 22, Catholic University of America Digital Collections. Page 29: Prayer to St. Agnes. Illustrations courtesy of Treasure Chest of Fun and Facts, Ostendorf, 1955, p. 3, Catholic University of America Digital Collections. Page 29: Model of Purity and Goodness. Illustrations courtesy of Treasure Chest of Fun and Facts, Ostendorf, 1955, p. 8, Catholic University of America Digital Collections. Page 49: Ball-and-stick Models of SCFAs. Illustration courtesy of molview.org/. Page 54: Pleiotropic Genes. Illustration courtesy of C.M. Jaenke and T.M. Williams, 2018. Page 54: CREs as Transcription Factor Binding Sites for Combinatorial Control. Illustration courtesy of C.M. Jaenke and T.M. Williams, 2018. Page 55: The Understanding and Challenges for Protein-coding and CRE Sequences. Illustration courtesy of C.M. Jaenke and T.M. Williams, 2018. Page 55: CREs Control Gene Expression Patterns. Photographs courtesy of C.M. Jaenke and T.M. Williams, 2018. Page 56: Reporter Transgene Assays: the “Gold Standard” Approach for Validating Putative CREs. llustration courtesy of C.M. Jaenke and T.M. Williams, 2018. Page 56: SCRMshaw’s Machine-learning Approach. Illustration courtesy of C.M. Jaenke and T.M. Williams, 2018. Page 57: The Abdomens of Male Fruit Fies Evolved Diverse Patterns of Pigmentation. Photographs and illustration courtesy of C.M. Jaenke and T.M. Williams, 2018. Page 57: Rudimentary GRN for D. melanogaster Tergite Pigmentation. Photographs and illustration courtesy of C.M. Jaenke and T.M. Williams, 2018. Page 61: Phospholipid Bilayer from Cell Membrane Containing Fatty Acids. Illustration courtesy of OpenStax Anatomy and Physiology, 2016. Page 61: Structure of Three Common Short Chain Fatty Acids. Illustration courtesy of Ben Mills, 2007. Page 63: Beta-lactam Ring in Ampicillin. Illustration courtesy of Wikimedia Commons, 2006. Proceedings 2018

CREDITS Page 66: Lifecycle of Drosophila melanogaster. Illustration courtesy of Creative Diagnostics, 2009-2018. Page 67: Tumor Expression in Drosophila. Illustration courtesy of Villegas, 2017. Page 67: Optical Projections of Whole Brain-ventral Nerve Cord Complexes from Late 3rd Instar Larvae. Illustration courtesy of Read, et al., 2009. Page 68: Overview of the PI3K/AKT/mTOR Pathway and Drug Targets. Illustration courtesy of Wikipedia.org Signal Transduction, 2018. Page 68: Mechanism of Action of Tyrosine Kinase. Illustration courtesy of Wikipedia.org Akt/PKB Signaling Pathway, 2017. Page 69: Structures of Important Kinase Receptors. Illustration courtesy of S. Grant, 2008. Page 70: Activation of Ras/Raf/MEK/ERK Signaling Pathway. Illustration courtesy of Wikipedia.org MTOR-ger.png., 2017. Page 71: Results from Clinical Trial of Patient Treated with Radiotherapy Plus Temozolomide. Photograph courrtesy of Wikipedia.org Archivo 2008.

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