Berry Summer Thesis Institute
Proceedings 2017
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 90-91 for credits Copyright Š 2017 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
LETTER FROM THE DIRECTOR
Dear Reader: The 2017 cohort of the Berry Summer Thesis Institute (BSTI) covers a wide range of majors and talents, which is why the research and creative work included in this fifth volume of the Proceedings includes everything from an analysis of the effects of ethanol on Listeria infections to a short story cycle-in-progress that explores the afterlife. Those students, staff and faculty who attended the 2017 Berry Summer Thesis Institute Symposium (including UD President, Dr. Eric Spina), were once again witness to the remarkable talents of our Honors students. This collection 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 Honors, I am especially grateful for the friends and alumni that continue to support the Berry Summer Thesis Institute. In particular, let me express my on-going gratitude to Mr. John W. Berry, Jr. — as well as his entire family — for many years of 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 the design and production of the Proceedings manuscript, and to Maria Ollier Burkett for facilitating the volunteer work with community partners that kept our students busy when they weren’t in the lab or library or studio. I would also like to thank Laura Cotten for enhancing the professional development of the BSTI cohort and to Jill Talley for her valuable administrative support. Please enjoy the work of our emerging scholars and artists! 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 eleven rising juniors the opportunity to participate in the 2017 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 ARTS AND HUMANITIES Page The Art of Feminism by Claire F. Bowman . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Perceiving Place: A Socially-Based Design Case Study by Mary A. Brinkman . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Pocahontas in the Land of Miscegenation: Persona and the Fantasy of Whiteness in Danzy Senna’s Caucasia by William E. Landers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Mortality in the Twenty-first Century: A Cycle of Short Stories about Death and the Afterlife by Rose A. Rucoba . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
ENGINEERING A Comparative Analysis of Breast Cancer Treatment and the Role of Neurotoxic Chemotherapy-induced Peripheral Neuropathy on Postural Stability by Paige L. Ingram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
HEALTH SCIENCE Breaking Down Barriers to Sustainable Eating at the University of Dayton by Lauren M. Murray . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
LIFE AND PHYSICAL SCIENCES An Examination of Bacterial Efflux Pumps and Their Role in Multidrug Resistance by Sarah P. Baxter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
Role of Immune System in Alzheimer’s Disease by Steven G. Borchers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
Determining the Effect of Ethanol on Listeria Infection by Ryan E. Restrepo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
Investigating Cell to Cell Interactions and Molecular Signals in Drosophila Glioma Models by Logan J. Roebke, Kirti Snigdha, Indrayani Waghmare and Madhuri Kango-Singh . . . . . . . . . . 76
SERCA2: A Gatekeeper of Calcium Homeostasis in the Brain by Joseph E. Saurine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
Photograph and Illustration Credits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
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The Art of Feminism Claire F. Bowman1,2,3 University of Dayton, 300 College Park, Dayton, OH, 45469 1. Department of Art and Design 2. Berry Summer Thesis Institute 3. University Honors Program
Advisor: Emily Sullivan Smith, MFA Department of Art and Design Corresponding Author: Claire F. Bowman Email: bowmanc4@udayton.edu
Introduction Growing up as a woman in today’s society poses unique challenges. From the moment we are born, we are defined through gender norms and stereotypes, from what colours we are expected to wear as toddlers to the very toys we play with. Another conflict arises as we age, when we get a full dose of society’s definition of feminine beauty — that the proper way to look and act is to fit within set patterns, and if you dare to embrace strength or independence you may be labeled as shrill, bitchy or at the very least undesirable or unattractive. Being a woman myself brings these issues close to my heart, even if it took me almost 20 years to realize it.
Background (Artists) First, I started by researching several feminist and political artists from the past several decades. I began my research with Barbra Kruger, one of my favourite contemporary artists. She focused mainly on graphic, bold prints, usually in black and white with eloquent phrases to get her message across. In her piece, Untitled (Your Gaze Hits the Side of My Face), she emphasizes the negative impact the male gaze can have on women, when they're seen solely as an object to be viewed by their male counterparts. I really like her brashness and bold style, especially her artful use of text. One of the most influential artists I studied was actually a group called the Gorilla Girls. Created in 1985, this group of anonymous women has taken a stand regarding the lack of women’s voices in the art world and have created highly visible mass media campaigns, including ads in large metropolitan areas, in an attempt to expose the injustices that female artists and artists of colour face. I love their humour and snarky attitude, as well as their urge to reach as many people as possible. I am also inspired by their scale, and hope to do something equally visible. Finally is the artist Felix Gonzalez-Torres. His art has a beautiful quality of intimacy to which I was instantly attracted. Gonzalez-Torres was an openly gay man who lived with his lover through the highpoint of the AIDS epidemic. When his lover, Ross, eventually died from complications brought on by this disease, his art was instantly affected. In his work, Untitled (Portrait of Ross in L.A.), he created a large pile of candy that was the exact weight of his lover, which he then encouraged the audience to take with them as they left, in effect perpetuating Ross’s memory and spreading him out over the world. I loved the connection this gave the artist to his audience, giving them something with which to physically walk away and remind them.
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ARTS AND HUMANITIES Background (Books) Another part of my initial research to broaden my contextual understanding of feminism was doing a lot of reading. With the help of my mentor, I collected a long list of books to read, all of them dealing with feminine issues and feminist ideology. The ones I have read so far have proven to be undoubtedly important in informing my project further. The first book I read was Cinderella Ate My Daughter by Peggy Orenstein. In it, Orenstein does her best to analyze and explain the ideas behind and driving the “girly girl” culture and how it informs young women to behave. It got me thinking about pink as a symbol for our gender, the Disney Princesses as a nationwide fad, and what we are telling our young girls about who we think they ought to be when we give them these gendered toys. Next were a series of essays titled The Culture of Beauty. Assembled as a selection of opposing arguments, this book helped me to further understand the effect society’s beauty ideals have on individual women. Whether it be the multitude of images that influence our body image, or the cosmetic industry that relies on making women feel inadequate, many women have unhealthy relationships with their body because of these unrealistic paradigms. Men Explain Things to Me is a collection of essays by Rebecca Solnit, a historian, journalist and feminist. Not only did I find her content both wonderfully enlightening and captivating, but her poetic language really drew me in. It was visceral and visual, already painting pictures in my mind. For example, when speaking on the widespread proliferation of sexual assault in this country, she said “We have dots so close they’re splatters melting into a stain, but hardly anyone connects them, or names that stain.” (Solnit 36).
Results As I continued my research, I kept detailed notes of ideas and phrases that struck me. With my mind almost overflowing with information and thoughts, I eventually reached a point where I began condensing my list to the ones that felt the most important to me. These are the products of that list, conveying several feminist themes I find the most important to me.
Mental Corset (Lauren). C. Bowman, 2017. Photography and mixed media.
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Mental Corset (Diane). C. Bowman, 2017. Photography and metallic pen.
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ARTS AND HUMANITIES The idea of the mental corset is meant to portray the unique, internal struggles all women face in an attempt to conform to some societal perception of beauty. The first image deals with the concept of being restrained by confining versions of femininity, and its effects on individual expression. The second image is meant to convey the struggles of women as they age, when society repeatedly tells them they are better off looking “younger�, and the resulting unhealthy relationship many women then have with their mature bodies. The next concept I investigated was the permeation of pink in modern female life. I wanted to address the way the colour pink tends to oppress individuality for the sake of colour coated gendering. The black and white portrait appears boring and less interesting than the pink floating around her, while the slash across her eyes serves to dehumanise her further, taking away the expressiveness of her eyes and blinding her vision with pink.
Involuntarily Rose-Coloured. Claire Bowman, 2017. Graphite and gauche.
Infestation. Claire Bowman, 2017. Mixed media.
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I also wanted to make a piece that spoke to the proliferation of the colour in our consumer culture. When marketing toward women, companies often take the “easy route” of just making the item pink, as though that makes it suddenly a women’s product (and it couldn't be used by a woman until the colour alteration). Therefore, I wanted to make a piece that spoke to this ridiculousness and unnecessary marketing strategy, where pink appears to be invading most aspects of normal life. ***
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ARTS AND HUMANITIES Between September 2001 and 2012, 11,766 people in the US died from domestic violence – that is more than those who died in the 9/11 tragedy, plus all soldiers killed in Iraq and Afghanistan, combined (Solnit 23). I wanted to create a piece that spoke to those people as unique individuals who were ultimately silenced while still creating a whole piece that can attest to the larger scale issue. Each key is unique, used for an individual lock and is also supposed to represent safety and protection, especially in the home, which is the greatest irony for those affected. Each key represents ten people, creating a total of 1,177 keys. ***
Memorial. 2017. Keys, fishing line and wood. Claire Bowman, 2017.
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The Baggage of Feminism. Claire Bowman, 2017. Large format print and ink.
This final piece is meant to represent the baggage of insulting terms that comes with publicly expressing feminist ideology. The anonymity of online insults and comments gives people an opportunity to divide themselves further from those who have differing opinions, closing their minds as they label the opposition as “other�. While from a distance, this large piece seems dominated by the cloud of insults, when one approaches it, they will notice several smaller signatures within the words, giving humanity back to those whom the labels were attempting to dehumanize.
Future Studies In the future, I plan to continue both the research and execution of feminist inspired art, whether it be through reading, exploring galleries, or talking to more women about their experiences. This in fact would be a more specific goal I have for the near future, especially to expand my knowledge and expression beyond my own circumstances and privilege. As a cis-gender, middle class white woman, there is still much I do not yet understand about the struggles countless other women face on a daily basis, and I hope my art can eventually be influenced by the people I aspire to talk with.
Acknowledgements I would first like to thank the Berry Family and University Honors Program for funding my research this summer. Without them, I would never have been able to dream up these concepts freely. Next, I would like to thank my mentor, Emily Sullivan Smith for her unyielding support, and her encouragement to enter the program in the first place. Finally, I would like to thank my parents and the rest of my cohort, for showing their continuous support to me and for my art through the summer, and making this program one of the best experiences of my life. 8
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The Baggage of Feminism. Claire Bowman, 2017.
References Gerdes, Louise I. The Culture of Beauty. Farmington Hills, MI: Greenhaven Press, 2013. Print. Gonzalez-Torres, Felix. Untitled. 1991. Gonzalez-Torres, Felix. Untitled (Portrait of Ross in L.A.). 1991. Guerrilla Girls. "Do Women Have to be Naked to get into the Met Museum? 2005. Kruger, Barbara. Untitled (Your gaze hits the side of my face). 1981. Photograph and type on paperboard. Orenstein, Peggy. Cinderella Ate My Daughter: Dispatches from the Front Lines of the New Girlie-girl Culture. New York, NY: HarperCollins, 2011. Print. Solnit, Rebecca. Men Explain Things to Me. Chicago, Illinois: Haymarket Books, 2014. Print.
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Perceiving Place: A Socially-based Design Case Study Mary A. Brinkman1,2,3 University of Dayton, 300 College Park, Dayton, OH, 45469 1. Department of Art and Design 2. Berry Summer Thesis Institute 3. University Honors Program
Advisor: Misty Thomas-Trout, MFA Department of Art and Design Corresponding Author: Mary A. Brinkman Email: brinkmanm3@udayton.edu
Abstract Space is one of the most basic foundational systems for living things. No matter what happens in the world, it happens in a spatial setting. The specific design of that setting or place plays a large role in the lives of those within it.1 Other factors within place — factors that might inform design choices — also affect perception of place. Broad consensus exists in scholarly literature about the general role that history, culture, environment and social factors play into the perception of place. It has been supported through past studies and research that our perceptions of reality before actually observing it, actively affect the reality we then observe.2 In other words, certain factors are constantly influencing the way in which we process information and interpret various spaces. However, a confirmatory analysis of this model, especially in regards to the specific categories influencing perception, has yet to be conducted. The purpose of this study is to expand upon and further explore the notion of such categories of place in informing graphic design. It is hypothesized that a person’s perception is influenced by a variety of factors such as history, culture, environment and social make-up. These actively affect what one will interpret as reality. This research and connected studies have led me to develop an in-depth understanding of what influences one’s perception of a place, specifically between contrasting neighborhoods within Dayton, Ohio. I am looking to find if the application of this knowledge will lead to the creation of more informed and effective design, especially in the formation and growth of positive social change. It is hoped that this research will educate designers, as well as the broader public, as to what is affecting their perception of place. This will then contribute to the understanding of what happens within that space and what goes into the larger make-up and language of place. Furthermore, I am working to create a body of information that can lend a hand in understanding how one can approach the make-up of place and harness qualitative data found within. Success with this approach will provide a powerful social model for advancing communication across various levels of perception. Outcomes from such research will steer future studies towards resolving misconceptions of place or will work to promote a better understanding of place through the medium of social based design.
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ARTS AND HUMANITIES Introduction Locations, or the separation of distinct areas, are defined by a multitude of factors. People are conditioned to think about a particular place through the association of these factors. Edward McMahon writes, “A sense of place is a unique collection of qualities and characteristics — visual, cultural, social, and environmental — that provide meaning to location. Sense of place is what makes one city or town different from another, but sense of place is also what makes our physical surroundings worth caring about”.3 Perception of place, and even the understanding of this concept is not common knowledge, but can be learned through direct contact with the people and issues at the heart of these areas. This research works to do just that: to gather an understanding of root characteristics and discover their relationship, meaning, and value within location.
Initial Research Initial research consisted of looking at the broader Dayton area and learning about the various neighborhoods within Montgomery County. After developing a basic knowledge of the city and its different neighborhoods, I started my work within the Twin Towers neighborhood. Twin Towers, also known as east Dayton, is a relatively small neighborhood, defined by its borders of Wayne Avenue, Wyoming Street, Steve Whalen Boulevard and U.S. Route 35. With consistently high immigration and poverty rates, Twin Towers is ranked barely livable. The area has a population of roughly 2,900 people and a median income of $17,000. Around 47 percent of the population has received a high school diploma and about 10 percent hold some sort of higher degree.4 The neighborhood began largely as an Appalachian community with a smaller presence of German and Jewish populations, but has transitioned into a largely white, Hispanic and African-American make-up.4,5 While this initial research shed some background on Twin Towers, it did very little in informing me about the specific characteristics of the neighborhood. Considering the topic of socially-based design influencing perception of place, I realized that experiential research would have the greatest outcomes. The reading of census reports, history books and newspapers could not provide the qualitative results that the interaction with those actually living within the spaces I was researching could. Therefore, in order to expand my research methodology, I partnered an in-depth literature review with an experiential and hands-on case study.
Twin Towers Neighborhood. Photograph courtesy of A. Brinkman, 2017.
Methods In response to initial research and past studies of perception, I decided to break down my methods into five segments: the study of a larger Dayton narrative, creation of Twin Towers narrative, analysis, development of future surveys and application of design. However, in this section of the text, we will only be looking at the background of narrative, the larger Dayton area, and the development of Twin Towers narrative. Results, analysis, future studies and application of design will follow in later sections.
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ARTS AND HUMANITIES Background: Use of Narrative “Stories breathe...they give life to people by establishing identities and situating experience within time. They endow us with a sense of self and give meaning to relationships.” (Irvine, Leslie).6 I began my research by using a means of qualitative data collection: narratives. After understanding that human dynamics lie at the heart of perception, I decided to analyze these dynamics through the study of people at the heart of place. Through the use of narrative, I began responding to the main goal of my work: to create meaningful connections with people and to gather the unique stories and voices of those within place — to design with the people and for the people. Research is increasingly pointing to the use of narratives as a successful way to anchor reality. By looking at people as living museums or as representations of place, I am studying the ways in which collective memory can be anchored not only in visual monuments and in works of art, but also in social narratives and the human construct.7 I decided to interview people from several neighborhoods — people living within the place, but also those viewing it externally. These narratives would allow for the diverse collection of a range of material. This material would reveal the ways in which people attribute meaning to their neighborhoods while living there, but would also look at how external sources are perceiving such places and why such differences in perception occur. The collection of narratives would reveal how each distinctive experience of place fits within the larger region, image and language of Dayton. I plan to juxtapose these experiences of place with my own personal perception of Dayton, Ohio.
The Facing Project — Getting to the Heart of the Gem City I began my study of narrative by analyzing a large set of stories already collected: a narrative body titled Facing Dayton: Neighborhood Narratives. The Facing Project is a story-telling venture that intends to bring awareness to the assets of a community and the human rights issues occurring within neighborhoods.8 The project’s main goal is to inspire social action. Run by the University of Dayton’s Fitz Center during the spring of 2017, UD students had the opportunity to go out into the city of Dayton and interview citizens within various neighborhoods. “The pairs meet, get to know each other and share stories of triumph and tragedy, of loneliness and community, of hate and happiness, of deep depression and lofty goals.”8 I read these narratives in order to gain a better perspective of what was occurring within Dayton and with its citizens. After thorough reading and analysis, I pulled out specific themes and factors within each story and at each place’s foundation. I looked at these themes across the board and gathered the overarching ones prevalent in almost every narrative. These overarching themes gave me a starting point for my own set of narratives. By analyzing the stories of the larger Dayton area, I was beginning to understand what aspects of history, culture, environment and social make-up were extremely prevalent in current notions of perception and that the influences of perception could indeed be broken down into such categories.
Twin Towers Narrative After analyzing the narratives of the broader Dayton area and finding data supporting my initial hypothesis, I narrowed my focus to the Twin Towers neighborhood. I began looking at the assets of the neighborhood and what services, businesses and programs are involved within it. I then reached out to these “community leaders” and asked them about their roles within Twin Towers and their perceptions about it. While some of these services and programs were externally based, I also interviewed people within the neighborhood, or internal sources. After collecting a large body of narratives, I repeated the process that I had used with the Facing Project narratives, analyzing the stories and interviews and pulling out the repeated factors and influences of perception.
Reflection Conducting my own set of narratives has not only allowed me to understand Twin Towers as a whole, but has allowed me to form relationships with the people living there and experience their lives and the factors of their community firsthand. I’ve built mutual trust with the neighborhood’s residents 12
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ARTS AND HUMANITIES and developed an awareness of the unique language of place. As seen in many social design cases and projects, “a few conversations with community leaders will rarely win over a community and might undermine the entire initiative, preventing any substantial social change.”9 This is why it is so important for a designer to physically interact with the spaces and people that they are creating design for — to interact and create with the goal of building a long-term relationship with place, rather than simply creating a short-term form of design. While graphic design is social by nature, it is easy for designers to lose touch with the implications of their work.9 Interaction, experience and awareness is crucial to making a difference through design. While I spent a large portion of my time interviewing people and listening to their stories, I also spent a great deal of time simply living within Twin Towers. I attended mass, sat in on recovery program board meetings, helped launch an outreach program, visited resident’s homes, shared meals, worked at an art studio, babysat, collaborated with businesses and services and participated in community events. For “when you build a thing, you cannot merely build [or design] that thing in isolation, but must also repair [and engage] the world around it and within it, so that the larger world at that one place becomes more coherent, and more whole; and the thing which you make takes its place in the web of nature, as you make it.”10
Results See Figure 1 for historically-based factors of influence. See Figure 2 for culturally-based factors of influence. See Figure 3 for environmentally-based factors of influence. See Figure 4 for socially-based factors of influence.
Figure 1. Historically-based factors of influence. Data compiled by A. Brinkman, 2017.
Figure 2. Culturally-based factors of influence. Data compiled by A. Brinkman, 2017.
Figure 3. Environmentally-based factors of influence. Data compiled by A. Brinkman, 2017.
Figure 4. Socially-based factors of influence. Data compiled by A. Brinkman, 2017.
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ARTS AND HUMANITIES Analysis and Discussion In order to understand the qualitative means of data collection and the results gathered, one must first understand perception at its most basic level and how perception can be broken down and grouped into various lenses of influence. This is done below in relation to direct results and specific findings from the methods above.
History Our perceptions of reality today are combined with evidence from the past. History and memory are constantly functioning in our lives; however, this functioning has traditionally been a controversial topic across all areas of study. Early philosophers often discussed the role of history. Descartes noted, “even the most faithful histories, if they do not change or augment the value of things in order to render them more worthy of being read, at least almost always omit the most base and least illustrious circumstances, so that what remains does not appear such as it is.”11 This notion prompts the question: how can we look at history as a means for research, when we are unaware of whether the past is being accurately represented? History, just as Descartes discusses, is almost always edited in some way. Kant and Nietzsche also addressed such notions. However differing the viewpoints of these philosophers and even current research, it is no secret that the past plays a significant role in the present. Rather, the question should be: how much of the past should we rely on, especially when differences in historical traditions give various contents to different realizations of the same structures?12 My research both acknowledges and supports the fact that people have different historical traditions, but specifically works to discover what these traditions are, and how they vary between groups, environments, and cultures. I also studied how different historical understandings lead to differing perceptions of place and sometimes tensions within space because of contrasting notions of the past. As we all bring our own content to a space, research has shown that it is valuable to study history in connection to memory and what methods can be used to approach this type of research.7 Differences in historical traditions give different contents to realizations of the same structures. For example, someone living with a developed historical background of an area will view the structures within the area very differently than another that has a limited historical understanding. Someone driving through Twin Towers with very little historical knowledge of the place might perceive the area as lacking, simple and barren. However, those that have an in-depth understanding of history’s role within the area would perceive the same buildings in a very different light, thus approaching space very differently than someone who doesn’t have similar historical context.
Historic Buildings in the Twin Towers Neighborhood. Photograph courtesy of A. Brinkman, 2017.
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Another example of history’s effect on perception can be seen in the neighborhood’s past with U.S. Route 35. The construction began in the 1960s and was predicted to take 5 years to complete, but ended up taking 10 years.13 The construction displaced over 20,000 people and led to the closure of many shops and local businesses. Its construction led to the first, and main, flight of people from Twin Towers. Many of the older generations currently living within the neighborhood were directly affected by its construction and still have strong feelings: some residents harbor anger due to the loss of their homes, some feel sadness because of Proceedings 2017
ARTS AND HUMANITIES the downfall of the neighborhood, and other residents compare what Twin Towers was to what it is now. The older generation’s historical understanding is very different than those currently moving into Twin Towers. They become angered when different cultures and people come into the area, with no historical knowledge and expect to be given homes that they feel like the government still owes them.13 When these newcomers have very little respect towards property and the up-keep of their home, tensions arise. Thus, history and its connection to the present is affecting how Twin Towers residents view the neighborhood’s current state. Lastly, we can look at perception through the study of perceptual constancy and reputation. More and more cities are finding that citizens have a desire for the historic — people are drawn to historic sites as they provide tangible evidence of the past. People want to place themselves in narrative and live within a functioning of history.7 This is seen in the Oregon District of Dayton. Once a red-light district, the area is now a thriving business district and growing hub for restaurants, activities and culture.13 However, the transition to such a state is not easy, as history is connected to memory, and thus tied to reputation. Perceptual constancy refers to a stableness in perception. Humans have a tendency to perceive the make-up of a place and associated objects as relatively stable and unchanging, despite possible changes in the imagery and data received.14 Perceptual constancy connects heavily to notion of place through reputation. For example, places will maintain consistent reputations because of perceptual constancy; thus, even if there are traits that arise in a space, contradictory of initial traits observed, these new traits or notions toward space will often be ignored and the space will be perceived in line with popular opinions or based on historical statuses. This makes it very difficult for places to break or change reputation.14 Even if an area is starting to grow and thrive, it can still face negative perceptions due to past reputation. While this connects greatly to a place’s history, it also reflects other current conditions such as culture, environment and social makeup, as will be discussed. In order to create effective design, it is necessary for the designer to understand that this constancy exists, the level to which it exists and functions, and in what ways such structures can be approached, especially if working to change reputation. In order to address the active role and influence of history in perception of place, one can study an area’s historical preservation. Historic preservation “encompasses the range of strategies by which historic structures are maintained, manipulated, and managed.”7 In working to understand how history affects perception, it is crucial to look at how a place harnesses historical preservation, or rather lacks such preservation. Such preservation provides tangible evidence of the past. Many people regard historical preservation as something that exists solely in the recognizable structures of space: churches, buildings and homes. Twin Towers has very limited historical preservation in the form of physical monuments; however, it does have a great deal of history even though not all of it is active or maintained through physical structures. Through my research and work, I challenge the notion that historical preservation can only be managed through physical monuments. Rather, I argue that people can be just as powerful or even more so in preserving such traditions and notions.
Culture “Even when small fragments of culture are elevated to awareness, they are difficult to change, not only because they are so personally experienced but because people cannot act or interact at all in any meaningful way except through the medium of culture.”1 Our perception and thought processes are not independent of the cultural environment we exist in. Culture affects one’s eating habits, clothing and actions. While culture shapes outward behavior, it also influences brain function and how memories are recalled and presented to others.15 A recent study of East Asians and Westerners suggest that culture actively affects the way people view the world. The effect is not so much seen in structural changes, but rather at a level of perception. It is not that a difference in cultures would change the make-up of the data being perceived, but that a difference in cultures affects how people respond to the data that they are perceiving. Different cultures will focus on different parts of an environment or respond differently to a place. The study found that east Asian cultures are more interdependent and individually spend more time monitoring Proceedings 2017
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ARTS AND HUMANITIES their environment and others, while westerners focus on the individual and central objects as these cultures tend to be more independent and focused more on self than others.15 Significant evidence has been found that people who are brought up in different cultures live in different perceptual worlds. “This can be found in their manner of orienting themselves in space, how they get around and move from one place to the next.”1 For example, my research revealed that Twin Towers has a very large immigrant population, but that many of these populations feel like they don’t fit well within the neighborhood because Dayton as a whole was not really designed for the integration and functioning of different cultures. Many feel like their cultural needs aren’t being met. Because of this, they perceive Twin Towers differently than a group who’s needs are being met. Culture affects your language, attention and subcultures, but how does this affect perception of place? Much of this cultural perception occurs in a three-step process: selection, organization, and interpretation.16 While people in different cultures are presented with the same data, different culture’s will select different information to process first. As mentioned above, westerners found themselves paying more attention to certain parts of a space over others. The second step in cultural perception is organization. One must organize the information they are presented. How will they group things within this space? Because of this step in cultural perception we should expect to find barriers to cross-cultural communication and understanding, as people associate certain factors within place differently based on their culture.17 My studies found that within the culture of Twin Towers there is a unique mindset, observed by both those living there, but also by external sources. Because the neighborhood has higher poverty rates, there is a great percentage of people living on welfare and supported by food stamps. Furthermore, Twin Towers has a great deal of services reaching out to those living there. This dynamic has led to what many residents call a mindset of entitlement. Because many of the residents have constantly been given items, community members are concerned that the younger generation has never seen their parents work and that they will be brought up with a similar mindset: that their basic needs will be met through these services and outreach programs.13 These interviews revealed a much larger challenge of the neighborhood — a challenge engrained in the culture of place. Because of this mindset, many view the neighborhood as a hub for service outreach, but also as a place for those in need of such services. In this example, there is a grouping of the people within place. The last step is interpretation. The same situation can be interpreted quite differently by diverse people. This can be understood through a simple hypothetical situation. Let’s say that you were wandering around the grocery store looking for meat and you came upon dog meat along with all the other options. Many reading this will have some reaction of disgust or confusion to this scene, maybe even feelings of intrigue. Your reaction of disgust to the idea of someone eating a dog as food is actually a culturally learned interpretation.17 This same idea is applied to place. The feelings we have toward different neighborhoods are not random, but exist based on interpretations rooted in culture. Twin Towers has historically been known as a place of drugs and mental diseases. Cultures that are not used to interpreting place with these additional social factors, often don’t know how to interpret or respond to such sensory data. Some cultures have a certain stigma towards these people and issues. However, so many fail to recognize or have the proper cultural understanding to realize that much of Twin Tower’s drug problems and mental health crisis lies in trauma. My research revealed that almost 100 percent of the cases that the Montgomery County Drug Courts process have drug addictions stemming from childhood trauma from violence, sexual assault or some other past negative treatment at their heart.13 However, regardless of one’s cultural understanding of drugs and mental disease, these challenges often bring negative stereotypes to a place, even if that place such as Twin Towers is a large advocate for growth and change in these areas, and actually has a better cultural understanding of what lies at the root of such structures. How we interpret certain elements of a place is founded in our cultural interpretation. Lastly, it is important to understand whether a neighborhood has a high or low context culture. A place with low context means there is very limited sharing, while a place with high context means that there is more sharing between people and a furthered understanding.17 Communication between strangers or different cultures would fall under the category of low context because there is limited 16
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ARTS AND HUMANITIES to no shared experiences between the groups; however, when there is communication with familiar groups, communication is less explicit because of shared context and a shared understanding of culture in regards to place. When looked at from the perspective of high and low context, it is easy to see how language and culture can be either a means of separation or a functioning of sharing.17 When designing, one of the most important steps is to understand what context a place falls under and how you can approach this. Twin Towers, with its large diversity, would fall on the scale closer to low context; however, some shared historical contexts with older generations, and shared cultures between newer groups and populations, does create more of a high context. I would argue that Twin Towers actually falls somewhere between the two. My research has shown that spaces might not only be one or the other and that the study of high and low context cultures should actually be looked at as a scale, rather than as separate categories.
Environment and Perception While our perception and thought processes are not independent of our culture, they are also not independent from our environment. As humans, we are always in an active relationship with our environment, and thus with the place we are within. This is understood through sensory input. Sensory input is the physical relationship we have with environment. Humans have cells in our mouths and noses that combine with those from the environment to send impulses to the brain, thus revealing the interrelationships of perception, thought and environment.17 However, the study of environment can also be looked at through transportation, architecture and the basic conditions that operate in place. But once again, how can environment’s influence on one’s perception be measured? Recent research has looked at the use of cognitive maps. Cognitive mapping is the process that humans use to think about space and the ways in which they reflect and act within place.18 Milgram and Jodelet found in their study with cognitive mapping that major elements of the city that they were studying emerged and that participants then linked these elements together through their everyday experiences. By having participants physically draw out a map of the area, they recognized differences in what environments people were drawing within spaces. Often time, the things that people were including on their map were places or characteristics of the area that held special value in their lives. This type of differing value is because of the various environments in which people grew up or with which they would associate.18 One’s environment allows a person to navigate and remember. An understanding of the specific cognitive mapping that people have towards a neighborhood would provide insight into what aspects of a place are being valued or undervalued. With this knowledge, designers can start designing from the perspective of those living there. The ability to inform design is often limited to architects, engineers and designers, but who knows the city better than its residents?18 By picking up on the feelings evoked by surroundings or environment, designers can reinterpret place. Cognitive mapping has shown that people place more value and importance on areas within the place in which they spend the most time or develop the strongest relationships — places like the home, school, church or work environment. In understanding this, there is great importance in realizing how these parts of environment are affecting how people regard the larger space. One might ask: how does the education Proceedings 2017
Homes on Xenia Avenue. Photograph courtesy of A. Brinkman, 2017.
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ARTS AND HUMANITIES of a neighborhood add to the larger perception of place? How is the housing situation within this space? Are there significant financial challenges? What are the environmental assets? Findings have also revealed that no two people see place in the same way, thus implying that not all men relate to the world around them the same due to differing relationships with environment. When studying perception, it is necessary to understand the legibility of environment — whether or not the factors of environment offer people insight about where they are, how to situate themselves,and what they can do within the place.18 One might look at how housing, education and amenities reveal a lens through which place can be perceived. Past map-making has actually found that people most quickly notice and remember environmental elements of an area as they play a crucial role in depicting and forming space. One of the biggest environmental influences on perception is housing. Twin Towers has a very large rental population. Home ownership has been decreasing within the past few years; once at 85 percent, it now has fallen to around 35 percent.13 A decrease in ownership has brought a loss of neighborhood pride and an increase in demographic diversity. Another main influencer of environmental perception is the role of Interstates 70 and 75. While roads are often seen as points of connection between groups of people, the crossing of 70 and 75 also brought in a large group of drug dealers to the area, creating an environment that would foster large social problems. Home ownership, an issue across many neighborhoods, is recognized by almost all groups and is actively affecting how people view Twin Towers. Moreover, it has been found that control over an environment or space creates positive feelings towards place. The manipulation, molding or decoration of space allows for a positive sense of self-identity within space. This opportunity to connect to environment ties heavily to identity and personal well-being.1 Because Twin Towers has such a low percentage of home ownership, there might be a decrease in such feelings of self-identity within space.
Social Factors and Perception Lastly, as you might imagine, one’s position in society and the factors occurring within society shapes the view of space and place. A person’s socio-economic background, family, occupational role and economic role not only influences day-to-day life, but influence the choices one makes towards space. After reviewing literature and past studies, it was apparent that while perception is affected by social factors, the act of separating and categorizing perceptions into the area of social factors may not be as effective. The various social factors of a space are ingrained within the other three areas of history, culture and social makeup. However, while this is true, it has been found across areas of study that social factors are actively affecting what people observe and perceive. Social scientist Timothy Pachirat’s book, Every 12 Seconds, discusses this. “In addition to the assumptions that surround my various jobs in the slaughterhouse, my self-presentation, appearance
Figure 5. Future Studies. A. Brinkman, 2017.
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ARTS AND HUMANITIES and mannerisms combined to create certain interpretations of me by others. Primary perhaps was my appearance…all of these factors and more affected how I was seen by others and consequently, what I was able to see.” Whether or not we recognize it, we have preconceived perceptions of people based on their social standing and tend to apply these perceptions to the places in which one resides.19 However, social factors can be looked at from another perspective. Social influence on perception of place can also be defined as the factors that affect lifestyle — factors such as generational cycles, town vs. gown, and emotions or evoked feelings. Even the role of health systems in place can be argued to be social factors as they are so affected by social determinants “such as income and wealth, education, occupation, and experiences based on racial or ethnic identification.”20 In analyzing place in regards to social factors, we cannot solely focus on the factors themselves, but should also study the institutions that are meant to address such factors. In Twin Towers, rather than simply acknowledge the factor of youth delinquency, it is necessary to understand what structures are addressing such social issues. Rather than focusing solely on the use of heroin, one must analyze the role that methadone clinics are playing in such use. It is not enough to simply point out social factors, but we must study them in relation to history, culture and environment.
Conclusion Understanding perception is critical in determining how we interpret reality and what factors are affecting interpretation of place. While I am aware of the dangers of the search for partial equivalences across levels of interpretation and place, I am still working to develop, within the limits of my knowledge, some guidelines and basic factors behind the specific institutions of a particular place — the structural invariants as seen in history, culture, environment, and social make up. Hopefully, based on such knowledge, I can begin to address the inequalities among place and in understanding it.
New Hope, Twin Tower's Art Studio. Photograph courtesy of A. Brinkman, 2017.
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ARTS AND HUMANITIES Future Studies In my initial proposal, I stated that I hoped to study several neighborhoods and compare their perceptions of place. However, because of the experiential means of research I used, and my goal to spend time with the people and within the space of each neighborhood, I was only able to study and analyze the Twin Towers neighborhood this summer. I plan to use my findings from the Twin Towers neighborhood as a foundational study. I hope to create a basic guide into interpreting place that social scientists, designers and the general public can use in analyzing any place or neighborhood at a basic level. I will continue my research within Twin Towers and further expand my methods of study. Figure 5 shows possible studies that I will be conducting within the next several months. These studies and surveys were created with the intention of expanding knowledge within the specific categories of perception. These methods will target the various areas within the categories of history, culture, environment and social make-up, but will also note their interrelated structure.
Applying Design Once I gather substantial data and knowledge of the area, I can address the final stage of my thesis work: applying design to place. I will do this in one of two ways. I can use a theory of design that isolates visual perception from linguistic interpretation and encourages indifference to historical, cultural, environmental and social meaning, or I can create design in a way that promotes the use of these factors for the neighborhood’s benefit. However, no matter my means of construction, I want to create something within the Twin Towers neighborhood that will target current negative perceptions, and promote a space open to growth.
Acknowledgements I would like to acknowledge the many people that were supportive in my research this summer and in writing this paper. I would like to thank Professor Misty Thomas-Trout, the University of Dayton Honors Program, the Berry Family, the Berry Foundation, the University of Dayton Art Department, New Hope Parish and Art Studio, all who were interviewed, and Kathy and Mark Brinkman. Additional thanks to the University Honors Program staff and Bro. Raymond Fitz, Ph.D., for their constant excitement, passion and guidance.
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ARTS AND HUMANITIES References 1.
Hall, Edward T. The Hidden Dimension. Peter Smith Pub, 1992.
2.
Bradley, Steven. “How Perception Alters Reality And Design Influences Content.” Vanseo Design, 7 Nov. 2013, vanseodesign.com/web-design/perception-and-reality/.
3.
Hsiao, Alesia. "Why Sense of Place Is Worth Caring About." Planetizen. N.p., n.d. Web. 20 Jan. 2017.
4.
Petrella, Bradley, and Lauren Breitenstein. The Facing Project: History of Dayton Neighborhoods. The Facing Project: History of Dayton Neighborhoods.
5.
Areavibes. "Dayton, OH Neighborhoods." Areavibes. N.p., n.d. Web. 07 Feb. 2017.
6.
Irvine, Leslie. My Dog Always Eats First: Homeless People and Their Animals. Lynne Rienner Publishers, 2016.
7.
Barthel, Diane. “Getting in Touch with History: The Role of Historic Preservation in Shaping Collective Memories.” Qualitative Sociology, vol. 19, no. 3, 1996, pp. 345–364., doi:10.1007/bf02393276.
8.
The Facing Project, facingproject.com/.
9.
Shea, Andrew. Designing for Social Change: Strategies for Community-based Graphic Design. New York: Princeton Architectural, 2012. Print.
10. Alexander, Christopher, Sara Ishikawa, and Murray Silverstein. A Pattern Language: Towns, Buildings, Construction. New York: Oxford UP, 1977. Print. 11. Descartes, Renee. Discourse on Method. Broadview Press, 2017. 12. Bourdieu, Pierre, et al. Distinction: A Social Critique of the Judgement of Taste. Routledge, Taylor & Francis Group, 2015. 13. Personal Communication, 2017. 14. Dias, Laura. Human Relations. Saylor Academy, 2012, saylordotorg.github.io/text_human-relations/s05-03-humanrelations-perception-s-e.html. 15. Burton, Kelli W. “Cultural Experience Affects Perception.” The Dana Foundation, 2007, www.dana.org/Publications/ Brainwork/Details.aspx?id=43697. 16. Chiao, Joan Y., et al. “Dynamic Cultural Influences on Neural Representations of the Self.” Journal of Cognitive Neuroscience, vol. 22, no. 1, 2010, pp. 1–11., doi:10.1162/jocn.2009.21192. 17. Ting-Toomey, Stella. Language, Communication, and Culture: Current Directions. Sage Publ., 1993. 18. “Human Perception and Environmental Experience.” The People, Place, and Space Reader, peopleplacespace.org/. 19. Pachirat, Timothy. Every Twelve Seconds: Industrialized Slaughter and the Politics of Sight. Yale University Press, 2013. 20. “U.S. Health in International Perspective: Shorter Lives, Poorer Health’ at NAP.edu.” Edited by Steven H Woolf and Laudan Aron, National Academies Press: OpenBook, www.nap.edu/read/13497/chapter/1.
Other Sources Cognitive Science and Design. Perf. Alex Faaborg. YouTube. Google Developers, n.d. Web. "Dayton, Ohio." Ohio History Central. N.p., n.d. Web. Gieseking, Jack Jen. "Where We Go from Here." Qualitative Inquiry 19.9 (2013): 712-24. Web. Simmons, Christopher. Just Design: Socially Conscious Design for Critical Causes. Cincinnati, OH: How, 2011. Print. “U.S. Health in International Perspective: Shorter Lives, Poorer Health’ at NAP.edu.” Edited by Steven H Woolf and Laudan Aron, National Academies Press: OpenBook, www.nap.edu/read/13497/chapter/1.
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Pocahontas in the Land of Miscegenation: Persona and the Fantasy of Whiteness in Danzy Senna's Caucasia William E. Landers1,2,3 University of Dayton, 300 College Park, Dayton, OH, 45469 1. Department of English 2. Berry Summer Thesis Institute 3. University Honors Program
Advisor: Thomas Morgan, Ph.D. Department of English Corresponding Author: William E. Landers Email: welanders24@gmail.com
Preface Racial violence in news headlines has dramatically returned racial injustice, once popularly thought to be a relic from history textbooks, to the national discussion. The tragedy in Charlottesville was a dramatic showcase of the seemingly outdated ideology of white supremacy. The national outcry against neo-Nazis and other overtly racist groups appears to confirm that most Americans have moved beyond their unjust past. It seems that racism is now unacceptable in our society. Unfortunately, the effects of racial injustice are actually accepted by Americans every day, and the sentiments that erupted in Charlottesville are not complete outliers in modern America. Conditions of racial discrimination are foundational to the United States, and they constitute a status quo for Americans. Everyday racism is subtle, not dramatic. Today, race usually operates through social conventions, unspoken agreements and implicit biases. Public violence may be less prevalent now than in previous times in our country’s history, but the logic of racism remains a potent force.
Introduction Persona defines the way that a society receives an individual. It is a purely external image for others to interpret. Americans often assume an intentionality in this visual and behavior presentation, as if each individual constructs the particular, external image that he or she desires. This understanding of persona as an agency of internal presence underlies many currents in our society, from greetingcard truisms to market-fueling consumer choices. It presumes the sovereign individuality of each person to posture, say, wear, drive or eat whatever allows him or her to express a quintessential “me.” However, socialization, the process by which dominant views and beliefs in American culture that imbed themselves in individual perspectives, defines the creation and limits of individuality. The philosopher Charles Mills describes this process as an “agreement to misinterpret the world” (The Racial Contract 18). Certain illusions, especially the fantasy of white supremacy, are continually perpetuated by a cultural echo-chamber effect. The assumed presence of race is implanted within each individual and is then validated within groups. Hence, the fantasy of whiteness can claim ideological universality in a white American society. The dynamic of racial perception imbues internal character with external judgement, empowering the dominant fantasy of whiteness to invent racialized, public personas and assign them to nonwhite individuals. In this way, persona actually functions as an external distortion of self-image. It displaces individuality.
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ARTS AND HUMANITIES Passing narratives illustrate the dissonance between the external persona and the internality of a person. This type of story follows phenotypically ambiguous characters, who are able to pretend, or “pass,” as members of another race. This often takes the form of a nonwhite person passing as white. This genre, which includes the likes of James Weldon Johnson’s The Autobiography of an Ex-Colored Man and Nella Larsen’s Passing, typically explores the realities of explicitly institutionalized racism. It reveals a material advantage to whiteness, which is implicit in a character’s desire and willingness to step beyond his or her private identity. However, slavery, segregation and the general concept of legalized racism seem the province of “different times.” In a contemporary frame, the author, Danzy Senna, explains passing narratives as a way to illustrate the “authentic self,” the identity that an individual personally constructs (Claudia M. Milian Arias, “An Interview with Danzy Senna” 449). She suggests a converse relationship between the internal personality and the public persona, which conceals truth for the sake of mobility. In Senna’s view, the passing character’s phenotypical ambiguity plays on the fluidity of racial identification. Ironically, this special transience plays on, and reveals, the persistence of stringent racial categorization as a social process.1 Passing narratives highlight the continuing rigidity of American racial difference within a supposedly multi-cultural, colorblind society. Danzy Senna’s novel, Caucasia, implements the historically relevant genre of the passing narrative to show how racial categorization produces cognitive dissonances between white and black persons in post-segregation America. Though past narratives tend to focus on the material advantages of whiteness, Senna approaches appearance as a matter of survival. Her contemporary novel deals with passing as a method of navigating potentially dangerous social terrain. It extends beyond some traditional passing narratives’ treatment of whiteness as a tool for profit, but it continues their central assertion that racial appearances and behaviors provide markers of operative privilege. Senna’s character, Birdie, plays the role of passer in this narrative. She dons a half-Jewish identity to inconspicuously blend with her white mother, Sandy, as the two characters flee from the threat of federal agents. As the girl and her mother discuss the journey ahead, Birdie is offered a spectrum of white or nearly white personas. In that moment, appearance becomes a natural resource. Together, Birdie and Sandy, the newly dubbed “Jesse and Sheila Goldman,” crisscross the backroads of New England. A pawn-store Star of David necklace and a copy of The Diary of Anne Frank cement the girl’s new persona in most eyes, but “Sheila” reserves a special nickname, “meshugga nebbish,” for skeptics’ ears (Caucasia 140). The chase leads to a small, rural New Hampshire town, where Sandy settles down and Birdie fully inhabits her nearly white persona. Here, the girl’s easy acceptance among white peers brings constant pressure to conform in appearance and behavior. Birdie employs her plausibly white persona to gain social acceptance as a covertly black interloper. However, she must sacrifice the expression of her authentic self to maintain this charade. Whatever her role, Birdie endures dissonance between her internal and external identities. The passing character balances the potential rewards of whiteness with the known detriments of blackness. Often functioning below conscious awareness, the fantasy of white supremacy and its accompanying invention of race inform social interactions and judgements. A persisting white gaze reifies racial difference to impose misidentifying personas on nonwhite individuals, restricting their ownership of personal expression. This process demonstrates the way that race, as a set of psychologically encoded expectations, simultaneously diminishes nonwhite personhood and white sensibility. Hence, racial persona restricts the humanity of nonwhite and white persons in different ways. The pervasive illusion of white supremacy fractures the dominant social perception of reality in America. Social fantasy produces an imaginary persona to displace individuality and expression of the authentic self.
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In her article, “The Mulatto Millennium,” Danzy Senna recalls acting as a “silent witness to [her white peers’] candid racism” (18). She also distinguishes “black” self-identification as a choice to associate with a “shared history” in America, as opposed to the white assignment of difference and inferiority (15). Together, these insights concretely reference a persisting social “othering” of racial appearance and differentiate between self-image and public persona.
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ARTS AND HUMANITIES Inventing Persona Race creates persona in the American social imagination. By crafting an illusory presence of difference, it projects undesirable feelings upon the nonwhite image. Mills approaches the perceptions and behaviors of racial difference as a “white moral cognitive dysfunction” (95). He links the project of European global domination, especially the conquest of the Americas, to a sociallyengineered callousing of empathy. These efforts produced a white colonial fantasy, which legitimated conquest by way of a collective desensitization to white violence against nonwhite populations. As a result, the foundational reasoning of American expansion enshrined the threat of violence with a promise of white innocence. In effect, racial socialization replicates a faulty social perception within each individual, and this lens produces specific responses, which are incongruent with reality. The law professor Derrick Bell, known for his work with Critical Race Theory, further explains this dissonance as he references a kind of race consciousness, which stunts the white ability to “‘imagine the world differently’” (Faces at the Bottom of the Well: The Permanence of Racism 8). Hence, an obsession with race actually impedes the white individual’s faculty of reason in the presence of the nonwhite individual. This imbedded divergence from reason and the image of nonwhiteness characterizes racism’s logic. Whatever its name, this fantasy maintains its distinctiveness by rendering difference to be repulsive. The social divisions of race propagate through a modern ideology of bodily and spatial image. This system creates raced categories, and it enforces group difference along visual definitions of normality. Charles Mills suggests that “one can pretend the body does not matter only because a particular body (the white male body) is being presupposed as the somatic norm” (53). It follows that the appearance of whiteness confers a raceless identity and mobility because the American “somatic norm” aligns with the white phenotype. The markers of difference reinforce Mills’ view of whiteness as a cognitive dysfunction. The needs for oppression and hierarchy create race, and the need for innocence creates fantasy. The rationalization between these and western systems of morality and religion results in a normality, which denies its own injustice. However, social normality defines itself against the traits of others. Derrick Bell touches on the oppositional definition of nonwhiteness when he discloses that “blacks can never be deemed the orthodox, the standard, the conventional” in America (155). The image of nonwhiteness becomes starkly apparent in America because whiteness acquires a racial invisibility as the accepted social norm. As a result, white persons may observe race in others, but not themselves. This supposed racelessness supplies the incentive for passing. A racialized person may shed the restrictive, social baggage of racial hierarchy through appearance. The particular geometry of race in America causes white persons to predicate their behavior on an imaginary, nonwhite figure. This figure removes the actual presence of phenotypically different individuals from social dialogue. Given its origin in a fantasy, the nonwhite figure may drastically conflict with the true personalities of the real people whom it pretends to describe. Specifically, the nonwhite figure exudes a wild and foreign persona, which provides conceptual boundaries to civility. Mills notices a relationship between savagery and race in the white social imagination, suggesting that “nonwhites may be regarded as inherently bestial and savage (quite independently of what they happen to be doing at any particular moment)” in white-normed societies (87). In this way, nonwhite bodies come to represent the dangerous and fearful places that lurk beyond the control or understanding of whiteness. The nonwhite figure acts as a perceptual mirage, which distances white responses from racialized individuals. Persona obscures the inner self by externally imposing a deviant status on nonwhite bodies, thereby partitioning those bodies as uncivil spaces. The American social imagination divorces normality from reality. Persona also displaces individuality in language. Just as the nonwhite image indicates the limits of civility in social spaces, the literary invention of race defines the qualities of Americanness. Venerated American author, Toni Morrison, asserts that race presents a “metaphor for the entire process of Americanization” and that “American means white” (Playing in the Dark: Whiteness and the Literary Imagination 47). She adds that an imaginary “Africanist other”2 provides the tools for crafting an American identity. In this way, nonwhite bodies became “surrogate selves” for early 24
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ARTS AND HUMANITIES white American writers to define the meaning of literary Americanness “through a self-reflexive contemplation of a fabricated, mythological Africanism” (Morrison 37, 47). The literary division between white and nonwhite bodies appears in the passing narrative’s stakes of identity. The passing character must manage each situation as a gamble between the benefits of abiding by a false racial pretense and the risks of revealing the authentic self. He or she actively manages a white façade in order to escape the prejudice of an imposed racial category. However, this survival mechanism requires the passer to constantly suppress his or her self-image. Passing leverages a constructed persona against a socially predetermined one, but it hazards the resulting alienation of internal identity. The nonwhite persona functions as a way to approach issues of identity and group membership in language. It allows writers to explore aspects of self, while distancing their persons from the object of fixation. Generally, the Africanist presence and its accompanying connotation of darkness provide diametric opposition to the qualities of whiteness. It embodies repulsion. To this end, fetishization, one of several literary strategies to mitigate the autonomy of Africanist characters, usually magnifies racial difference and the “categorical absolutism of civilization and savagery” by attaching “erotic fears or desires” to otherwise insignificant features (Morrison 67-68). Race becomes an object of reviled fixation. However, the nonwhite image and its association with savagery may be transformed into a mark of desire by a white actor. In this case, the white personality’s willful appropriation of nonwhiteness reverses the traditional abhorrence. The fetishized appearance creates an exoticism around the nonwhite persona, and the desire’s forbidden nature amplifies the thrill of pursuit. Whether avoided or hunted, the Africanist persona reduces the status of nonwhites from person to object. The fantasy of race imperils the authentic self.
White Norms and the External Displacement of Persona During the middle third of the novel, the section entitled “From Caucasia, with Love,” Birdie encounters the consequences of persona. Senna’s characters demonstrate the way that racial persona displaces nonwhite individuality and defines white behavior. In the predominantly white space of New Hampshire, Birdie can adapt her “authentic racial self” because she displays racially ambiguous features, sometimes appearing Italian, Jewish or Indian, among other labels for human phenotype. Yet ambiguity does not empower Birdie to escape racial categorization. Instead, it highlights the enduring influence of racial persona and a “white gaze” as influencers of social reception. Each observer assigns Birdie one of several different personas, meaning that the girl’s appearance externally defines her image. Even when Birdie passes as white or off-white, she faces the stakes of misidentification, just like any racialized individual would. Even a semblance of whiteness fails to provide a sovereign individuality. In addition, the character of Nicholas demonstrates the way that the logic of racial fantasy operates through persona to separate white persons from nonwhite objects. Just as the appearance of difference between racial categories invents the issue of racialized misidentification, it also causes white behavior to respond to a false persona, rather than a real person. The socialization of whiteness overrides Nicholas’ self-control, mitigating his power of judgement. Difference embeds fantasy between apparently unlike individuals. Derrick Bell notes that the fantasy of white supremacy subtly operates through personal “preferences,” which act as gatekeepers of social inclusion (7). Recalling an early encounter with Nicholas, when the boy had noticed her speaking the secret language of Elemeno, Birdie narrates that “Nicholas ruffled my
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Morrison defines “American Africanism” as “the ways in which a nonwhite, Africanlike (or Africanist) presence or persona was constructed in the United States, and the imaginative uses this fabricated presence served” (6). She argues that this presence supplied an opposing image for white self-definition.
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ARTS AND HUMANITIES hair and said, ‘I think I’ll call you Pocahontas… Because you turn all brown in the sun. Like a little Indian’ ” (Caucasia 192-193). Implicit in preferences, white norms distinguish beauty from deviancy along variations in racial phenotype. As a result, “ruffled,” which conveys physical intrusion, comes to foreshadow an external meddling in identity. Following in this tension, “turn” suggests change and distinguishes between two states of body. Because “turn” specifically references Birdie’s newly “brown” skin, these two states logically align with somatic normality and deviancy. They reveal the body as a neutral, accommodating canvas to be defined by external assumptions.3 This sense of change invites a transformation of image, with its accompanying connotation of racial status. The dynamic of Birdie’s alternate image manifests in a new name, “Pocahontas,” and new heritage, “Indian.” Ordinarily, nicknames suggest an emotional intimacy between individuals. These informal, specialized monikers usually provide a sense of familiarity and exclusivity between friends. However, “Pocahontas” recalls a mythology of tribal savages and noble explorers, while “Indian” invokes a history of white racial conquest in North America. This white colonial fantasy recalls a false history of white innocence, a rationalization or outright denial of white atrocity in the name of expansion. It diverges from reality and frames Birdie’s transformation within an invented heritage of racialized inferiority. Rather than mutually reinforcing a human bond, the nickname, “Pocahontas,” creates a “brown” savage, then alienates it from white civility. Moreover, “little” often confers endearment, eliciting a familial relationship like the guardian bond between a parent and child. However, it also assumes a relationship between superior and inferior partners. When applied to the invented presence of the savage “Indian,” “little” loses its affectionate quality, instead reinforcing an unequal power dynamic. Together, “little Indian” illustrates that way that a white gaze minimizes an alienated presence. In this way, a character’s status alters as a function of her skin hue, externally imposing the fantasy of white supremacy by way of an invented persona. Birdie’s transformation from the white, or nearly white, persona of Jesse to the nonwhite persona of “Pocahontas” displaces the relative equality of shared racelessness with a racialized submission. The girl’s new position reflects not only a mythology of inferiority, but also phenotypical abnormality. As a result, phenotypical appearance defines identity in the white gaze, regardless of an individual’s own intentions. Here, the invention of difference initiates a transition from bodily normality to deviancy, thereby embedding a fantasy of superiority within the invented image. Fantasy fabricates persona by magnifying bodily differences. As Birdie’s skin darkens, Nicholas escalates his imaginary Pocahontas by investing the girl’s appearance with an exotic attraction. This illusion reinforces what Derrick understands as the “unspoken” nature of nonwhite somatic deviancy (152). It also begins to mirror what Toni Morrison describes as a fetishization of race. As a result, the appearance of race comes to externally define nonwhite identity, but it also unconsciously determines the way that white-socialized individuals imagine nonwhite presence. Even as Nicholas seeks physical intimacy with Birdie, a socialized fantasy creates distance between the white boy and the brown girl: “Then I felt it, wet and grainy against my face, like a dog’s kiss, but not a dog. It was Nicholas’s tongue against my cheek, and I opened my eyes to see him chuckling over me. “‘You have a mustache.’ “‘Shut up,’ I said, rolling over to hide my stinging eyes. I tried to get up, but he pulled me back down. “He whispered, ‘I like it. It makes you look dirty, like I could lick you clean’” (Caucasia 200).
An embedded white gaze constructs the nonwhite persona. It controls the white character’s perception, particularly in the fetishization of female facial hair. During an examination of the codes that govern appearance, desire, and deviancy in Ernest Hemmingway’s The Garden of Eden and To Have and Have Not, Toni Morrison illuminates the forbidden taboo of blackness. She documents the way that white characters come to fetishize darkness in one story, while they contrarily seem repulsed by blackness in another. The difference, what makes blackness occasionally attractive but otherwise repugnant, appears to reside in a “voluptuous illegality,” the thrill of consuming the forbidden fruit of black deviancy (Morrison 87). This same thrill shapes Nicholas’ fixation on 26
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ARTS AND HUMANITIES Birdie’s upper lip. A “mustache” is a traditional marker of masculinity (Caucasia 200). On a female character, this feature would normally undermine the certainty of gender differences and confuse a character’s identity, thereby conveying an intensely uncomfortable undesirability. However, in the fantasy of Pocahontas, a “mustache” seems to heighten the thrill of deviancy. It provides a dark taboo, which inflates the forbidden undertone of encounter. Rather than marking a character for avoidance, this female “mustache” confers an alien exoticism upon the object of fantasy, further displacing the object character’s own identity. Such difference not only excites the white gaze, it stirs desire. Senna’s Nicholas displays a similar logic of acquisition to Hemmingway’s Catherine.4 The opposing traits of “dirty” and “clean” further construct the nonwhite persona. Charles Mills observes that American social and political systems view nonwhites as “incarnating wildness and wilderness in their person” (87). In this vein, “dirty” invokes a kind of wilderness in the nonwhite body and, specifically, the female mustache (Caucasia 200). Moreover, the text’s phrasing implies an association of “dirty” with the nonwhite persona, encoding wildness as impurity. This connotation sharply contrasts with the trappings of a white middle-class home. Voicing this tension, “clean” calls images of purity and civility to mind. Hence, the dichotomy of “dirty” and “clean” marshals the underlying assumptions of normality to suggest a dynamic that resembles the relationship between a lion and its tamer. It provides the taboo of savagery, a proximity to danger, but with the control of a white master. In other words, the supposed animalistic savagery of the nonwhite persona elicits exotic desire, rather than fear or repulsion, only with the establishment of white dominance.5 Once rendered exotic, the nonwhite persona becomes an object of fixation for the white gaze. Nicholas is overcome by the prospect of consuming this forbidden fruit. His behavior turns animalistic, despite his elite background. A “lick” conveys both devouring and purification. As devouring, this action suggests a symbolic ingestion of the deviancy, an acquirement of exoticism. It displays a racial fetishization by facilitating the appropriation of a reified darkness by the white gaze. As purification, it reinforces difference between the white subject and the nonwhite object, equating the project of racial appropriation to a removal of nonwhite deviancy. In a single term, these components link a white appropriation of exoticism to a destruction of the nonwhite persona. They echo the desire for conquest, thereby revealing a bodily colonization and a role for fetishization in the white normative fantasy. Moreover, the likening of “lick” to a “dog’s kiss” serves to invert the white invention of savagery upon itself, showing animalistic desire within white behavior. Ordinarily, a “kiss” conveys feelings of affection and emotional intimacy. It would usually suggest a genuine bond between two amorous, familial or, at least, fully human, partners. However, describing the “kiss” as a “dog’s” conflicts this traditional connotation. “Dog’s” carries animalistic undertones. Though domesticated canines are often considered to be relatively civil as family members, friends or even coworkers, they do remain hierarchically below humans. Sharp teeth, fur and a quadruped gait visually mark dogs in proximity to wilderness, rather than personhood. Seemingly irrational behavior and lacking communication also mark them as animals in human society. It follows that defining a “lick” as a “dog’s” likens a human to the family pet, with all the accompanying undertones of irrationality and unintelligence.
3.
Another physical trait seems to define racial categorization alongside skin. Hair symbolizes identity throughout the novel, particularly in the way that Samantha Taper’s “confused, half-nappy” ponytail reflects a growing, but limited, search for self-confidence as the only black girl in a white town (Caucasia 250). Just as “nappy,” a characteristic that traditionally describes only the hair of African peoples, marks Samantha as a black girl among white peers, generally straight locks associate Birdie with white appearance, regardless of the girl’s internal identity.
4.
Toni Morrison observes the white, desire-obsessed character of Catherine’s quest to appropriate dark appearances as concrete traits in Hemmingway’s The Garden of Eden (87).
5.
Nicholas establishes a physical dominance in relation to Birdie by posturing “over” her and by “pulling” her into the position that he prefers her to take. These actions demonstrate both psychological and physical assertions of superiority, following the incursion of an actor upon an object. They also convey a sense of roughness or near-violence. As a result, they mirror one of Charles Mills’ theses within The Racial Contract, which states that white supremacy must be “enforced through violence and ideological conditioning” (81).
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ARTS AND HUMANITIES This difference inserts a barrier to full interaction between the animalistic and the human, nullifying all but the purely physical aspect of “kiss.” In a twist, it confers wildness on the white gaze, rather than the nonwhite persona. This suggests that white fixation on the taboo of savagery reveals unhuman thought in the behavior of the white, rather than the nonwhite character. As a result, “lick” comes to betray a loss of self-control by the white character, who is fully consumed by the wild fantasy. Without this control, the fetishization of female mustaches and brown skin collapses, the appearance of white domination evaporates, the lion eats its tamer. Not in the nonwhite persona, but through the white gaze does the danger of savagery cross from illusion to reality. The nonwhite savage exists only as a figment of the white imagination. It inhabits a perceptive space between the nonwhite individual and the white observer, obscuring visual communication between the two. This image displaces the internal identification of the nonwhite individual with an object of fixation, and it undermines white self-control through a socialized obsession with race. This ultimately diminishes genuine human exchange in subtle and unconscious ways, especially in narrative authority and ownership. The fantasy of racial normality externally defines the nonwhite persona, attaching a false image to bodily deviant figures. It also controls white behaviors and obsessions in nonwhite presence. As a result, white somatic norms displace racialized individuals in the American social hierarchy, thereby alienating them from civility.
The Stakes of Misidentification The fantasy of white superiority displaces personal identities and alters social behaviors. To the white observer, an object image eclipses the nonwhite personality. This effect produces dissonance between the nonwhite person’s appearance and internal self. Birdie’s thoughts reveal stark contrast to the layers of persona that a white gaze constructs around her appearance. Even as Nicholas’ behaviors toward the girl begin to address the construct of Pocahontas, Birdie continues to mediate her particular set of personalities. The tension between persona and self illustrates how the invention of nonwhiteness elicits misidentification. Birdie tacitly accepts her identities by acknowledging their names. As Danzy Senna explains, the girl’s names represent tactical decisions, “the self she’s chosen” (Arias 449). Even the name, “Birdie,” and the identity that it represents are consciously mantled. In New Hampshire, the character’s public self, the one that she acknowledges, reflects a white Jewish girl, named Jesse.6 Ordinarily light skin and straight hair allow Birdie to pass as Jesse without challenge, and none of the white locals suspect her as the black interloper in their midst.7 Though the girl initially selects her identities, an external image eventually overwhelms her choices. An object persona haunts the nonwhite individual, even in passing: “The pictures were horrible, making the Congolese into hideous caricatures, but I laughed anyway at the absurdity of it. “ ‘They’ve made us look like animals,’ I said, holding my belly… “He giggled into his hand and said, ‘You said ‘us.’ You said it made us look like animals.’ The hilarity of my statement sent him into hysterics, and he rolled over, silently quaking beside me.” (Caucasia 204).
The white imagination’s opposition between civility and savagery confounds the nonwhite individual. The illustration of Africanist characters appears “hideous.” This description conveys utter aesthetic repulsion. A “hideous” object must be avoided or destroyed, due to its complete undesirability and opposition to decency. In this vein, white body norms define raceless, European features as the standard for visual allure, and they comparatively polarize physical desirability against the nonwhite body. Beauty is whiteness, and whiteness is not African, Asian or indigenous to the Americas. A “caricature” drastically exaggerates the cultural and physical characteristics of a subject, representing him or her as the sum of these reified components. This reduction of personality to a collection of differences is usually illustrated for comical effect. It follows that a “hideous 28
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ARTS AND HUMANITIES caricature” exaggerates the visually displeasing aspects of its subject and rearranges them into a humorous persona. However, the humor of the piece also depends upon the observer. The nonwhite person, who associates with the image’s subjects, may find amusement in the ridiculous and blatant inaccuracy of such animalistic depiction. In this way, the illustration itself is seen as “absurdity.” It is funny in an ironic way and only because it showcases the creator’s otherworldly detachment from reality. By contrast, the white viewer might be “sent…into hysterics,” overwhelmed by comedy, in response to the illustration’s apparent confirmation of his or her preconception of nonwhite wildness. To the white gaze, “absurdity” lies in the situational irony of a nonwhite figure, who naturally harbors a nascent savagery, claiming full personhood. The nonwhite observer laughs at the reality behind the image, but the white viewer snickers at the illusion of persona. The passer reveals tension between these alternate perspectives. She bears the weight of both, wrestling with the pull of multiple identities. The appearance of whiteness shields her from immediate ridicule, but an internalized blackness highlights the white perceptual dysfunction. The spontaneous line, “they’ve made us look like animals,” betrays the passer’s self-image, thereby placing her white persona in jeopardy. “Us” communicates group ownership, and “they” conveys distance. The use of “us” to reference the subjects of the “caricature” reveals the passer’s internal association with nonwhiteness, as does the use of “they” to describe the illustration’s creators, who are assumed white. The act of whiteness balances desirability against the risk of exposure, an intentional fabrication of identity against the constant shadow of racialized persona. This threat manifests in Nicholas’ reassurance, “ ‘I was just kidding about you looking colored… You’re pretty’ ” (Caucasia 205). “Kidding” is often elicited to superficially nullify insulting comments as humorous. This phrase’s direction toward appearing “colored” invokes white somatic norming, and the use of humor explicates an oppositional relationship between nonwhite appearance and the attractiveness that “pretty” conveys. As when viewing the illustration of Africanist savagery, the white observer gawks at the absurdity of nonwhite beauty. He also reveals perceptual contrast between normal attraction and the Pocahontas fantasy’s exoticism. Just as Hemmingway’s character, Catherine, seeks a black taboo, Senna’s Nicholas chases the thrill of danger by constructing a persona of Africanist-style savagery around an ordinary girl. The fantasy’s exotic allure constitutes nothing more than a cheap thrill, a quick and dirty diversion from normality. The status quo of desirability reasserts itself when the thrill fades. To the white observer, Birdie’s serious beauty rests in her white appearance, not her deviant persona. A black girl in white skin, the passer observes candid disdain for her true identity. Nonwhite socialization confers an internal outsider status, regardless of any external resemblance to the white norm. Even when she is not specifically targeted, the passer struggles against the crushing weight of the nonwhite persona. She, alone, recognizes the cognitive dissonance between white perception and the individual. In this way, the nonwhite figure must navigate the misidentification of false personas at the peril of losing the self.
6.
I continue to refer to the character as “Birdie” for consistency, even though this name is technically just one of the several personas that she adopts.
7.
However, there is evidence to suggest that the Jewish persona, which Birdie eventually dilutes into a generic whiteness with the declaration, “well, [I’m] not really Jewish. I mean, only my dad was, and he’s dead,” carries its own off-white difficulties (Caucasia 247).
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ARTS AND HUMANITIES Conclusion The passing narrative illustrates a social discourse between external image and internal identification. It reveals the way that a white gaze and its judgements function as imaginary devices, rather than universal realities. The complete permeation of racial categorization underwrites the collective validity of a white bodily normality, which governs patterns of inclusion and rejection. However, the establishment of a created whiteness relies on definition by a created nonwhite presence. Assuming this bodily normality, white observers project revulsion upon the different cultures and phenotypes of persons, who become marked as deviant. This division may be encoded along lines of civility and savagery, whiteness and nonwhiteness, or a number of other distinctions between familiarity and alienation. The dominant position of white norming in America invents an artificial nonwhiteness around supposedly foreign populations. This persona defines white perceptions and behaviors toward nonwhite individuals. It imposes racist expectations of character and history on these persons, regardless of their actual dispositions. In this way, the invention of persona impairs white rationality with regard to race and displaces nonwhite individuality. A haunting persona restricts the nonwhite individual’s choice of identity. In the white-dominated space of a small New Hampshire town, the character of Birdie intentionally obscures her black inner self behind a white persona. She accepts a fictitious identity and furnishes it with the outward appearance of authenticity. This duality informs her encounters with white fantasies. Birdie chooses to inhabit the role of Jesse because a raceless appearance grants her the means of survival, but she never accepts the image of Pocahontas. Instead, this other persona reimagines Birdie as the object of a white colonial fantasy. In Nicholas’ eye, Birdie’s dark summer tan marks a significant, phenotypical change. The effect is so strong that it prompts the white observer to notice a deviancy from the bodily norm. A moral cognitive dysfunction, the modern product of historical European domination, subtly alters the white observer’s perception. The boy ceases to address Jesse or Birdie as he begins to address Pocahontas. This visual difference erects an additional, cognitive barrier between the passer’s authentic self and her external image. The fantasy eventually consumes the white perception and behavior toward the object of the nonwhite persona. The particular brand of deviancy that Nicholas attaches to Pocahontas reverses the usual pattern, reflecting an exotic attraction, rather than a savage repulsion. Interestingly, the dissonance between Birdie’s internal and external selves continues after the Pocahontas illusion fades. A comic book’s illustration of animalistic, Africanist characters elicits laughter from both Nicholas and Birdie. The secretly black Birdie mocks the ridiculous and inaccurate premise of such an illustration, but Nicholas finds humor in the assumed reality of the depiction. The girl’s position as a passer reveals the white gaze’s historically maintained detachment from reality. Nicholas remains unaware of Birdie’s internal self, just as he fails to see beyond the nonwhite persona. This leads him to define beauty and bodily deviancy, even in exoticism, as mutually exclusive qualities. Whether beneath a wild taboo or an implicit normality, the passer’s authentic self struggles against a constructed figure. The Pocahontas fantasy showcases a reified, but exotically desirable, body. The illustration of Africanist savagery highlights a casual repulsion for a black self. Birdie combats external misidentification on multiple fronts in this confusion of personalities. Moreover, the way that a white social fantasy commandeers Nicholas’ behavior demonstrates a subversion of self-control for white persons in nonwhite presence. Passers wager their personalities for the incentives of profit or survival. Even if they succeed, they remain haunted by the social baggage of race and the imperative to suppress their authentic selves. The fantasy invents persona to displace the nonwhite individual in the white imagination. The result produces a collective illusion, which blockades truly equal interaction between the normal and the deviant, the raceless and the racialized.
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ARTS AND HUMANITIES Acknowledgements I am thankful to the Berry Family and the University Honors Program for supporting my research and experiences. I especially owe this project to the Honors Program staff members for their advice and feedback throughout the program. I am also very grateful to my thesis mentor, Dr. Tom Morgan. This project would not have taken shape without his perspective, guidance and sarcasm.
Works Cited Arias, Claudia M. Milian. “An Interview with Danzy Senna.” Callaloo, vol. 25, no. 2, 2002, pp. 447-452. Bell, Derrick. Faces at the Bottom of the Well: The Permanence of Racism. New York: Basic Books, 1992. Print. Johnson, James Weldon. The Autobiography of an Ex-Colored Man. Boston: Sherman, French & Company, 1912. Print. Larsen, Nella. Passing. New York: Oshun Publishing Company, 2013. Print. Mills, Charles. The Racial Contract. Ithaca: Cornell University Press, 1997. Print. Morrison, Toni. Playing in the Dark: Whiteness and the Literary Imagination. New York: Vintage Books, 1993. Print. Senna, Danzy. Caucasia. New York: Riverhead Books, 1998. Print. Senna, Danzy. “The Mulatto Millennium.” Half and Half: Writers on Growing Up Biracial and Bicultural. New York: Pantheon Books, 1998. pp. 12-28.
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Mortality in the Twenty-first Century: A Cycle of Short Stories about Death and the Afterlife Rose A. Rucoba1,2,3 University of Dayton, 300 College Park, Dayton, OH, 45469 1. Department of English 2. Berry Summer Thesis Institute 3. University Honors Program
Advisor: Albino Carrillo, M.F.A. Department of English Corresponding Author: Albino Carrillo Email: acarrillo1@udayton.edu
Summary In a cycle of short stories, I portray death and the afterlife through a collection that follows a rich variety of characters, as each struggles with his or her own beliefs and experiences with death and the afterlife. I examine religious, non-religious, traditional and non-traditional beliefs in this collection, providing a wide range of perspectives on mortality. In addition to these values and perspectives, I draw on authors Elizabeth Evans, Flannery O’Connor and Stephanie Vaughn, who all influence this collection with their various and unique portrayals of death and the afterworld. The outcome is a series of vastly different and intriguing stories that come together to illustrate the great mystery of death and what, if anything, comes after. The short story below, Forbearance, tells the story of Alex, a teenager who witnesses a suicide while on vacation in Paris, France. Alex’s gender and identity go unknown throughout the story as he or she recovers from the incident and comes to his or her own conclusions about death and the afterlife. The story is told in first person and explores how adolescence and family background can affect one’s perception of death. Alex’s struggle with mortality is intertwined with Victor Hugo’s The Hunchback of Notre Dame and its themes of suffering and justice. The story is also influenced by O’Connor’s distant narrative viewpoint, combined with Evans’ portrayal of strong young protagonists.
Forbearance “Elbows off the table!” I pressed my elbows into the ridges of the table, and then slid them off and inspected the red indents they left on my skin. “We’re at a nice restaurant, Alex. Keep your hands in your lap. And sit up straight for God’s sake.” Calvin said this without so much as glancing up from his Le Parisien. He reclined back in his chair and went back to pretending I didn’t exist. I didn’t like Calvin, but he had the unprecedented privilege of being my father, so I guess I had to like him a bit, or at least pretend like I did.
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ARTS AND HUMANITIES I took a drink of my water, chewing on the straw between sips and staring into space. Semiconsciously, I blew into my water and heard bubbles form at the bottom. Calvin didn’t seem to notice. I blew a bit harder and watched the bubbles rise to the top that time. Then harder. I watched it overflow and felt the ice-cold drips of water hit my jeans through the cracks in the table. “Stop that!” he said, his voice rising, but his eyes never left page 5 of the Politique section. I fought every urge to lay my head on the table. Instead, I slid down in my chair, laid my head back with my eyes closed, and listened to the sound of my stomach grumbling. *** When my mom saw me off, she tucked the newest Stephen King novel in my carry-on bag. “Forbearance, Alex,” she said. Then she kissed the top of my head. I read the entire book twice on the flight over. I started to regret it no more than twenty minutes after I landed, on the cab drive over to the hotel. My mother must have had more forbearance than a monk to put up with Calvin for thirteen years. I couldn’t even put up with him for twenty-four hours! He dragged me here for “a cultural immersion experience.” And because he said my recent behavior was giving Mom migraines again and I needed to give her a break and going to France would make me a better person or something. Or maybe it was a smarter person. A more artistic person? It hadn’t done any of that for me, though. In fact, by the time Calvin had carried my things up to the hotel room and I saw the three presents wrapped and waiting on my bed — so poorly wrapped that I could see the Wii logo peeking out in the corner of one box — I realized this trip was nothing more than Calvin’s sad attempt to win me back after nine months of Wednesday night phone calls, alimony checks, and absenteeism. The presents, the promises, the suite with two balconies, a Jacuzzi, and a king-size bed all to myself were just part of the act so that he would pass the test when I went home and told mom everything. None of it was ever meant to last. See, the thing is that Calvin is like a giant turtle. He will only move if there’s something in it for him — food, money, a promise that I’ll put a good word in for him with mom. When he wants something, he is on the move, and he will even snap at you if necessary. But otherwise, he spends his time in his shell, and woe to the man that dares disturb him. This is why I was so surprised when Calvin woke me up at eight the next morning with a rare, and oddly creepy, smile on his face. He promised to take me out to this really cool breakfast place if I endured a few hours of sightseeing with him for some bonding time. Turns out “sightseeing” was taking an hour and a half bus tour through the congested city streets to look at, but not actually go and see, the top 10 tourist attractions of Paris. And “breakfast” turned out to be at this really snooty restaurant where you get to sit on this balcony overlooking all of Paris. I normally wouldn’t complain about this, but the waiter took one look at my Fall Out Boy concert t-shirt and jeans and stuck us in the back of the balcony behind this huge shrub. Once we sat down, the only thing I could find eatable or pronounceable on the menu was a croissant with jam, for which I had been waiting for almost forty minutes. *** I suddenly had an idea. “Hey, dad,” I said, opening my eyes and slowly sitting back up. “Hmmm,” came the response. “So I was talking to my friend Kayla. You know Kayla—blonde hair, freckles—we’ve been friends since we were like five?” Silence. Proceedings 2017
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ARTS AND HUMANITIES “Anyway, Kayla is really into all this philosophical stuff and she said that in the olden days criminals used hide out in churches and claim ‘sanctuary’ like Esmeralda did in The Hunchback of Notre Dame. And we got to talking and things got really deep and we thought about what would happen if like all the terrorists and white supremacists and criminals of the world starting doing that again. What if they all starting running into churches when being chased by the cops and started claiming ‘sanctuary’? I mean it’s not like the cops could really do anything. I mean they could, but then you’re shooting someone in front of Jesus and that’s really bad. Even if you’re not a religious person, you have to think about how that’s gonna affect the nation and all the religious people out there.” “Alex…” Calvin said through a sigh. “Well, I was just thinking, with Notre Dame right around the corner and with all the terrorist attacks that have been happening in Europe, I thought all it would take is one, just one guy to claim ‘sanctuary’ and then a whole copycat frenzy would take place. I mean just think about it, that would be the ultimate F-you to the Western world because most of us are Christian and we’ve gotten so out-of-touch with our Catholic Christian roots and to go into one of our places of worship and claim ‘sanctuary’ and test both our morals and our faith is pretty messed up.” Calvin set his paper down. “And then I was thinking about how that would spiral out of control. I mean could a physically abused kid run into a church and claim ‘sanctuary’ the way a criminal could? Or what about someone on the run from the IRS? I mean the possibilities are endless!” “Alex, please,” Calvin said as he rubbed his temples. “Well, I was just thinking about how you could accuse anyone of anything by claiming ‘sanctuary’ and hold whoever it is accountable and then I started thinking about what I would claim ‘sanctuary’ for —” “Alex,” he said, working his forehead like he was rolling dough. “Well, what I was really wondering is what you would claim sanctuary for. Like if it be from work, or credit card debt or mo —” “ALEX!” And the whole table shook. People at a nearby table looked at us. I had awoken the snapping turtle. Calvin collected himself, then took off his glasses and massaged his temples again. “Alex, pleeeease, this talking, this obsessive, constant talking, can’t you see this is why your mother can’t put up with you?” It was a statement, not a question. “Please, go now and take a break,” Calvin said, eyes closed, hands on his face, “I need a minute to myself. Go. NOW!” I crossed my arms over my chest, and then rolled up and out of my chair with all the grace of the angsty fourteen-year-old that I was. I dragged my feet across the balcony to the railing. I figured I had the right to enjoy the view at least once while our waiter was busy and before my breakfast came. And before I had to spend the rest of the day with Calvin. Sometimes I didn’t get Calvin. Or really anybody for that matter. People are always talking about how we need all these great thinkers and great ideas to save the world, and I have a lot of them because I spend a lot of time thinking and a lot of time philosophizing and a lot of time talking about what I think about to Kayla and other people who care. But when I tell people like Calvin or my mom
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ARTS AND HUMANITIES or most other people, they really don’t want to listen and most of the time they either pretend they have a headache or just walk away. I rested my arms on the railing and then rested my chin on top of them. I gazed over the city. I spotted the Eiffel Tower and wondered, ironically, if Calvin’s little “cultural immersion trip” would ever involve us actually getting to climb to the top of it. Then, I looked straight down and watched the little toy cars find their way through the city traffic. I watched all the little ants meander into the city’s museums, churches, cafes, and tourist traps. I took a few steps back and made a frame with my thumbs and pointer fingers. Then I squinted through it at the city. I tried to imagine what the city would have looked like during the late 1400’s, when Quasimodo was rescued by Esmeralda’s kindness and, in turn, rescued her from being hanged. I tried to imagine Frollo in all his lust and Phoebus in all his longing. Then I leaned my head back and turned my frame to the sky. The hanging flower troughs, ivy, and white stone of the restaurant fell into view. I looked all the way up to the top of the building, squinting against the sun. And I that’s when I saw him. He fell gracefully. Oddly enough, that’s the first thing that came to mind when I saw him. Actually, the first thing I thought was that someone had dropped a coat off the roof. Kinda silly if you think about it. I dropped my hands and my chin and followed him with my eyes rolling down like they do when studying a huge painting. I analyzed the fabric of his black coat with all its waves and wrinkles as he passed the balcony railing. Then I took a few steps closer and looked down. As he tumbled down to earth, I watched the ants scatter. I followed him all the way down until he was nothing but a mere speck of black, until he stopped. *** When my mom finally grew some balls and asked Calvin for a divorce and they made it official, I was sent to the school psychologist to “sort out my feelings.” The psychologist was a very pretty lady with thin brown hair and a nice smile. She sat me down and handed me a box of tissues “just in case.” She said she heard my parents were “separating,” and then said through a big, wide smile with a hand on my shoulder “It is not your fault.” And I said “I never said that it was.” Then she told me it was perfectly okay to be angry and said she was sorry to assume my feelings and then asked me straight: “Alex, how do you really feel about the divorce?” Without skipping a beat I told her. “It’s been a long time coming. I’m just glad they finally both got the balls to go through with it. Maybe now there will be some goddamn peace in our house.” The look of utter and complete shock on her face is forever cemented in my mind. It might sound a bit diabolical, but sometimes I find myself grinning when I think about it. I wasn’t sorry I said it. It was the truth. I saw my parents’ divorce coming since the day I was born, or really since the day I learned to distinguish between talking and yelling…and screaming. But never did I ever think I would get to witness that look ever again. Not until I looked up from watching the man and found not one, but eight other people with that same look on their faces. They were on either side of me, some leaning over the rail of the balcony, others standing a bit further back, just staring.
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ARTS AND HUMANITIES “Was that a man that just fell?” I turned to see a young woman in a purple dress looking at me. Her accent told me she was probably a British tourist. My Fall Out Boy t-shirt must have given me away as an American. I looked into her pleading eyes that conveyed all the confusion and innocence of that moment and suddenly the reality of what had just happened set in. But before I had time to respond, Calvin came up behind me and put his hand over my eyes. “Don’t look, Alex!” came his voice. He pulled me back and I squirmed a little, but I found that I had suddenly lost my voice. “It’s okay, honey. I got you. It’s all over now. It’s okay.” Slowly, he walked me backwards, then turned me around and guided me out of the restaurant, all the while saying, “It’s alright Alex. It’s all over now. It’s all over. Let’s go home, kiddo.” *** The moment I got in the cab I knew the inevitable was to come: Calvin would take me back to the States and tell mom everything (if he hadn’t already) and they would want to talk about it with me and quite possibly I would be sent back to see a psychologist. Quite possibly the same woman I had to see during the divorce. And all this just because I looked down when he fell. Calvin didn’t even have to drag it out of me. He just asked how much I saw and I told him “everything.” And that was enough for him to know that even though I didn’t see all the blood and gore, I saw enough to “affect” me and he told me he was booking the next flight home. The weird thing was, I didn’t feel “affected” at all by what happened. The truth was I felt very far away from it. I had seen this man fall and die and still I hadn’t really seen him. To me, he was an ant squashed on the sidewalk, not a man who had committed suicide. That’s not a very nice way to put it, but that’s the best way I can describe it. This man could have very well been an inanimate object to me, and yet…at the same time, he felt very real to me. I suppose in the same way you see an ant squashed on the sidewalk and you feel a small sadness in your heart because you know he’s one of God’s creatures. I know that doesn’t sound a whole lot better, though. Against Calvin’s wishes I searched the news, desperately wanting to know more about the man, but between the mix of French and English subtitles, and a few different news channels, I really couldn't decipher who he was. He was no one really. No one important I mean. Left behind no one. Left behind no legacy. Left behind no note or clue as to why he jumped. He was a stranger. *** On the cab drive to the airport the next morning, I watched the rain slide down the window, giving my view of Paris a kind of tear-soaked look to it. The little rivulets on the glass would run when the cab went fast and when it stopped the window collected a new accumulation of drops. It was an endless cycle of running water and new teardrops, running water and new teardrops. It was stop-go for over a half hour after we left the hotel. Between the rain and the weekend traffic and the investigation at the restaurant closing down a whole street, I figured we would get to the airport by my 21st birthday. “Can you go any faster?” Calvin half-yelled. “I am trying, sir. The roads…too blocked up. This fastest I can go,” the driver said in broken English.
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ARTS AND HUMANITIES “Honk at them. Make them move up a little more,” he said, craning his neck to try to see ahead on the road. Then he turned to me. “We’re gonna get there soon, Alex. Don’t you worry. If you’re getting carsick from all this I can give you some aspirin,” he said, a half-hearted smile on his face. “No thanks,” I said, staring out the window. Calvin was on edge. I could tell that on any other day he would have been cursing left and right, screaming at the cab driver. Ever since “the incident,” however, Calvin had been walking on eggshells around me. Like my witnessing the suicide made me a different person. As if I was so “affected” by what I saw that I was now too fragile and disturbed to be treated like normal. Calvin had always ignored me and, to use the cliché adults say, “was never really there for me,” but now I had become the center of the universe, and my fragile state combined with the road rage made him a ticking time bomb. We moved up a little more. Through streams of rainwater I could see the two towers of Notre Dame illuminated by spotlights. The cathedral looked magnificent, menacing even in the dark of the rain. It pulled me in. “Hey, dad, I uh…really have to pee. Do you mind in I run in real quick?” “What, in there?” he said, gesturing with his head toward the cathedral. “Yeah, I’ll just be a minute.” He looked at me funny. I decided to play the victim card. “Come on. It’s the only restroom near enough. And…well we haven’t really had the chance to do any sightseeing since…you know…” Calvin sighed, and his whole body relaxed with it as he looked at me. “Okay, but don’t be any longer than five minutes. If we move up, I’ll text you, but try to be quick.” “I will,” I said, opening the door. *** Once inside, the silence drowned out the rain and my senses took hold. It was magnificent, astounding, divine, absolutely resplendent! All the words you know and don’t know in the English dictionary could never come close to describing what it looked like. It was the essence of beauty, and yet…it was exactly the kind of beautiful I had imagined it to be. Not underwhelming or un-climactic by any means, but expectedly unexpected. If that makes any sense. I took it all in, absorbed every detail, memorized each and every arch, and window, and crucifix that I could. I looked to the right and left of me, noticing the two rows of pillars that stood resolute on either side of the aisle. I pictured Frollo hiding in one of the pillar’s shadows. Then, as if on que, the bells rang and I jumped a little in my skin. I couldn’t help but think it was Quasimodo up there ringing them for me. That maybe he would go to meet Esmeralda in secret somewhere in Paris tonight. Taking a seat in one of the back pews, I crossed myself and sat on the kneeler. I gazed up at the golden castle behind the alter and felt like I had stepped back in time; a time when religion was just that — religion — not political, not plastered all over the news, not corrupt, but was something people truly believed in and gave their lives to. It was a time when people built places like this out of sheer fear of the Lord, to please a power they couldn’t see, but loved beyond all measure. I bowed my head then and said a silent prayer for the world, that we might have revitalization. Then I got up and crossed myself again. Bending down to push the kneeler up, I noticed two words carved into the back of the pew in front of me. I didn’t have to look twice to know what they said. Proceedings 2017
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They said it all. I didn’t have to think twice about where they came from. They came from here. They were the silent answer to my prayer.
SPIRA SPERA Breathe. Hope.
Acknowledgements There are so many people that have guided and inspired me throughout my experience with the Berry Summer Thesis Institute program. I would first like to thank the Berry Family Foundation and the Berry family for their support of the BSTI program and for giving me this amazing opportunity in the first place. Second, I would like to thank those who are part of BSTI and the Honors Program — Dr. Nancy Miller, Dr. John McCombe, Mrs. Maria Burkett, Ms. Laura Cotten, Ms. Ramona Speranza and Ms. Jill Talley. Their insight and knowledge helped me grow both as a student and a person during my experience with BSTI and I am extremely grateful for their direction of the program. Finally, I would like to thank my mentor, Dr. Carrillo. His honesty, wisdom and guidance helped me grow tremendously as a writer. Without his direction and advice I would not be the writer or person I am today. The Berry Summer Thesis Institute program has changed me for the better and I will be forever grateful to all those who were a part of the program and helped make it such an impactful and positive experience for me.
Hope. Photograph courtesy of R. Rucoba, 2017.
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A Comparative Analysis of Breast Cancer Treatment and the Role of Neurotoxic Chemotherapy-induced Peripheral Neuropathy on Postural Stability Paige L. Ingram1,2,3,4 and Kimberly E. Bigelow1,2,3 University of Dayton, 300 College Park, Dayton, OH, 45469 1. Department of Mechanical and Aerospace Engineering 2. Engineering Wellness through Biomechanics Lab 3. Berry Summer Thesis Institute 4. University Honors Program
Advisor: Kimberly E. Bigelow, Ph.D. Department of Mechanical and Aerospace Engineering Corresponding Author: Kimberly E. Bigelow Email: kbigelow1@udayton.edu
Abstract Twelve percent of women suffer from breast cancer each year, but survivorship is increasing due to improvements in treatments. However, it appears there are lasting effects after treatment due to the toxicity of chemotherapy compounds. One of the most severe side effects is peripheral neuropathy which results in decreased sensation in the nervous system. With this loss, an individual’s balance and postural stability is likely impacted, leading to an altered quality of life. Monfort et al. at The Ohio State University are among the first to identify balance deficits in breast cancer patients during treatment, even after the first treatment cycle. We recently joined with them as they extended this work to include long-term follow-up testing after the completion of treatment. Our efforts specifically investigate postural stability, range of motion and balance control while individuals stand on a force plate, looking at variances between individuals receiving different treatments and experiencing diverse outcomes. Preliminary data analysis from the limits of stability balance tests suggest there are differences between the three subject groups, with those not receiving neurotoxic chemotherapy showing the most consistent improvement six months after treatment. Breast cancer survivors that were treated with neurotoxic chemotherapy had varied individual responses six months post-treatment. Further 40
data analysis is required; however, our results suggest that interventions to improve the postural stability of those treated with neurotoxic chemotherapy may be warranted.
Introduction According to current statistics, 1 in 8 U.S. women will develop breast cancer over the course of their life. In just 2017 alone, 255,180 U.S. women will be diagnosed [1]. While women continue to be diagnosed with breast cancer, survival rates have increased more than ever recorded before [1]. In fact by 2026, there will be an estimated 4,571,210 breast cancer survivors living in the U.S. [1]. Survivorship has increased drastically due to ground-breaking discoveries in cancer treatment and chemotherapy drugs such as taxanes and platinum compounds [1]. While survivorship has increased, research suggests that there are long lasting symptoms after cancer treatment that can affect one’s quality of life, leading to a relatively new characterization of cancer as a chronic illness [2][3]. Although not all patients go through chemotherapy, many of these lasting side effects come from chemotherapy due to the toxins that are distributed throughout the human body. Some toxins are classified as neurotoxic because they have the potential to deteriorate nerve tissue [4]. One of Proceedings 2017
ENGINEERING the most severe side effects induced by neurotoxic chemotherapy drugs is chemotherapyinduced peripheral neuropathy (CIPN). CIPN is characterized by damage to peripheral nerves causing sensory and motor nerve impairment, numbness, pain and muscle weakness [5]. CIPN is a common condition in cancer patients going through aggressive chemotherapy, with 30-70% reporting CIPN symptoms [6]. Peripheral nerves essentially connect the central nervous system (CNS), consisting of the brain and spinal cord, to the rest of the body. They do this by carrying impulses to the CNS from other parts of the body through sensory nerves and carrying impulses to other parts of the body to the CNS through motor nerves [7]. The central nervous system is the control center for all of our bodily functions and sensations. Due to nerve damage, those with CIPN tend to have pain and lack of sensation throughout their nervous system, often starting at the feet and move superiorly toward the head [6]. This lack of sensation results in a loss of proprioception due to the damaged peripheral nerves by neurotoxic compounds. Several studies also found that the neurotoxic agents of chemotherapy may cause vestibular deficits making it difficult to maintain balance equilibrium [8][9]. The CNS relies on somatosensory (proprioception), vestibular (ear) and visual sensory systems to maintain balance and postural stability [10]. With a loss of proprioception, CIPN patients may be faced with significant balance, gait and functional impairments [6][11]. This loss of sensation and proprioception can greatly alter one’s quality of life, functional independence and increase one’s risk of falls, which appears to be one of the most
problematic consequences of chemotherapy [8][5]. In fact, breast cancer survivors without CIPN have a 15% higher risk of falls than their healthy counterparts [8]. Falls can lead to severe injury, activity limitation due to fear of falling and eventual functional decline [9][8]. Although most symptoms often subside after 6 months, some breast cancer patients report numbness and discomfort 6 years after their last treatment [12]. Even more shocking is that we do not know why some cancer patients develop CIPN and others do not [13]. Limited work has been done to investigate CIPN, however, research at The Ohio State University is working to change that. Previous research at OSU, detailed in Figure 1, found that throughout treatment breast cancer patients experience balance and postural deficits as early as their first treatment and impairments intensify throughout treatment [4]. There is research to suggest that breast cancer survivors experience postural deficits long after treatment as well [9]. The same researchers at The Ohio State University have expanded their efforts and created a new study where they have invited Dr. Bigelow and her biomechanics lab to collaborate. The objective of this larger study is to determine the cognitive, functional and postural impairments that continue or develop after treatment during the rehabilitation period, which is often three to six months after their last treatment, and recovery period, which is more long-term survival. Furthermore, we plan to investigate how and why these deficits might differ between people who develop CIPN and those who do not. To the determine the potential lasting effects of chemotherapy, testing was done at two separate
Figure 1. Results from Monfort et al.’s research showcases the gradual progression of balance deficits as patients with taxane-based chemotherapy continue their treatment [4]. S.M. Monfort et al., 2016.
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ENGINEERING longitudinal time points, one up to six weeks after treatment and the second three to six months after treatment. This paper details our summer efforts at the University of Dayton which focused on analyzing dynamic postural control from the study participants. Using data from limits of stability and functional reach tests, the postural control of breast cancer survivors after chemotherapy treatment can be investigated. For this data from the limits of stability and functional reach tests, we hypothesized that significant dynamic balance impairments three to six months after treatment will occur in all those who received neurotoxic chemotherapy, however more intensified impairments will occur in those who had chemotherapy and report with signs of moderate to severe CIPN.
Methods Participants As of now, 17 females and 3 males with stage I-III breast or colon cancer are included in the study. Twelve of the current study participants have completed testing at both time points. Inclusion criteria to participate in this study were: individuals (men and women) greater than 18 years old diagnosed with breast stages I-III or stage I-IV colon or other GI malignancies, have never received adjuvant or neoadjuvant therapy with paclitaxel, docetaxel or oxaliplatin or have completed treatment with one of these agents within six weeks of enrollment. For patients who have completed chemotherapy, those with symptomatic CIPN or asymptomatic CIPN with no dose reductions or dose holds are considered. Patients with functional limitations due to musculoskeletal conditions can be included, due to their relatively stable condition over the course of the study. Exclusion criteria included: pregnant or nursing women, preexisting lower extremity amputations, inability to give informed consent, incapacity to walk or stand without assistance due to any condition, needing an ambulatory assistive device, back or lower extremity surgery in the last 6 months, or back or lower extremity surgery at any time point that interferes with gait and balance. The eligible study participants were divided into three subject groups. The first was the control group (n=6, 5 having completed testing) which are those who have stage I-III breast or colon 42
cancer but did not go through neurotoxic chemotherapy with paclitaxel, docetaxel or oxaliplatin. However, the individuals in the control group may have gone through some other type of cancer treatments. The second is the asymptomatic group (n=8, 3 having completed testing), which are those who completed neurotoxic chemotherapy but did not present with symptoms of CIPN. The third group is the symptomatic group (n=7, 3 having completed testing), which are those who completed neurotoxic chemotherapy and present with symptoms of moderate to severe CIPN.
Procedure The procedure of the overall study involved testing multiple biomechanical parameters, including quiet-standing posturography tests, limits of stability tests, functional reach test, proprioception tests on treadmill using motion capture system, single and dual task walking, overgound six-minute walking, qualitative surveys and cognitive tests. Additionally, a nerve conduction tests and skin biopsy test were added to the procedure to test for the physical existence of CIPN, which is often associated with a lack of cutaneous nerve receptors and demyelinated la afferent muscle fibers [6]. The cognitive test is a Cogstate computerized neurocognitive testing battery. The focus of this paper will center around the dynamic postural control assessments. To test for dynamic postural control, the limits of stability (LOS) and functional reach (FR) assessments were conducted. LOS and FR tests both try to simulate everyday balance challenges cancer patients encounter in daily activities and task. LOS and FR tests are considered reliable measure of functional stability [14]. These assessments can give insight into the functional impairments that people would encounter every day and the risk involved [15]. LOS requires a participant to stand on a balance plate that records their center of pressure (COP) movement. In dynamic balance tests, a larger excursion of COP indicates more postural control and better balance. The force plates measure downward forces and moments that allow one to find the location of ground reaction forces acting on the person’s feet (centers of pressure) [11]. In LOS assessments, these measures can be analyzed in Bertec balance plate Acquire 4 software to determine one’s range of motion and Proceedings 2017
ENGINEERING controlled shift of their center of mass. The LOS test measures the maximum range that someone can comfortably maintain balance in forward, backward, left and right directions on a force plate [16]. The test is dependent upon one’s range of motion and confidence in completing the task. If a person has large limits of stability, then they are considered to have more control of their center of mass and greater COP excursion, thus better balance control and range of motion. Similar to the LOS assessment, FR tests are a simplified limits of stability test that measures the maximum forward control of balance in the upper and lower extremities [17]. In the test, subjects are asked to stand upright and make a fist and extend their arm forward, then the placement of their third metacarpal is recorded along a yard stick and marked as Position 1. Then the subject is asked to reach as far forward as they could without losing their balance or taking a step and the placement of the end of the third metacarpal is recorded and marked as Position 2. The test is also run similarly for the feet, but the subject pushes a plastic slider as they move their dominant foot. The difference between the two positions is one’s functional reach or margin of stability. A larger margin of stability indicates more postural control in the forward or anterior direction [15]. The FR assessment of the extremities can give an indication of limb strength and range of motion. Decreased lower extremity strength has been found to be a risk factor for increased falls and balance deficits [11]. The results of functional arm reach tests in other populations have reliably predicted the risk of falling, which is considered one of the most challenging outcomes from chemotherapy [17]. This study’s functional reach tests are run while the participant is on a balance plate, which offers more information on the control of one’s center of mass while performing dynamic balance motions in the anterior direction.
Data Analysis COP excursion and margin of stability data from limits of stability and functional reach tests were used to quantify and measure the controlled shift of someone’s center of mass and dynamic balance. COP excursion is characterized by the displacement of one’s center of pressure from the natural mean position of stance [14]. COP excursion is the most commonly used measure of postural control [14]. COP data collected Proceedings 2017
from the Bertec balance plate was filtered and buffered through Bertec Acquire 4 software at 500 Hz. Once data was collected through Acquire, it was processed through a Matlab code and exported to Excel. Once exported, sway range values, which are the maximum travel of the COP throughout each trial, were calculated in the anterior-posterior(y) and medial lateral (x) direction. Sway range was calculated by taking the maximum COP excursion in one direction and subtracting it from the minimum COP excursion in the same direction (COPy_max – COPy_min). To analyze the results from the LOS assessment for both directions, the sway ranges were averaged separately across all trials in each direction. To compare values across the three subject groups, the averaged values for each individual were plotted on a line graph at time point 1 and time point 2. As of now, data analysis has been completed with all current study participants for LOS data in the anteriorposterior direction while the medial lateral LOS data is almost near completion. For the FR assessment, the data was recently received, therefore no data analysis has been conducted as of now. However, the data will be analyzed by calculating and averaging the margins of stability of each individual throughout all five trials for the dominant upper and lower extremity. The margins of stability are calculated by finding the difference between the individual’s maximum reach and neutral position, which is their natural standing position. The margin of stability gives an indication of one’s maximum range of motion in the forward direction [15]. Like the LOS data, we will average the margin of stability values of each individual across all trials and plot their averaged values on a line graph at time point 1 and time point 2 to compare. The analysis of force plate data from the FR assessment will be very similar to the LOS analysis, except for the sway range is only analyzed in the anterior or forward direction, meaning we will only look at the maximum COP travel in the anterior direction throughout each trial.
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ENGINEERING Results Due to limited sample size, only trends were observed between the three main groups. Many of the study participants have not completed testing at the second time point six months after their last treatment. Sway range values from the LOS assessment represent the maximum travel of one’s COP throughout a trail, so it is considered healthiest for an individual to be able to lean as far as possible in this task. When comparing the sway ranges from the LOS assessments, a higher sway range value indicates more controlled balance, good range of motion and/or overall confidence. Lower sway range values indicate the individual had limited ability and/ or confidence to move away from their neutral standing position while comfortably maintaining their balance. When analyzing LOS data in the anterior-position direction, which is an important direction for everyday tasks like reaching, walking and stepping, some different and interesting trends emerged across the subject groups. Results from the three subject groups, detailed in Figure 2, indicated that all five individuals in the control group, which are those who did not go through neurotoxic chemotherapy but may have been through other treatments, showed a consistent pattern of improvement in postural control at the six-month post treatment time point. This consistent change seems to indicate that strength and/or confidence improved as these got further away from their treatment, enabling them to control their balance and lean farther in the anterior-posterior direction six months after treatment. For those individuals who received neurotoxic chemotherapy but did not report with signs of CIPN (asymptomatic),
there is a less consistent pattern of change, with one individual who improved in postural stability, one worsened, and one remained at about the same level from first time point testing six weeks after treatment. For those who had neurotoxic chemotherapy and reported with signs of CIPN (symptomatic), three of the four individuals remained the same or slightly worse, while one individual appeared to greatly improve. The majority of the individuals in these subject groups at the six-month time point tended to either worsen or stay around a similar level of postural control from the first time point.
Discussion and Future Direction The objective of this paper was to determine the changes that occurred in cancer survivors during the rehabilitation period after chemotherapy. This is the first pilot study to investigate a variety of changes that could occur during this three- to six-month period. Currently, limited work has been done to understand the changes or deficits experienced during this period and clinicians often do not act to reduce the lasting symptoms of cancer treatment until after 6 months [18]. As more information is gathered and learned during this rehabilitation period, significant improvements to post-chemotherapy treatment and rehabilitation strategies could occur. With improvements to treatment and rehabilitation, the quality of life, functionality and overall confidence of cancer survivors could be impacted. While there are some limitations to the current study due the small sample size, study participants are continually added or completing their
Figure 2. The results from preliminary data analysis of the anterior-posterior direction of LOS assessment. Each line represents a study participant. On the y-axis, the overall COP excursion, also known as sway range, is displayed. On the x-axis, the number one represent results from testing immediately following treatment and the number two represents the second time point testing 6 months after treatment. P. Ingram, 2017.
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ENGINEERING six-month follow-up testing. Additionally, due to Institutional Review Board delays in collaboration between The Ohio State University and the University of Dayton, only the LOS data was analyzed thus far. When more participants have completed their follow-up and their balance data is collected, general conclusions can be made. With the current data, interesting and insightful trends were found and suggest there are differences between the three subject groups. The control group showed the most consistent improvement after treatment, where each individual increased in their range of motion between time Point 1 and time Point 2. While we do not have baseline data from the subjects before they were diagnosed with cancer or exposed to any treatment, this pattern of improvement suggests that something is consistently occurring during the rehabilitation period causing these individuals’ range of motion and postural control to improve. It is possible that the treatments that these individuals went through, even though not a neurotoxic chemotherapy, affects postural stability. If future work supports this notion, it may be beneficial to investigate the postural and functional stability of various cancer patients with different cancer treatments to detect if there are any underlying mechanisms of postural instability, aside from neurotoxic chemotherapy. Alternatively, while LOS assessments evaluate one’s range of motion, they are also dependent on one’s confidence in the activity. Therefore, this consistent improvement could be due to the changing confidence of individuals as they get further away from treatment and have the energy and motivation to improve. Unfortunately, since we do not know the baseline balance data of these study participants, we do not know for certain that their balance and postural control were impaired due to cancer. However, the trends seem to imply that those who do not go through neurotoxic chemotherapy may naturally improve in postural control on their own and may not need rehabilitation or other interventions. In contrast, the asymptomatic and symptomatic group did not show a consistent pattern of improvement post treatment. While we don’t know exactly why or how to justify this variation, we do see greater differences in responses among the individuals six months after treatment. A majority of the individuals in these groups either stayed about the same level or worsened in their range of motion at their six-month follow-up testing. As of now, there Proceedings 2017
appears to be a difference between the control group and the symptomatic and asymptomatic group, but no obvious difference between the symptomatic and asymptomatic group. Based on these preliminary results, intervention strategies and rehabilitation may be especially warranted for the asymptomatic group and the symptomatic group so that they are able to improve their balance and overall functionality to obtain the level observed in the control group by time Point 2. The overall desire of this work is to better understand and determine if there is a need for rehabilitation and intervention in cancer survivors during the rehabilitation period. Considering the practical importance of the anterior-posterior direction in everyday life, mentioned previously, the results from the LOS assessment have the potential to give a realistic indication of cancer survivor’s level of postural control and areas of risk in their everyday activities. Based on our initial findings, for those undergoing neurotoxic chemotherapy some rehabilitation during the rehabilitation period may be warranted to help improve balance and everyday functionality. If future work supports this notion, we will investigate the use of existing rehabilitative techniques for the asymptomatic and symptomatic group who did not seem to improve in LOS on their own after treatment. If rehabilitation is rendered as useful based on future data analysis, we would look into introducing interventions after treatment or potentially during treatment if manageable by the individual. Rehabilitation strategies, like exercise programs, have appeared to show promising improvements for cancer survivors. One study found that exercise and strength training can reduce symptoms, minimize fatigue and increase physical function and strength after cancer treatment. The overall quality of life of these cancer survivors seemed to improve after exercise as well [19]. Another study found that introducing exercise intervention programs immediately following treatment can help reduce some of cancer’s long-lasting symptoms [18]. Lastly, long-term exercise training programs have shown to improve stability and cognitive function in diabetic populations with peripheral neuropathy, which is a similar condition to CIPN [20]. Introducing exercises after treatment that target specific areas of weakness for breast cancer survivors shows promise for improving 45
ENGINEERING strength and overall quality of life based on previous research. Particularly, long-term exercise programs for those with CIPN could help manage their long-lasting symptoms such as muscle weakness and fatigue based on Balducci’s study of the diabetic population with peripheral neuropathy. Additionally, ankle-foot orthotics (AFOs), are a common clinical assistive device used to reduce the risk of falls and assist in gait by enhancing proprioceptive and physical cues to the lower limbs [21]. They have been used in populations that experience a loss of sensation in the distal limbs, such as individuals who suffered a stroke. In the stroke population, AFOs facilitated significant improvements in balance and gait [22]. The utilization of these devices have been associated with static postural control improvements in the diabetic population with peripheral neuropathy [21]. Although more research is needed to investigate the benefits and disadvantages of AFOs, based on current research, the use of AFOs may result in significant postural improvements for cancer survivors, specifically those with CIPN. Moving forward, data analysis of limits of stability data will continue as more study participants finish testing at both time points. We will also initialize analysis of data from the functional reach tests of upper and lower extremities. Once more individuals complete testing, we can begin to explore large scale statistics and come to more general conclusions. If interesting trends continue to emerge, we will look into publishing results in various biomechanics journals. To expand the study, we will look into funding opportunities to create a multi-center breast cancer study that can further analyze the effects of chemotherapy during and after treatment.
and support throughout the Berry Summer Thesis Institute, as well as all the undergraduate researchers in her Engineering Wellness through Biomechanics lab this summer. Lastly, I would like to thank all the other members of the Berry Summer Thesis Institute cohort for making my summer full of rewarding and meaningful experiences.
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S.M. Monfort, X. Pan, R. Patrick, J. Singaravelu, C.L. Loprinzi, M.B. Lustberg, A.M.W. Chaudhari, Gait & Posture Natural history of postural instability in breast cancer patients treated with taxane-based chemotherapy: A pilot study, Gait & Posture. 48 (2016) 237–242. doi:10.1016/j.gaitpost.2016.06.011.
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S. Kneis, A. Wehrle, K. Freyler, K. Lehmann, B. Rudolphi, B. Hildenbrand, H.H. Bartsch, H. Bertz, A. Gollhofer, R. Ritzmann, Balance impairments and neuromuscular changes in breast cancer patients with chemotherapy-induced peripheral neuropathy, Clinical Neurophysiology. 127 (2016) 1481–1490. doi:10.1016/j. clinph.2015.07.022.
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J. Hopkins, About the Peripheral Nervous System | Nerve Injuries and Symptoms | Johns Hopkins Peripheral Nerve Surgery Center, (n.d.). http://www. hopkinsmedicine.org/neurology_neurosurgery/ centers_clinics/peripheral_nerve_surgery/conditions/ peripheral_nerve_system.html (accessed June 21, 2017).
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K.M. Winters-Stone, B. Torgrimson, F. Horak, A. Eisner, L. Nail, M.C. Leo, S. Chui, S.W. Luoh, Identifying factors associated with falls in postmenopausal breast cancer survivors: A multi-disciplinary approach, Gait & Posture. 92 (2011) 646–652. doi:10.1016/j.apmr.2010.10.039.
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M.A. Wampler, K.S. Topp, C. Miaskowski, N.N. Byl, H.S. Rugo, K. Hamel, Quantitative and Clinical Description of Postural Instability in Women With Breast Cancer Treated With Taxane Chemotherapy, Gait & Posture. 88 (2007) 1002–1008. doi:10.1016/j.apmr.2007.05.007.
Acknowledgements I wish to thank Dr. Ajit Chaudhauri and Dr. Scott Monfort and the other researchers at The Ohio State University, who envisioned, initiated and carried out the larger part of this study on which my work is based. Their efforts have been supported by a National Institutes of Health R03 grant award. I would also like to express my appreciation to the Berry family and the Honors Program staff for not only creating such a beneficial research program but for funding and supporting my research efforts this summer. I would also like to thank Dr. Bigelow for her constant guidance 46
10. Central Nervous System (CNS) Function, Parts, Diagram & Charts, WedMD. (2017). http://www.emedicinehealth. com/anatomy_of_the_central_nervous_system/article_ em.htm (accessed June 22, 2017).
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ENGINEERING 11. S.M. Monfort, X. Pan, R. Patrick, B. Ramaswamy, R. Wesolowski, M.J. Naughton, C.L. Loprinzi, A.M.W. Chaudhari, M.B. Lustberg, Gait, balance, and patientreported outcomes during taxane-based chemotherapy in early-stage breast cancer patients, Gait & Posture. 164 (2017) 1–9. doi:10.1007/s10549-017-4230-8. 12. K.M. Winters-Stone, F. Horak, P.G. Jacobs, P. Trubowitz, N.F. Dieckmann, S. Stoyles, S. Faithfull, Falls, Functioning, and Disability Among Women With Persistent Symptoms of ChemotherapyInduced Peripheral Neuropathy., Journal of Clinical Oncology: Official Journal of the American Society of Clinical Oncology. 35 (2017) 2604–2612. doi:10.1200/ JCO.2016.71.3552. 13. T. Bao, C. Basal, C. Seluzicki, S.Q. Li, A.D. Seidman, J.J. Mao, Long-term chemotherapy-induced peripheral neuropathy among breast cancer survivors: prevalence, risk factors, and fall risk, Breast Cancer Research and Treatment. 159 (2016) 327–333. doi:10.1007/ s10549-016-3939-0. 14. G.K. Juras, F. Wychowania, G. Sobota, B. Bacik, Evaluation of the Limits of Stability ( LOS ) Balance Test, Journal of Human Kinetics. 19 (2008) 39–52. doi:10.2478/ v10078-008-0003-0. 15. P.W. Duncan, D.K. Weiner, J. Chandler, S. Studenski, Functional Reach: A New Clinical Measure of Balance, Journal of Gerontology. 45 (1990) M192–M197. doi:10.1093/geronj/45.6.M192. 16. M.H. Cameron, S. Lord, Postural Control in Multiple Sclerosis: Implications for Fall Prevention, (2010). doi:10.1007/s11910-010-0128-0. 17. S.M. Lynch, P. Leahy, S.P. Barker, Reliability of measurements obtained with a modified functional reach test in subjects with spinal cord injury., Physical Therapy. 78 (1998) 128–33. http://www.ncbi.nlm.nih.gov/ pubmed/9474105. 18. R.R. Spence, K.C. Heesch, W.J. Brown, Exercise and cancer rehabilitation: A systematic review, Cancer Treatment Reviews. 36 (2010) 185–194. doi:10.1016/j. ctrv.2009.11.003. 19. K.S. Courneya, C.M. Friedenreich, Physical exercise and quality of life following cancer diagnosis: a literature review., Annals of Behavioral Medicine: A Publication of the Society of Behavioral Medicine. 21 (1999) 171–179. doi:10.1007/BF02908298. 20. S. Balducci, G. Iacobellis, L. Parisi, N. Di, E. Calandriello, F. Leonetti, F. Fallucca, Exercise training can modify the natural history of diabetic peripheral neuropathy, Journal of Diabetes and Its Complications. 20 (2006) 216–223. doi:10.1016/j.jdiacomp.2005.07.005. 21. K.E. Bigelow, Immediate Influence of Carbon Composite Ankle-Foot Orthoses on Balance and Gait in Individuals with Peripheral Neuropathy: A Pilot Study, JPO Journal of Prosthetics & Orthotics October. 26 (2014) 220–227. http://ovidsp.ovid.com/ovidweb. cgi?T=JS&CSC=Y&NEWS=N&PAGE=fulltext&D=ovf tp&AN=00008526-201410000-00010. 22. S.F. Tyson, R.M. Kent, Effects of an ankle-foot orthosis on balance and walking after stroke: A systematic review and pooled meta-analysis, Archives of Physical Medicine and Rehabilitation. 94 (2013) 1377–1385. doi:10.1016/j.apmr.2012.12.025.
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Breaking Down Barriers to Sustainable Eating at the University of Dayton Lauren M. Murray1,2,3 and Diana Cuy Castellanos1,2 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
Advisor: Diana Cuy Castellanos, Ph.D. Department of Health and Sport Science Corresponding Author: Diana Cuy Castellanos Email: cuycastellanos1@udayton.edu
Abstract Sustainability is at the forefront in many conversations and innovations at the University of Dayton (UD), ranging from the Hanley Sustainability Institute to the Sustainability Club. However, sustainability extends beyond water and energy conservation. In addition to these, students and faculty at UD can impact the carbon footprint through sustainable eating. The purpose of this research was to define sustainable eating, determine the carbon footprint of foods served in the UD dining halls, examine its importance, consider the barriers to sustainable eating, and develop and evaluate an educational program for UD students and faculty within the dining halls. In this study, sustainable eating is defined as food with the lowest carbon footprint. First, we hypothesize that animal foods will have higher carbon emissions compared to plant foods per pound. Secondly, we hypothesize that Virginia West Kettering (VWK) dining hall customers will select foods with a smaller carbon footprint once provided with education surrounding sustainable eating.
Introduction Due to the urgency of climate change, now is an imperative time to assess the carbon footprint of food. The carbon footprint measures environmental impact through greenhouse gas emissions — greenhouse gases (GHGs) include carbon dioxide, methane, nitrous oxide, water vapor and others. Further, it is well known that food production and distribution contribute to GHGs. 48
The Western diet is defined as a diet high in domestic meats, refined sugars, cereals, oils and nonhuman milk (Carraro-Bastos et al. 2011). Research suggests the Western diet results in 20-30% of all GHGs in the US (Beverland, 2014). Further, 37% of methane and 65% of nitrous oxide emissions are due to animal agriculture (Conrad, 2012). Methane and nitrous oxide are particularly potent gases because of their ability to absorb energy. The Global Warming Potential and the lifetime of these gases are two key factors in their environmental impact. The Global Warming Potential (GWP) compares the ability of GHGs to the ability of carbon dioxide to trap energy. The lifetime of GHGs measures how long each GHG stays in the atmosphere. The GWP of methane is 28 to 36 times that of CO2 and nitrous oxide is 265-298 times that of CO2. Carbon dioxide stays in the atmosphere for thousands of years, while methane lasts approximately 10 years and nitrous oxide over 100 years (US EPA, 2017). Research indicates the reduction of methane and nitrous oxide emissions will affect the atmosphere faster due to their shorter lifetimes (Conrad, 2012). Although meat is one of the main pressures on the environment, the human demand for meat is constantly increasing (Welin and Van der Weele, 2012). Reasons for this increase include the subsidization of animal products in developed countries, cultural influences, and institutional factors (Conrad, 2012 and Beverland, 2014). Therefore, it is important to develop interventions that encourage sustainable eating to decrease GHGs while still promoting the Proceedings 2017
HEALTH SCIENCE consumption of a balanced and nutrient-dense diet. Encouraging reduced meat and dairy intake and increased fruit, vegetable and grain intake could reduce the environmental impact of food production, which calls for a change in consumer behavior. Pieniak and colleagues suggest change in behavior is more likely to occur when a person has: (1) strong intention, (2) no environmental constraints (such as pressures from friends or family, money restrictions, etc.), and (3) the skills and knowledge to change their behavior (Pieniak, Zakowska-Biemans, Kostrya, and Raats, 2016). Therefore, the purpose of this research is to: (1) determine the greenhouse gas emissions of the foods served in Virginia West Kettering (VWK) dining hall and (2) develop, implement and evaluate the effectiveness of a sustainable eating education intervention directed at sophomores at the University of Dayton.
Methodology and Results Carbon Emission Calculations The first phase of the research consisted of data collection and carbon footprint calculations of the foods served at VWK dining hall. UD food service providers supplied reports containing information about food products purchased for Marycrest and VWK dining halls. The reports included the type of product, the product region and occasionally the distance travelled. If the distance travelled was not included in the report, the manufacturing plant was mapped and the distance between the plant and the foodservice provider was determined.
The Food Carbon Emissions Calculator by Clean Metrics, a web-based LCA software tool for the modeling and analysis of life cycle greenhouse gas (GHG) emissions, energy use and water use in food and beverage products, was utilized to determine the greenhouse gas emissions from each product. The GHGs examined within the Calculator include nitrous oxide, methane and carbon dioxide, which are in terms of CO2 equivalents. The production and transportation GHG emissions were calculated by inputting the product and distance travelled into the Food Carbon Emissions Calculator. This estimated the kilograms of CO2e emissions per pound of product, which was converted into pounds of CO2e emissions per pound of product. Once the carbon footprint of each individual product was assessed, the weighted average emissions were calculated for the products. These calculations were entered into an excel spreadsheet and analyzed to evaluate how products compared in regard to their emissions. UD dining services supplied the recipes for meals served at VWK. The average product emissions and the weight of products within meals was combined to calculate the average pounds of CO2e emissions per pound of meal. The meal emission calculations were also put into an excel spreadsheet and analyzed regarding amount of emissions. Ingredients and meals were placed in order from lowest to highest CO2e emissions for further analysis.
Result of Carbon Emissions Calculations Over the course of six months, the products in Marycrest and VWK produced over 2,517,277 lbs of CO2e emissions, and the top 10 products bought by Marycrest and VWK constitute 92.97% of these GHG emissions (See Figures 1 and 2). After comparing products, it was discovered that lamb contributed the most GHGs per pound of product whereas mushrooms contributed the least. Generally, meat products like pork, salami, bacon and seafood had the highest average emissions per pound of product. On the other hand, vegetables and fruits had significantly lower average emissions (See Figures 3 and 4). After comparing meals, it was found that hot dogs contributed the most GHGs per pound of meal whereas cream of asparagus soup contributed the least (See Figures 5 and 6).
Figure 1. The sources of all GHG emissions of food products bought by Marycrest and VWK in a 6-month period. Over 48% of all emissions came from chicken products. L. Murray, 2017.
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Figure 2. The total emissions from the top ten products bought by Marycrest and VWK over the course of six months. These emissions comprised over 92% of all GHG emissions from this time. L. Murray, 2017.
Figure 3. Ten ingredients that emitted the most GHGs per pound of product. Lamb emits the most GHGs — over 24 lbs of CO2e emissions per pound of lamb. L. Murray, 2017.
Figure 4. Ten ingredients with the lowest GHG emissions. Mushrooms emit the least GHGs — a little over 0.06 lbs of GHGs per pound of mushrooms. L. Murray, 2017.
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Figure 5. Ten meals with the highest GHG emissions per pound of meal. Hot Dogs emitted the most GHGs — over 12 lbs of GHGs per pound of hot dog. L. Murray, 2017.
Figure 6. Ten meals with the lowest GHG emissions per pound of meal. Cream of Asparagus Soup emitted the least GHGs — 0.1 lbs of GHGs per pound of soup. L. Murray, 2017.
Sustainable Eating Education The sustainable eating education program will be eight weeks in duration and implemented in the fall of 2017. It will promote the understanding of the carbon footprint as related to food choices of those who dine at VWK. The educational program is split into two parts: (1) a food carbon footprint ranking system and (2) a sustainable eating education program.
Ranking Scales Each station within the dining halls will receive a carbon footprint scale that applies to the food being served that day. The goal of the ranking system is to help dining customers visually Proceedings 2017
conceptualize the carbon footprint of their food options. The ranking scales are color coded to aid customers in assessing the carbon footprints: the top 25% of emitters are colored red, the second 25% colored yellow, and the bottom 50% colored green. There are two different ranking systems: (1) comparing ingredients and (2) comparing meals. At certain dining stations within VWK the consumer has the ability to choose the ingredients that comprise their meal. The ranking scales located at these stations compare the ingredients available so that consumers can choose ingredients based on GHG emissions (See Figure 7). The ingredient GHG emissions tend to be much lower than the GHG emissions of pre-determined meals. Therefore, the dining 51
HEALTH SCIENCE Ingredient Scales
Meal Scales
Figure 7. The Ingredient Scales used at dining stations where students have the ability to choose which ingredients comprise their meal. L. Murray, 2017.
Figure 8. The Meal Scales at dining stations where students do not have the ability to choose the ingredients that comprise their meal. L. Murray, 2017.
stations that offer pre-determined meals have a separate ranking scale. The ranking scales located at these stations compare the GHG emissions of the meals available (See Figure 8).
outlines the themes of the seven educational poster boards. •
The first barrier that surfaces is lack of pre sustainability literacy. The goal of the first educational poster will be to provide education about sustainability and why it is important. It will answer the questions: What is sustainability? What are the possible consequences of not eating sustainably? And what are the benefits of being sustainable?
•
The second barrier that was identified is lack of knowledge about sustainable eating (Schosler, 2016). The second poster will address the following questions: Which foods count as sustainable? Which foods are not sustainable? How can individuals
Sustainable Eating Education Program The second part of the educational program includes an intervention to educate students and faculty about sustainable eating through monthly poster boards located in the VWK dining hall. The poster boards will address specific barriers that students and faculty may have to sustainable eating, and they will provide information to negate these barriers. They will also provide definitions of sustainable eating and information on different foods. The following 52
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HEALTH SCIENCE be more sustainably minded regarding food consumerism? •
•
The third barrier is lack of knowledge about the impact of sustainable eating (Conrad, 2012). The third poster will respond to the following questions: How does sustainable eating impact greenhouse gas emissions? How does sustainable eating impact the environment? How does the effect of sustainable eating compare to other sustainability measures? Another barrier that surfaced during preliminary research is the concept that diet is a private matter and any criticism of food choice is a restriction of liberty (Conrad, 2012). This poster will answer the questions: How does diet affect more than just the individual? How do our obligations to others and the environment affect the way we view our food choices?
•
An additional barrier identified is the inconvenience of lifestyle changes such as diet (Schosler, 2016). The poster addressing this barrier will satisfy the questions: What are the personal/health benefits of eating sustainably? What is the cost of eating sustainably? What is the accessibility of sustainable food on campus? Does the taste of sustainable foods compete with the taste of unsustainable foods?
•
The sixth barrier identified was the diversity in motivations behind food choices. The Regulatory Focus Theory states that there are two basic motivators: promotion and prevention. Promotion oriented individuals value obtaining positive outcomes, whereas prevention oriented individuals value avoiding negative outcome (Schosler, 2012). Therefore, the poster for this barrier will focus on the positive and negative outcomes of sustainable eating, and it will answer the questions: How do food choices align with values? What are the values that influence food choices? And what are the consequences of these choices?
•
The final barrier is the concern about how sustainable eating affects personal health (Conrad, 2012). The poster regarding this barrier will answer: Is it healthy to eat less meat and animal products? Is it healthy to eat a plant-based diet?
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Effectiveness of the educational program will be measured utilizing a food waste methodology, sustainable eating student questionnaire and student focus groups.
Food Waste Protocol Food waste studies will be performed in VWK before and after the program is implemented. Foods considered most and least sustainable will be measured before and after meals are served with a food scale. The data collected will determine which foods are increasing and which foods are decreasing in dining hall sales in relation to the program implementation. This quantitatively determines if the educational intervention significantly affects consumer food choices.
Sustainable Eating Questionnaire In addition, a questionnaire will be created to assess students' attitudes and beliefs about sustainable eating at baseline and eight weeks. The questionnaire will be framed around the social cognitive theory, which states that behavior is based on interaction between the person and the environment. It also assumes that a person’s behavior is affected by skills, self-efficacy and outcome expectancies. Some environmental factors include peer modeling, availability and accessibility of the behavior (Wayne, 2010). The questionnaire will examine consumers’ skills to choose sustainable foods, self-efficacy around sustainable eating and outcome expectancies for choosing sustainable foods.
Focus Groups Focus groups will be administered to explore students' perceptions of sustainable eating at baseline and eight weeks. Focus groups will continue at baseline until data saturation is reached. Each group will consist of six to ten students who will be asked questions regarding skills, self-efficacy and outcome expectancies about sustainable eating as well as ways to improve the intervention.
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Bibliography
The methods above provide a way to analyze the carbon footprint of food and a possible intervention program to encourage sustainable eating. This can be utilized in other areas that involve consumer food choices such as restaurants and grocery stores. It can also serve as a model for other universities that desire to educate consumers about sustainable food choices. The VWK education intervention will be a pilot for the extension of the program in the Marycrest first-year dining hall. Eventually, this can also be extended to upper division students who make their food choices within their own homes instead of dining halls. Providing guidelines to these students could potentially affect their consumer behaviors at grocery stores or restaurants.
Beverland, M. (2014). Sustainable Eating: Mainstreaming Plant-Based Diets In Developed Economies. Journal of Macromarketing, 34 (3), 369-382. doi:10.1177/0276146714526410.
The results of the Carbon Emission Calculations are consistent with previous research that suggests that animal products have higher GHG emissions (Conrad, 2012). However, the analysis of the carbon footprint does not include the packaging and cooking of the food, which would be an important consideration within grocery stores. Further research and more information is required to assess this aspect of the carbon footprint. In addition, the carbon footprint of highly processed and packaged foods could not be calculated using the Clean Metrics calculator. This limited the food that was included in the study to fresh foods and foods prepared by UD dining staff. The use of different databases and the continuing of research could relieve this limitation.
Acknowledgements
Conrad, S. (2012). Are we morally obliged to become vegans? Climate Change and Sustainable Development: Ethical Perspectives on Land Use and Food Production, 367-372. doi:10.3920/978-9-8686-753-0-55. Davies, A. R. (2014). Co-creating sustainable eating futures: Technology, ICT and citizen–consumer ambivalence. Futures, 62, 181-193. doi:10.1016/j.futures.2014.04.006. Jallinoja, P., Niva, M., & Latvala, T. (2016). Future of sustainable eating? Examining the potential for expanding bean eating in a meat-eating culture. Futures, 83, 4-14. doi:10.1016/j.futures.2016.03.006. . Pieniak, Z., Zakowska-Biemans, S., Kostyra, E., & Raats, M. (2016). Sustainable healthy eating behaviour of young adults: towards a novel methodological approach. BMC Public Health, 16, 1-9. doi:10.1186/s12889-016-3260-1. Ruiz, F. (2012). Food, sustainability and ecological responsibility: hunger as the negation of human rights. Climate Change and Sustainable Development: Ethical Perspectives on Land Use and Food Production, 342-347. doi: 10.3920/978-9-8686-753-0-51. Sargant, E. (2014). Sustainable Food Consumption: A Practice Based Approached, 11. 13-21. Schosler, H., de Boer, J., Boersema, J. (2012). A theoretical framework to analyse sustainability relevant food choices from a cultural perspective: caring for food and sustainability in a pluralistic society. Climate Change and Sustainable Development: Ethical Perspectives on Land Use and Food Production, 335-341. doi: 10.3920/978-9-8686-753-0-50. United States Environmental Protection Agency (2017). Greenhouse Gas Emissions: Understanding Global Warming Potentials. Retrieved from https://www.epa.gov/ ghgemissions/understanding-global-warming-potentials. Wayne, C., Miller. (2010). Chapter 7: Behavior Models. Helping the Obese Patient Find Success, 101-114. Retrieved from https://www.westernschools.com/Portals/0/html/N1253/ X73Fsj_files/OEBPS/Text/Section0003.html. Welin, S., Van der Weele, C. (2012). Cultured meat: will it separate us from nature? Climate Change and Sustainable Development: Ethical Perspectives on Land Use and Food Production, 348-351. doi: 10.3920/978-9-8686-753-0-52.
I would like to thank the University Honors Program and the Berry Family for funding my research. I would also like to thank my advisor, Dr. Diana Cuy Castellanos, for offering guidance. Finally, I would like to thank Joan Bauman and Dining Services for providing information about recipes and dining hall products.
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An Examination of Bacterial Efflux Pumps and Their Role in Multidrug Resistance Sarah P. Baxter1,2,3 and Matthew E. Lopper1,2 University of Dayton, 300 College Park, Dayton, OH, 45469 1. University of Dayton Department of Chemistry 2. Berry Summer Thesis Institute 3. University Honors Program
Advisor: Matthew E. Lopper, Ph.D. Department of Chemistry Corresponding Author: Sarah Baxter Email: baxters2@udayton.edu
Abstract Multidrug resistant bacteria have become a great concern in the world of medicine. Antibiotics are not being discovered at a fast enough rate to fight this resistance, leaving many bacterial infections left unable to be treated with the current antibiotics. The efflux of drugs out of cells is one of the mechanisms contributing to this resistance. Transporter proteins, called efflux pumps, located along the cytoplasmic membrane of bacterial cells are responsible for this uncontrollable ejection of antibiotics out of cells. In drug resistant bacterial cells, efflux pumps have been found to be very problematic in the introduction of antibiotics and other drugs, often expelling the desired chemicals out of the cell. This makes it difficult for the bacteria to receive the intended effects of those drugs. Efflux pumps, their role in conferring multidrug resistance, and ongoing efforts to block their activity will be investigated.
Antibiotic Resistance Antibiotic resistance has become a major threat to human health. According to the Center for Disease Control, over 2 million people were infected with resistant bacteria in 2013. As a result, 23,000 people died because the bacteria were resistant to the antibiotics available (Centers for Disease Control, 2013). Microbes have provided many of the antibiotics and derivatives used today, but as resistance spreads, antibiotics become ineffective (Lewis, 2013). No new class of antibiotic has been discovered since 56
the 1960s and the derivatives of current drugs are not eliminating the problem of antibiotic resistance (Lewis, 2013). Pharmaceutical companies are struggling to afford the $800 million and the 10 or more years that the process of developing a new drug demands (Conly & Johnston, 2005). Furthermore, we typically only get a few years of the antibiotic use before resistance is observed, leaving little time for pharmaceutical companies to realize a return on their investment. Resistance to an antibiotic has been observed in as little as one year after its discovery (Lewis, 2013). Research has been aimed at finding solutions to the problem of bacterial resistance to antibiotics, especially against the six resistant ESKAPE bacteria: Enterococcus faecium, Staphylococcus aureus, Klebsiela pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter species (Prasch & Bucar, 2015). These pathogens are common causes of hospitalbased infections that harm ill individuals with an already compromised immune system and have been found to reject available antibiotics (Santajit & Indrawattana, n.d.). There are four main mechanisms of resistance. The first is the modification of the target site, preventing the antibiotic from binding. The second is the inactivation of the antibiotic by bacterial enzymes. The third is the cell’s overproduction of the target enzyme as a means to compensate for the effects of the inhibitor, and the fourth is the prevention of the penetration of antibiotics within the cell or the efflux of the antibiotic out of the cell (Lewis, 2013). β-Lactams, Proceedings 2017
LIFE AND PHYSICAL SCIENCES Aminoglycosides, Tetracyclines, Macrolides, Lincosamides, Streptogamins, Oxazolidinones, Phenicols, Quiolones, Pyrimidines, Sulfonamides, Rifamycins and Cationic peptides are classes of antibiotics greatly impacted by the drug efflux and altered target mechanisms of resistance (Davies & Davies, 2010).
Bacterial Efflux Pumps In 1980, the tetracycline pump (TetA) in E. coli was the first efflux pump to be discovered, making efflux pumps a relatively new area of study (Prasch & Bucar, 2015). Efflux pumps are large transporter proteins located along the membrane of a bacterial cell. These transporter proteins are divided into two groups based on their source of energy for efflux. Primary transporters, also known as ABC multidrug transporters, hydrolyze ATP as a source of energy (Prasch & Bucar, 2015). Secondary transporters use the proton gradient that results from the bacterial cell’s metabolism as a source of energy (Marquez, 2005). Secondary transporters can be further divided into four different families: the major facilitator (MF) superfamily, the resistance-nodulation-division (RND) family, the small MDR (SMR) family, and the multiple antibiotic and toxin extrusion (MATE) family (Prasch & Bucar, 2015).
2007). The pump spans from the cytoplasm to the outside of the cell, penetrating the surface of the cell, and is responsible for ejecting antibiotics out of bacteria cells, preventing the accumulation of the drug within the cell. This causes the antibiotics to be ineffective. In order for current antibiotics to be effective again, the efflux pumps must stop pumping the drugs out of the bacteria cells. The AcrAB-TolC system is the major pump contributing to antibiotic resistance in E. coli, leading to much research on the structure and function of the AcrAB system (Coldham, Webber, Woodward, & Piddock, 2010). Efflux Pumps have been found to play roles in biological processes outside the efflux of antibiotics. Multidrug resistant pumps have also been found to play roles in transportation of glucose and bile salts, cell homeostasis, intercellular signaling, stress adaptation and more (Prasch & Bucar, 2015). AcrAB-TolC and MexAB-OprM are very important for the survival of bacteria as they allow bacteria to survive in the presence of toxic agents (Santajit & Indrawattana, n.d.). The genes that code for the overexpression of efflux pumps are now being selected for as efflux pumps continue to play a critical role in the survival of bacteria, contributing to greater antibiotic resistance.
Organisms often code for multiple efflux pumps. MexAB-OprM and AcrAB-TolC are two major tripartite systems studied, but other efflux pumps discovered in the ESKAPE bacteria are presented in Table 1 (Stavri, Piddock, & Gibbons,
Figure 1. A mechanism of antibiotic resistance that prevents the accumulation of antibiotics within the cell. The bacterial cell on the left does not overexpress its efflux pumps and it dies due to the accumulation of the antibiotics within the cell. The bacterial cell on the right overexpresses its efflux pumps, has less accumulation of antibiotics within the cell, and resists cell death. S. Baxter, 2017.
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LIFE AND PHYSICAL SCIENCES Table 1. The ESKAPE bacteria are listed below along with the efflux pumps found in each pathogen The pumps overexpressed in clinically-relevant multidrug resistant strains are noted with an asterisk (*) (Piddock, 2006).
Structure of the Tripartite AcrAB-TolC Efflux Pump The tripartite AcrAB-TolC efflux pump system can be found in gram-negative bacteria and is a known contributor of resistance (Symmons, Bokma, Koronakis, Hughes, & Koronakis, 2009). There are three main components to the efflux pump complex (Coldham et al., 2010). AcrB is the inner membrane pump located in the cytoplasm and periplasm that ejects the drugs out of the cell. AcrB uses the energy of the proton motive 58
force to power conformational changes in the pump's subunits that help propel compounds through the central passageway and out of the cells.(Symmons et al., 2009). The TolC channel is the hollow outer membrane channel that antibiotics travel through in order to leave the cell. This channel is the portion that penetrates the surface of the bacteria cell, allowing the antibiotics to leave. These pumps are relatively large in comparison to the antibiotics they eject out of the cell. The TolC protein made of 471-amino acids spans the outer membrane, forming a Proceedings 2017
LIFE AND PHYSICAL SCIENCES tunnel about 100 Å long into the periplasm and the AcrA protein of 373 amino acids spans from 100 Å to 200 Å in length (Yu, Aires, & Nikaido, 2003). AcrB composed of 1,049 amino acids is much larger than the other two components ("UniProt," n.d.). The pumps span from the cytoplasm to the outer membrane of the cell, even penetrating the surface of the cell. The structure of TolC was found to be a stable trimer in 2000 (Hayashi et al., 2016). AcrA is the remaining component. These are the adaptor proteins that connect the AcrB pump to the TolC channel. The interaction between AcrB and TolC has been investigated. Some have found that AcrB and TolC have no direct interaction, making the adaptor proteins an essential component to the structure of the pump (Du et al., 2014). Others, however, have found through direct disulfide cross-linking experiments that AcrB and TolC have a direct connection (Hayashi et al., 2016). The interactions between these two components has been questioned along with the stoichiometry of the pump components. Understanding the interactions between pump components may provide information about the mechanism of
multidrug export from these pumps and lead to discoveries in the inhibition of these pumps. The stoichiometry of the AcrAB-TolC pump is still under investigation. There is evidence supporting a 3:6:3 ratio of AcrB:AcrA:TolC (Du et al., 2014). Dr. Dijun Du from the University of Cambridge has found the pump to be made up of an “AcrB trimer, an AcrA hexamer and a TolC trimer,” consistent with the proposed 3:6:3 stoichiometry (Du et al., 2014). Other research has also been found to support a 3:3:3 ratio. An in-vivo cross-linking experiment showed a 1:1 stoichiometry of AcrA: ArcB, supporting the 3:3:3 ratio hypothesized (Hayashi et al., 2016). It has been found that a 3:3:3 pump can function as a drug exporter, but the function of 3:3:3 and 3:6:3 complexes must be further investigated (Hayashi et al., 2016). Figure 2 provides the structure of the tripartite efflux pump complex courtesy of Symmons et al. The AcrB pump has been found to be formed by three monomers, each playing a role in the efflux of drugs (Seeger et al., 2006). The “access” monomer allows the substrate or drug to enter the pump, the “binding” monomer opens the binding site for the substrate once it enters the pump, and the “extrusion” monomer forces the substrate out of the cell (Murakami, 2002). The pocket in which the substrate enters is expanded, allowing the substrate to bind and causing the access and extrusion binding pockets to shrink. The bound substrate is then squeezed out of the cell through the TolC channel by the shrinking of the binding pocket (Murakami, 2002).
Efflux Pump Inhibitors from Edible Plant Sources The overexpression of efflux pumps can result in little or no accumulation of an antibiotic within the cell due to the efflux of the drug out of the cell (Marquez, 2005). Figure 1 presents the antibiotic accumulation in a nonresistant and resistant bacterial cell. Blocking the activity of these efflux pumps has become an area of interest for researchers. Inhibiting these pumps could be a solution to the multidrug resistance phenotype seen in clinically-important bacterial strains. Figure 2. The structure of the AcrAB-TolC efflux pump along the membrane of the bacteria cell. The three components are labeled and colored. Reprinted from “The assembled structure of a complete tripartite bacterial multidrug efflux pump,” by M. F. Symmons, 2009, PNAS, vol. 106, pg. 7173, 2009.
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Edible plants are promising for inhibiting ability because they contain organic compounds that are chemically diverse and are likely to be less toxic to humans than other synthetic inhibitors 59
LIFE AND PHYSICAL SCIENCES (Prasch & Bucar, 2015). A look at edible plant sources provides a possible solution to the problem of toxicity in inhibitors. Twenty-three different plants have already been identified as containing compounds capable of inhibiting efflux pumps (Prasch & Bucar, 2015). Plants may provide a great solution to the problem of antibiotic resistance as they are renewable and yet to be explored (Prasch & Bucar, 2015). Less than 10% of all plant species have been investigated, leaving a great number of diverse plant species that could contain interesting compounds. The plant species yet to be investigated may contain clinically relevant compounds when used in conjunction with existing antibiotics (Sana, Jameel, & Rahman, 2015). A number of natural plant extracts have already demonstrated inhibitory activity including extracts from garlic, ginger, turmeric, rosemary, mint, clove, neem, and coriandrum (Kumar, Jandaik, & Patial, 2016). An investigation of more plant species may yield more promising compounds. While the mechanism involved in the inhibition of multidrug resistant efflux pumps is still under investigation, it has been proposed that inhibitors either bind directly to the pump or deplete the source of energy for the pump (Marquez, 2005). Some inhibitors may bind in a competitive manner while others may bind in an allosteric, or non-competitive manner (Poole, Zgurskaya, Misra, Opperman, & Nguyen, 2015). The hope is that compounds may be derived from edible plant sources that will effectively block the efflux pumps and allow the existing antibiotics to accumulate within the cells. These inhibitors may allow the antibiotic to be more effective at low concentrations due to the increased accumulation of the antibiotics within the cells (Sana et al., 2015). Reserpine is a plant alkaloid that has been found to block drug resistance, but concentrations necessary to inhibit the pump make the inhibitor not clinically relevant (Marquez, 2005). Finding a plant source that inhibits multiple MDR efflux pumps at nontoxic concentrations could provide a natural solution to the multidrug resistant efflux pumps.
Acknowledgements Thank you to the University Honors Program, the Berry Foundation and the Chemistry Department for funding my research this summer. I would also like to thank Dr. Lopper and the members of the Lopper lab — Erich Auer, Venicia Hawach, Donny McKenna, CJ Moellering, and Bo Leszcynski — for their support and valuable insight throughout the project.
References Aeschlimann, J. R. (2003). The role of multidrug efflux pumps in the antibiotic resistance of Pseudomonas aeruginosa and other gram-negative bacteria. Insights from the Society of Infectious Diseases Pharmacists. Pharmacotherapy, 23(7), 916–24. Retrieved from http://www.ncbi.nlm.nih.gov/ pubmed/12885104. Andersen, J. L., He, G.-X., Kakarla, P., K C, R., Kumar, S., Lakra, W. S., … Varela, M. F. (2015). Multidrug efflux pumps from Enterobacteriaceae, Vibrio cholerae and Staphylococcus aureus bacterial food pathogens. International Journal of Environmental Research and Public Health, 12(2), 1487–547. https://doi.org/10.3390/ijerph120201487. Bialek-Davenet, S., Lavigne, J. P., Guyot, K., Mayer, N., Tournebize, R., Brisse, S., … Nicolas-Chanoine, M. H. (2015). Differential contribution of AcrAB and OqxAB efflux pumps to multidrug resistance and virulence in Klebsiella pneumoniae. Journal of Antimicrobial Chemotherapy, 70(1), 81–88. https://doi.org/10.1093/jac/dku340. Centers for Disease Control and Prevention. (n.d.). Antibiotic / Antimicrobial Resistance. Retrieved August 20, 2017, from https://www.cdc.gov/drugresistance/index.html. Coldham, N. G., Webber, M., Woodward, M. J., & Piddock, L. J. V. (2010). A 96-well plate fluorescence assay for assessment of cellular permeability and active efflux in Salmonella enterica serovar Typhimurium and Escherichia coli. Journal of Antimicrobial Chemotherapy, (May), 1655–1663. https:// doi.org/10.1093/jac/dkq169. Conly, J., & Johnston, B. (2005). Where are all the new antibiotics ? The new antibiotic paradox. The Canadian Journal of Infectious Diseases & Medical Microbiology, 16(3), 159–160. Coyne, S., Courvalin, P., & Périchon, B. (2011). Effluxmediated antibiotic resistance in Acinetobacter spp. Antimicrobial Agents and Chemotherapy, 55(3), 947–953. https://doi.org/10.1128/AAC.01388-10. Davies, J., & Davies, D. (2010). Origins and evolution of antibiotic resistance. Microbiology and Molecular Biology Reviews : MMBR, 74(3), 417–33. https://doi.org/10.1128/ MMBR.00016-10. Du, D., Wang, Z., James, N. R., Voss, J. E., Klimont, E., Oheneagyei, T., … Luisi, B. F. (2014). Structure of the AcrAB – TolC multidrug efflux pump. Nature, 509(7501), 512–515. https:// doi.org/10.1038/nature13205. Hayashi, K., Nakashima, R., Sakurai, K., Kitagawa, K., Yamasaki, S., & Nishino, K. (2016). AcrB-AcrA Fusion Proteins That Act as Multidrug Efflux Transporters. Journal of Bacteriology, 198(2), 332–342. https://doi.org/10.1128/ JB.00587-15.Editor.
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LIFE AND PHYSICAL SCIENCES Ines Borges-Walmsley, M., Mckeegan, K. S., & Walmsley, A. R. (2003). Structure and function of efflux pumps that confer resistance to drugs. Biochem. J, 376, 313–338. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1223791/ pdf/13678421.pdf.
Spengler, G., Martins, A., Schelz, Z., Rodrigues, L., Aagaard, L., Martins, M., … Amaral, L. (2009). Characterization of intrinsic efflux activity of enterococcus faecalis ATCC29212 by a semi-automated ethidium bromide method. In Vivo, 23(1), 81–88.
Jang, S. (2016). Multidrug efflux pumps in Staphylococcus aureus and their clinical implications. Journal of Microbiology, 54(1), 1–8. https://doi.org/10.1007/s12275-016-5159-z.
Stavri, M., Piddock, L. J. V, & Gibbons, S. (2007). Bacterial efflux pump inhibitors from natural sources. Journal of Antimicrobial Chemotherapy, (December 2006), 1247–1260. https://doi.org/10.1093/jac/dkl460.
Koronakis, V., Sharff, A., Koronakis, E., Luisi, B., & Hughes, C. (2000). Crystal structure of the bacterial membrane protein TolC central to multidrug efflux and protein export. Nature, 405 (June), 914–919. Kumar, R., Jandaik, S., & Patial, P. (2016). Screening of medicinal plants of Himachal Pradesh for efflux pump inhibitory activity against Escherichia coli. Journal of Pharmacognosy and Phytochemistry, (July), 96–100. Lewis, K. (2013). Platforms for antibiotic discovery. Nature Reviews. Drug Discovery, 12(5), 371–87. https://doi. org/10.1038/nrd3975. Marquez, B. (2005). Bacterial efflux systems and efflux pumps inhibitors. Biochimie, 87, 1137–1147. https://doi. org/10.1016/j.biochi.2005.04.012. Masi, M., Barbe, J., & Page, J. (2005). Inhibitors of efflux pumps in Gram-negative bacteria. TRENDS in Molecular Medicine, 11(8), 382–389. https://doi.org/10.1016/j.molmed.2005.06.006. Mikolosko, J., Bobyk, K., Zgurskaya, H. I., & Ghosh, P. (2006). Conformational Flexibility in the Multidrug Efflux System Protein AcrA. Structure, 14(March), 577–587. https://doi. org/10.1016/j.str.2005.11.015.
Symmons, M. F., Bokma, E., Koronakis, E., Hughes, C., & Koronakis, V. (2009). The assembled structure of a complete tripartite bacterial multidrug efflux pump. PNAS, 106(17), 7173–7178. UniProt. (n.d.). Retrieved September 12, 2017, from http:// www.uniprot.org/. Viveiros, M., Martins, A., Paix, L., Couto, I., Eva, F., Kern, W. V, & Amaral, L. (2008). Demonstration of intrinsic efflux activity of Escherichia coli K-12 AG100 by an automated ethidium bromide method. International Journal of Antimicrobial Agents, 31, 458–462. https://doi.org/10.1016/j. ijantimicag.2007.12.015. Yu, E. W., Aires, J. R., & Nikaido, H. (2003). MINIREVIEW AcrB Multidrug Efflux Pump of Escherichia coli : Composite Substrate-Binding Cavity of Exceptional Flexibility Generates Its Extremely Wide Substrate Specificity. Journal of Bacteriology, 185(19), 5657–5664. https://doi.org/10.1128/ JB.185.19.5657.
Murakami, S. (2002). Crystal Structures of a Bacterial Multidrug Transporter Reveal a Functionally Rotating Mechanism. Life Science: Structural Biology, 14–15. Murakami, S., Nakashima, R., Yamashita, E., & Yamaguchi, A. (2002). Crystal structure of bacterial multidrug efflux transporter AcrB. Nature, 419(October), 587–593. Pagès, J., & Amaral, L. (2009). Biochimica et Biophysica Acta Mechanisms of drug efflux and strategies to combat them : Challenging the efflux pump of Gram-negative bacteria. BBA—Proteins and Proteomics, 1794(5), 826–833. https://doi. org/10.1016/j.bbapap.2008.12.011. Piddock, L. J. V. (2006). Clinically relevant chromosomally encoded multidrug resistance efflux pumps in bacteria. Clinical Microbiology Reviews, 19(2), 382–402. https://doi. org/10.1128/CMR.19.2.382-402.2006. Poole, K., Zgurskaya, H., Misra, R., Opperman, T. J., & Nguyen, S. T. (2015). Recent advances toward a molecular mechanism of efflux pump inhibition. Frontiers in Microbiology, 1–16. https://doi.org/10.3389/fmicb.2015.00421. Prasch, S., & Bucar, F. (2015). Plant derived inhibitors of bacterial efflux pumps : an update. Phytochemistry Reviews, 14(6), 961–974. https://doi.org/10.1007/s11101-015-9436-y. Sana, M., Jameel, H., & Rahman, M. (2015). Miracle Remedy : Inhibition of Bacterial Efflux Pumps by Natural Products. Infectious Diseases and Therapy, 3(2), 1–6. https://doi. org/10.4172/2332-0877.1000213. Santajit, S., & Indrawattana, N. (n.d.). Mechanisms of Antimicrobial Resistance in ESKAPE Pathogens. BioMed Research International. https://doi.org/10.1155/2016/2475067. Seeger, M. A., Schiefner, A., Eicher, T., Verrey, F., Diederichs, K., & Pos, K. (2006). Structural Asymmetry of AcrB Trimer Suggests a Peristaltic Pump Mechanism. Science, 313(September), 1295–1299.
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Role of Immune System in Alzheimer's Disease Steven G. Borchers1,2 and Amit Singh1,2,3,4 University of Dayton, 300 College Park, Dayton, OH, 45469 1. Department of Biology 2. Berry Summer Thesis Institute 3. Center for Tissue Regeneration and Engineering at Dayton (TREND) Indiana State University, Terre Haute, IN 4. Center for Genomic Advocacy (TCGA)
Advisor: Amit Singh, Ph.D. Department of Biology Corresponding Author: Amit Singh, Ph.D. Email: asingh1@udayton.edu
Introduction Alzheimer’s Disease (AD, OMIM 104300), is a fatal progressive neurodegenerative disorder which slowly and inexorably kills neurons, affects memory, causes difficulty in performing simple tasks, changes personality and affects cognitive functions. The brain overall undergoes a massive loss of tissue. More specifically, this disorder encompasses neurological alterations such as cortical shrinkage, enlarged ventricles and a diminished hippocampus. AD is named after the German physician Alois Alzheimer. In 1906, Dr. Alzheimer and his colleagues examined the brain of a 52 year old patient named Frau Auguste D after her death, and found unusual plaques and tangles. The patient exhibited memory loss and difficulty in talking (Shriver et al., 2011; Michael, 2012). These plaques and tangles are classic markers of AD, which could only be detected by a brain biopsy. Although the symptoms of this disease were not referred to as Alzheimer’s until 1906, its description has been observed in writings dated back to Egypt in ninth century B.C. Another account of AD comes from 14th century England, describing a verbal test to check for forgetfulness (Michael, 2012). Despite all these accounts and references, the disease did not get a formal diagnosis until the beginning of the 20th century. AD is the most common form of dementia and is the third leading cause of death following cancer and heart disease. The actual cause of 62
AD is not fully understood. Furthermore, there is no cure available for AD to date. The most recent estimates show that more than 700,000 deaths in US every year can be attributed to AD (Alzheimer's Association, 2014). The numbers are striking. It has been estimated that more than 5 million Americans and 46 million people worldwide are currently dying from Alzheimer’s disease. The number is staggering and it is proposed that this number is expected to triple by 2030. It has been estimated that frequency of AD increases with age. The number of AD cases almost doubles every 10 years for individuals after 65 years of age (Figure 1). These AD patients are cared for by almost 16 million caregivers in the U.S. and more than 100 million worldwide. Therefore, the cost of caregiving to AD patients is astronomical and is increasing every year. It is putting significant economic and emotional burden on families and society. To further compound the issue, there is no cure available for this disease. It is therefore aptly called the Silver Tsunami of the 21st century (Sarkar et al., 2016). Although the symptoms are clearly outlined for Alzheimer’s disease, the process by which neurodegeneration occurs is unknown. The brains of affected patients show two common pathological hallmarks in terms of clinical manifestation: the accumulation of neurotoxic extracellular Aβ42 protein plaques and the development of intracellular neurofibrillary tangles Proceedings 2017
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Figure 1. The frequency of Alzheimer’s cases. Source:http://www.usagainstalzheimers.org/crisis?gclid=Cj0KCQjwub7 NBRDJARIsAP7wlT9oupe4YL61VarmAKMG_Qes1uNMcfpo5JJkD4PGPp5Hwc0SyBQM07UaAjUCEALw_wcB.
comprised of hyperphosphorylated tau (Kumar and Singh, 2015). A large body of evidence has been accumulated suggesting Aβ42 mediated neurodegeneration is the primary pathological mechanism behind disease progression (Mullard, 2017). The amyloid hypothesis explains how the cleavage of the transmembrane Amyloid Precursor Protein (APP) causes the formation of an abnormal amyloid β42 peptide rather than a normal amyloid β40 peptide, resulting in the generation of extracellular amyloid plaques and the onset of neurodegeneration (Hardy, 2009, Sarkar et al., 2016).
Animal Models to Study AD Fruit flies and humans have a high degree of genetic conservation between them and because of this, basic biological pathways of these two organisms are nearly identical (Bier, 2005). Therefore, Drosophila have been extensively used to model human disease and to carry out drug screens (Rincon-Limas et al., 2012; Pandey and Nichols, 2011). We and other researchers have used the Drosophila eye model system to further build upon this hypothesis and understand the genetic mechanism of Alzheimer’s disease, because of the high degree of genetic conservation between flies and humans (Tare et al., 2011; Cutler et al., 2015; Moran et al., 2013; Steffensmeier et al., 2013; Sarkar et al., 2016). Our approach is to overexpress the human Aβ42 encoding polypeptide by a GMR enhancer Proceedings 2017
in developing retinal neurons. Misexpression of human Aβ42 triggers progressive retinal neuron degeneration/death (Figure 2). Our model exhibits the AD progressive neurodegenerative phenotypes in the Drosophila eye. One of the major areas in the field of Alzhiemer’s Disease research is the understanding of the genetic underpinnings and immune system response to this disease. It is known that when humans age, their immune system gets compromised or weak and as a result this dreadful disease can manifest. This may be one of the reasons that it takes time for the disease to develop and get worse with the passage of time (Figure 1). Since the plaques are formed extracellularly, it has been hypothesized that these plaques send a signal to the neurons to initiate the cell death machinery or cell death response. In this review, we have directed our efforts to discuss some of the signaling pathways involved in the Drosophila immune system.
Immune System Response in Alzheimer’s Disease One of the main causes of Alzheimer’s disease is the accumulation of a mis-cleaved form of the amyloid precursor protein (APP) (Hardy et al., 2002). This protein is normally forty amino acids in length but in a patient which will develop Alzheimer’s disease, the protein 63
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Figure 2. GMR-Gal4/UAS System for targeted misexpression of Aβ42 in Drosophila melanogaster eye. Targeted misexpression of human Aβ42 is achieved using the Gal4/UAS system. Gal4, a general transcription factor borrowed from yeast, binds to the upstream activating sequence (UAS) of a gene of interest, in our case Aβ42, and initiates transcription only when under control of the Glass Multiple Repeat (GMR) enhancer. This leads to targeted misexpression of human Aβ42 (hAβ42) amongst the differentiating photoreceptor neurons in the eye of the fly, beginning in the late third instar larval stage and continuing into adulthood. Extracellular accumulation of Aβ42 around retinal photoreceptor neurons leads to progressive neurodegeneration of the eye, as seen in the fly on the right. Images provided by A. Singh, 2017.
will also be cleaved into a 42 amino acid long strand. APP is normally cleaved by alpha and gamma secretases which leads to the 40 amino acid protein. However, in Alzheimer’s disease, APP is cleaved by alpha and beta secretases which leads to the 42 amino acid long peptide. With the addition of these last two amino acids, the APP will change to a hydrophobic nature (Speretta et al., 2012). The change in nature causes these protein strands to begin to clump together, starting as oligomers leading to microtubules and finally to plaques. These plaques will send signals to the healthy neurons and activate pro-inflammatory responses in these cells. The inflammatory response in mammals is composed of two major kinase pathways. These include nuclear factor-kappa B (NF-kB) and mitogenactivated protein (MAP) pathways (Gilmore, 2006) (Tanji et al., 2005). The Toll pathway contains the dorsal-ventral polarity protein (Dorsal) and dorsal-related immune factor (DIF) which are two NF-kB homologues. This leads to a possible connection between the inflammatory response in Alzheimer’s disease and the immune response pathways seen in Drosophila. More 64
evidence comes from the MAP kinase pathway because this inflammation induced pathway is homologous with the highly conserved Jun-NTerminal kinase (JNK) pathway described in the Imd pathway. Alzheimer’s disease’s characteristic neuronal cell death is a possible outcome of the activation of the JNK pathway. This is because one of the downstream targets of the JNK pathway in Drosophila is the activation of Drosophila apoptosis factors leading to cell death (Hetru et al., 2009; Tare et al., 2011; Sarkar et al., 2016; Ren et al., 2017). There is ongoing research in both our lab and others to understand how regulating these immune response pathways in Drosophila or other animal model systems can affect onset and progression of AD, or if members of these pathways can be biomarkers or therapeutic targets for this disease.
Toll Pathway One of the major immune response pathways of Drosophila is the Toll pathway (Figure 3). This pathway is activated in response to gram-positive Proceedings 2017
LIFE AND PHYSICAL SCIENCES bacteria and fungal infections (Michel et al., 2001). Starting with gram-positive bacteria, this class of microorganism’s membrane is the activating ligand which will bind to two different recognition proteins. Drosophila melanogaster’s genome codes for thirteen different PeptidoGlycan Recognition Proteins (PGRPs). Each of these PGRPs specialize in recognizing different molecules present in varying cellular membranes (Werner et al., 2000). The two PGRPs that begin the gram-positive cascade are PGRP-SA and PGRP-SD. These are considered short recognition proteins in comparison to other immune response pathways later discussed in this review. Although there are two separate PGRPs to respond to gram-positive peptidoglycan, both of these proteins work together to activate the next portion of the pathway (Tanji et al., 2005). The fungal approach to this pathway is different in that it does not utilize PGRPs but instead uses two molecules that are present in the extracellular matrix. The serine protease Persephone activates the next step of the pathway as long as the second molecule, the inhibitor for Persophone, Necrotic, becomes inactive (Ligoxygakis et al., 2002). Both the gram-positive and the fungal pathways will
then converge on the cytokine Spaetzle (Spz). This cell signaling protein will be cleaved by the zymogen Spz processing enzyme to produce fragments with carboxy-dense regions. These fragments are the key ligands which will bind to the Toll transmembrane protein, activating the intracellular portion of this pathway (Weber et al., 2003). The next targets of the pathway are three proteins considered to contain death domains. Death domains of proteins are specific helical structures which act as conduits for information exchange between proteins, especially ones which also have death domains. The reason for the name “death domain” stems from its similarity to caspase’s structural morphology (Feinstein et al., 1995; Weber et al., 2001). After the interactions of the three proteins dMyD88, Tube, and Pelle, researchers have not identified which kinase, enzyme or molecule leads to the next stage of the Toll pathway. What is known is that the pathway continues with the degradation of Cactus, an inhibitor cytoplasmic protein, which binds to Dorsal and DIF preventing their translocation into the nucleus. Once Cactus is degraded, Dorsal and DIF are able to cross the nuclear membrane and bind to target genes to produce antimicrobial peptides and other
Figure 3. The Toll immune response pathway. S. Borchers, 2017.
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LIFE AND PHYSICAL SCIENCES immune response peptides (Brody, 1998; Tanji et. al., 2005). After this pathway has been activated and target genes are being expressed, a negative feedback loop is created by inducing the necrotic inhibitor which deactivates Perephone (Irving et al., 2001). The Toll pathway is only one of the immune response pathways utilized by Drosophila and more research is still being conducted on negative feedback loops and kinase interaction with Cactus.
Immune Deficiency (Imd) Pathway Another immune response pathway that Drosophila use is the Imd pathway (Figure 4) and it specializes in reacting to gram-negative bacterial infections (Kleino et al., 2014). In contrast to the Toll pathway discussed above, the Imd pathway does not have an extracellular signaling pathway to activate before the transmembrane protein. This pathway is entirely intracellular and contains many internal branches in comparison to the linear approach of the Toll pathway. To begin this pathway, the transmembrane PGRP-LC and PGRP-LE, which are in the long recognition protein class, work in tandem to create the signal cascade (Takehana et al., 2004). Gram-negative peptidoglycan fragments are effectively the ligand that binds
to the PGRPs, and these fragments can come from whole gram-negative cells and fragments of these cells (Kaneko et al., 2004). After the activation of the transmembrane proteins, the first intracellular protein is the Imd protein itself. This protein contains a death domain similar to the kinases described in the Toll pathway. The death domain part of this protein also alludes to future downstream targets which may cause apoptosis in the afflicted cell (Georgel et al., 2001; Hay et al., 1994). The Imd protein leads to the activation of the Fas-associated protein with death domain (FADD) giving another example of the information sharing capabilities of death domain contain proteins. The first branching point of this pathway occurs at this point in the pathway. The FADD activates both the protein DREDD and the TAK1 complex. The TAK1 complex is the source of the second branching point of the Imd pathway. The TAK1 complex activates both the IKK complex and the JNK (c-Jun N-terminal kinase) pathway (Tanji et. al., 2005). The IKK complex is a protein specializing in phosphorylation which plays a tandem role with the DREDD protein to specifically cleave the Relish protein (Hetru et al., 2009). The DREDD protein is another intracellular protein containing a death domain that cleaves one half of the Relish complex. This protein has a human homolog caspase 8 which directly relates to cell
Figure 4. The Imd immune response pathway. S. Borchers, 2017.
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LIFE AND PHYSICAL SCIENCES apoptosis in cases of extreme disease (CASP8, 2017). The second half of the Relish complex is phosphorylated by the IKK complex, leading to the release of the trans-nuclear section of the protein. This section contains an amino-terminal transcriptional regulatory domain which will form a dimer as it attaches to the target genes in the nucleus (Hetru et al., 2009). Regulation of this pathway can possibly be controlled by the activation of Relish protein as it has been linked to the degradation of the TAK1 complex. This negative feedback loop will essentially shut down the Imd pathway and prevent the cell from developing an overactive immune response (Park et al., 2004).
Conclusion The immune response to microorganisms in Drosophila is mainly controlled by two cellular pathways. These pathways are induced by reacting either extracellularly, in the case of Toll, or across the membrane, in the case of Imd, with PGRPs with peptidoglycan fragments. The Toll pathway utilizes both an extracellular and intercellular pathway to react to fungal and gram-positive bacteria. This pathway transmits signals until reaching the inhibitor Cactus, which will fully degrade to release the trans-nuclear proteins Dorsal and DIF. The Imd pathway reacts to gram-negative peptidoglycan fragments directly with transmembrane PGRPs. Relying on a branched, intracellular pathway leading to the specific cleavage of the Relish protein complex, the Imd pathway has potential for intracellular negative feedback loops which could deactivate partial or complete sections of the pathway. The inflammation caused by Alzheimer’s disease activates homologous response pathways in mammalian models. By experimenting on the Drosophila’s immune pathways, researchers can better understand the molecular genetic basis of the cellular responses in mammalian models. This review was meant to briefly explain these two pathways’ activations and internal responses to the presence of foreign microorganisms as well as analyze the connections these pathways can have to the response pathways activated by Alzheimer’s disease.
Acknowledgments I would like to thank Mr. Berry and the Berry Summer Thesis Program for funding my research this summer. Without their financial aid, I would not have progressed in my research as much as I have. I would like to thank my research mentor, Dr. Amit Singh, for his guidance and support though my summer research process. I would like to thank Dr. Singh for providing me the reagents to carry out my experiments. I would also like to thank my lab coworkers, family and friends for working with me this summer and helping me achieve my potential as a researcher. I would also like to thank the University of Dayton Honors Program and the University of Dayton College of Arts and Sciences for allowing me to use their facilities and for making my summer research possible.
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Tare, M., Modi, R., Nainaparampil, J., Puli, O., Bedi, S., Fernandez-Funez, P., Kango-Singh, M., Singh, A. (2011). Activation of JNK Signaling Mediates Amyloid-β-Dependent Cell Death. PLOS. v.6(9): e24361.
Michel, T., Reichhart, J. M., Hoffmann, J. A., Royet, J. (2001). Drosophila Toll is activated by Gram-positive bacteria through a circulating peptidoglycan recognition protein. Nature. 414:756–759. Moran, M. T., Tare, M., Kango-Singh, M., Singh, A. (2013). Homeotic gene teashirt has a neuroprotective function in amyloid-beta 42 mediated neurodegeneration. PLoS ONE. 8(11). http://doi.org/10.1371/journal.pone.0080829. Moses, K., Rubin, G.M. (1991). Glass encodes a site specific DNA binding protein that is regulated in response to positional signals in developing Drosophila eye. Genes Dev. 5: 583-593. Mullard, A. (2017). Alzheimer amyloid hypothesis lives on. Nat Rev Drug Discov. 16(1), 3–5. Retrieved from http://dx.doi. org/10.1038/nrd.2016.281.
Weber, A.N., Tauszig-Delamasure, S., Hoffmann, J.A., Lelièvre, E., Gascan, H., Ray, K.P., Morse, M.A., Imler, J.L., Gay, N.J. (2003). Binding of the Drosophila cytokine Spätzle to Toll is direct and establishes signaling. Nat. Immunol. 4:794–800. Weber, C.H., Vincenz, C. (2001). The death domain superfamily: a tale of two interfaces?. Trends Biochem. Sci. 26 (8): 475-481. Werner, T., Liu, G., Kang, D., Ekengren, S., Steiner, H., Hultmark, D. (2000). A family of peptidoglycan recognition proteins in the fruit fly Drosophila melanogaster. Proc. Natl. Acad. Sci. 97:13772–13777.
Pandey, U., Nichols, C. (2011). Human Disease Models in Drosophila melanogaster and the Role of the Fly in Therapeutic Drug Discovery. Pharmacological Reviews. 63(2): 411-436. Park, J.M., Brady, H., Ruocco, M.G., Sun, H., Williams, D., Lee, S.J., Kato, T. Jr, Richards, N., Chan, K., Mercurio, F., et al. (2004). Targeting of TAK1 by the NF-κ B protein Relish regulates the JNK-mediated immune response in Drosophila. Genes Dev. 18:584–594. Plontz, M. (2012). "A Brief History of Alzheimer's Disease." A Brief History of Alzheimer's Disease. Today's Care Giver, Nov. 2011. Web. June 2012. <http://www.caregiver.com/ channels/alz/articles/a_brief_history.htm>. Ren, P., Li, W., Xue, L. (2017). GLYAT regulates JNK-mediated cell death in Drosophila. Scientific Reports. 7(1):5183. Rincon-Limas, D.E., Jensen, K., Fernandez-Funez, P. (2012). Drosophila Models of Proteinopathies: the Little Fly that Coulds. Curr Pharm Des. Sarkar, A., Irwin, M., Singh, A., Riccetti, M., Singh, A. (2016). Alzheimer’s disease: the silver tsunami of the 21st century. Neural Regeneration Research. 11(5): 693-697. Shriver, M, Skelton, K, Fetherling, D., Hickey, M. (2011). Alzheimer's in America: The Shriver Report on Women and Alzheimer's: A Study. New York: Simon & Schuster, 2011.
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LIFE AND PHYSICAL SCIENCES
Determining the Effect of Ethanol on Listeria Infection Ryan E. Restrepo1,2,3,4 and Yvonne Sun1,2,3 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
Advisor: Yvonne Sun, Ph.D. Department of Biology Corresponding Author: Yvonne Sun Email: ysun02@udayton.edu
Abstract
Introduction
The effect of alcohol consumption on the human body has long been a subject of research investigations because of its health benefits as well as potential hindrances to human development and function. Alcohol is widely consumed across the world but few cultures compare to the level of consumption witnessed at university campuses in the United States. Excessive alcohol consumption often exhibited by young college students has been shown to disadvantage learning and mental development. The long-term goals of our research are to establish the effects of alcohol consumption on immune health and to better educate students on the possible dangers of excessive alcohol consumption. For the Berry Summer Thesis Institute, our project focused on determining the effects of alcohol on the pathogenesis of a foodborne pathogen, Listeria monocytogenes. Using standard hemolytic assays and cell culture infection models, we observed a significant impact of ethanol on L. monocytogenes toxin production and cellular invasion. Using high alcohol preferring mice as a novel model of animal infection, we demonstrated a gender-based difference in susceptibility to L. monocytogenes infections in response to alcohol treatments. These important results provide the preliminary evidence to continue my thesis research to further investigate how ethanol perturbs L. monocytogenes and host interactions. From the research training, we will also become more informed advocates for safe drinking on campuses.
Alcohol is a substance commonly found on college campuses across the United States. The culture associated with excessive alcohol consumption has been known to negatively affect school performance. In fact, according to National Institute on Alcohol Abuse and Alcoholism, about â&#x20AC;&#x153;1 in 4 college students report academic consequences from drinking, including missing class, falling behind in class, doing poorly on exams or papers, and receiving lower grades overall.â&#x20AC;? (College Drinking, 2015). Furthermore, alcohol consumption has been known to impair inhibitions, motor function and critical reasoning â&#x20AC;&#x201D; increasing the risk of sexual assault, theft and unintentional injury such as motor vehicle accidents while intoxicated.
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When a human consumes alcohol, the environment of the stomach neutralizes a portion of the alcohol from the beverage. However, after the stomach releases its contents into the duodenum of the small intestine, most of the alcohol present in the original beverage remains to be absorbed (Cederbaum, 2012). The effect of alcohol absorbed in the small intestine is the subject of my research. Alcohol, more specifically ethanol, increases the permeability of the intestinal epithelium to certain organic molecules (Elamin et al, 2013). This increased capability of gut content to cross the intestinal barrier presents a possible danger to excessive alcohol consumers as it could allow a pathogen to more easily enter the blood stream in larger numbers, increasing the severity of infection. 69
X LIFE AND PHYSICAL SCIENCES In the environment of the intestinal epithelium, cells form tight junctions in order to prevent foreign particles from crossing the intestinal barrier into the blood stream. These tight junctions are the primary defense of epithelial cells in the intestines. Maintaining these tight junctions is crucial for normal functioning of the small intestine as it also prevents the diffusion of bacteria that is normally symbiotic from infecting the human body through the blood stream. The introduction of ethanol compromises these tight junctions that prevent diffusion of dangerous macromolecules (Draper, 1983). The model pathogen used in this investigation is the common foodborne pathogen Listeria monocytogenes (Lm). Lm is found in a wide variety of foods such as ice cream, salads, vegetables and cheeses. When a human consumes Lm-nfected food, the bacteria can survive the acidic environment of the stomach. When the stomach contents are released into the duodenum, Lm relies on its ability to cross the intestinal barrier into the blood stream, where it uses its toxins to inflict Listeriosis on the human body. Pathogens orally consumed often initially infect the human body by crossing the intestinal epithelial barrier so Lm is an adequate model for infection (Portnoy, 2002). The presence of alcohol in the intestinal epithelium can increase the permeability of the cells lining the intestines, allowing for greater diffusion of gut content into blood. This compromisation of the intestinal barrier can be attributed to the weakening of the tight junction, which provides the main defense for intestinal cells to prevent infection. With these defenses marred, Lm and other pathogens can more easily cross the intestinal barrier increasing the severity of infection, as well as increasing the likelihood of infection when alcohol and pathogens are consumed together in very short periods of time.
Materials and Methods Hemolytic Assay Listeria monocytogenes cultures were prepared by inoculating 1mL of BHI media with 1 colony of WT from a spread plate. Prior to inoculation, BHI and ethanol were combined to form concentrations of 0.05%, and 0.20% ethanol (v/v). After inoculation, cultures were then placed in either an aerobic or anaerobic incubators 70
for no more than 18 hours. After incubation, cultures were removed from their incubators and were measured for their optical density to quantify their growth. Samples of the cultures were then placed in a centrifuge to separate the bacterial cells from supernatant, where the toxin of interest is. After centrifuging, each supernatant sample (100μL) was added to the top row of a 96-well plate, along with 5μL of 0.1M DTT, a reducing agent. While incubating at room temperature with DTT, the remaining wells were filled with 100μL hemolytic assay buffer (HAB). After 10 minutes of incubation, 100μL of HAB was added to the supernatant sample, pipetted up and down repeatedly to mix, and 100μL was transferred to the next row down to continue to dilute the supernatant samples in 1:2 ratio. After dilutions, 100μL of defibrinated sheep blood was added to each well to a final concentration of 1% hematocrit. The plates were placed in an aerobic incubator for 30 minutes. After incubation, plates were placed in the centrifuge to separate the blood cells from the supernatant. The supernatant was then transferred to a flat-bottom 96-well plate and the optical density was taken to determine the toxin activity of the Listeria monocytogenes culture.
Caco-2 Colonic Epithelial Cell Infections Caco-2 colonic epithelial cells were grown in DMEM media with 10% Fetal Bovine Serum for several days in order to harvest the maximum amount of cells for the infection procedure. Cells were then treated with 0.1% Trypsin to dissociate them from the bottom of the flask. The cells were then placed in the centrifuge along with 10mL of fresh media. After centrifuging, the cell media was poured out and replaced by 10ml of new media. The pellet of cells was then resuspended in the new media. Using a hemocytometer, the concentration of cells was determined. A total of 6 million cells (in 1 mL) were added to each of the 24-well plate. The cells were then placed in an aerobic incubator at 37° C for 24 hours to allow for the cells to adhere to the bottom of the well plate. After 24 hours, the Caco-2 cells were treated with media supplemented with ethanol. After another 24 for the cells to acclimate to the new conditions, the cells were then infected with 108 Listeria monocytogenes colony forming units (CFU). After 1 hour of incubation with Listeria, media infused with gentamycin was added to each well to eliminate the intercellular bacteria. Proceedings 2017
LIFE AND PHYSICAL SCIENCES Following another hour of incubation, the cells were then lysed using 0.1% Triton-X, plated onto LB spread plates, and placed in an aerobic incubator at 37° C for at least 48 hours until the plates could be counted for CFU. This experiment was used to determine the differences in susceptibility to Listeria infection between untreated Caco-2 cells and ethanol treated cells.
HAP2 Mice Infection High alcohol-preferring mice were acquired from Dr. Nicholas Grahame and placed in single cages in order to acclimate them to the environment of the vivarium at University of Dayton. For this period of time, all mice received water and food ad libitum. After 12 days, the experimental mice were given bottles filled with 10% ethanol. At 3, 6, 9 and 12 days following the initial treatment of ethanol, experimental mice were given water as a substitute. At 15 days after the initial treatment with ethanol, all mice were infected with 108 CFU of Listeria monocytogenes. At 3 days after infection, all mice were sacrificed in order to harvest the liver and spleen. These samples were homogenized and plated at varying dilutions in order to quantify the number of colony forming units in order to determine the bacterial burden in these organs as an indicator for animal susceptibility to Listeria monocytogenes infection.
Figure 1. Normalized toxin activity of aerobically-grown Listeria monocytogenes. A significant difference between ethanol treated Listeria monocytogenes cultures and controls was found. Cultures grown in 0.05% ethanol produced less toxin than the control. “*” represents p value of <0.05. R. Restrepo, 2017.
Proceedings 2017
Results Hemolytic Assay We first analyzed the effect of ethanol on listeriolysin O (LLO) toxin production of Listeria monocytogenes (Lm) grown in Brain Heart Infusion (BHI) media under both aerobic and anaerobic conditions. Under aerobic conditions, supplementation of 0.05% ethanol resulted in a small but significant decrease in LLO production compared to control cultures without ethanol (Figure 1). However, this effect was not observed in the anaerobic cultures. Under anaerobic conditions, supplementation of 0.05% ethanol did not significantly alter LLO production (Figure 2).
Caco-2 Cell Infections Next, we investigated the effect of ethanol on the susceptibility to Listeria monocytogenes infection using Caco-2 colonic epithelial cells as a host model. First, we treated the Caco-2 cells with 0.05% ethanol prior to infections and removed ethanol during infections. We observed that at 1-hour post infection, there was a significant increase in the number of intracellular Lm in cells pretreated with ethanol. At 2 hours post infection, there were no significant differences in the number of intracellular Lm between control and pretreated Caco-2 cells (Figure 3). Next, we included the ethanol treatment both prior to infections and during infections. We
Figure 2. Normalized toxin activity of anaerobically-grown Listeria monocytogenes. No significant difference between control and ethanoltreated cultures was found. R. Restrepo, 2017.
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Figure 3. Differences in intracellular CFU of Listeria monocytogenes between ethanol treated Caco-2 cells and controls taken at 1 and 2 hours post infection. A highly significant difference was shown between the ethanol treated Caco-2 cells and the control cells at time point 1. Ethanol has successfully increased Caco-2 susceptibility to Listeria monocytogenes infection given that there is no ethanol during the infection procedure. â&#x20AC;&#x153;***â&#x20AC;? represents a p value <0.001. R. Restrepo, 2017.
observed that at 1 hour post infection, there was a decreasing trend in the amount of intracellular Lm with correlating to the increasing ethanol concentrations. However, this trend was not observed at 2 hours post infection (Figure 4).
HAP2 Mice Infection Finally, we wanted to explore the effect of ethanol on the susceptibility to Lm infection of High Alcohol Preferring (HAP) mice. Out of the 39 total mice, 20 were subjected to treatment with 10% ethanol. The remaining 19 mice received water for the duration of the experiment. Using a one-tailed, unequal variance t-test, we found a significant increase in bacterial burden in the spleens of ethanol-drinking females versus their male counterparts (Figure 5). When infection was plotted based on the average daily ethanol or water consumption, no significant correlation was found (Figures 6-9).
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Figure 4. Differences in intracellular CFU of Listeria monocytogenes between ethanol treated Caco-2 cells and controls taken at 1 and 2 hours post infection. Unlike the previous figure, ethanol was present during the infection procedure. In these conditions, an increasing concentration of ethanol correlated with a decreasing susceptibility to Listeria monocytogenes infection at 1-hour post infection. R. Restrepo, 2017.
Discussion Does Ethanol Affect the Toxin Production or Growth of Listeria monocytogenes? When Lm was grown in cultures with varying concentrations of ethanol, only in aerobic conditions did the Lm produce less toxin with ethanol present. By contrast, no significant difference was found in the anaerobic cultures. Although this is an interesting finding, the environment of the intestinal epithelium is most often anaerobic. Furthermore, the results obtained mean that any increase or decrease in Caco-2 or HAP2 mice susceptibility must be attributed to the host, not Lm. Thus, the results attained from the hemolytic assay suggested that in the environment of the intestinal epithelium, Lm is not affected by the presence of alcohol. However, I will continue to investigate Lm response to alcohol at higher concentrations.
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LIFE AND PHYSICAL SCIENCES X
Figure 5. Colony-forming units found in each mouse spleen and liver. Using a one-tailed, unequal variance t-test, female spleens were more susceptible to Listeria monocytogenes infection than males. R. Restrepo, 2017.
Figure 6. Colony-forming units of Lm found in male spleens based on the amount of ethanol and water consumed (ml/day). R. Restrepo, 2017.
Figure 7. Colony-forming units of Lm found in male livers based on the amount of ethanol and water consumed (ml/day). R. Restrepo, 2017.
Figure 8. Colony-forming units of Lm found in female spleens based on the amount of ethanol and water consumed (ml/day). R. Restrepo, 2017.
Figure 9. Colony-forming units of Lm found in female livers based on the amount of ethanol and water consumed (ml/day). R. Restrepo, 2017.
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X LIFE AND PHYSICAL SCIENCES Does Ethanol Increase Caco-2 Susceptibility to Listeria monocytogenes infection? Under the generalized procedure of the Caco-2 infection with Lm, two sub-experiments were performedâ&#x20AC;&#x201D;with ethanol present during the infection process in one experiment but absent in the other experiment. Interestingly, these two experimental setups yielded different results. In the experiment where ethanol was not present during infection, the results illustrated that prior treatment with ethanol rendered the Caco-2 cells more susceptible to Lm infection. Thus, Caco-2 cells are compromised by ethanol, increasing their susceptibility to Lm infection. One possible mechanism by this increase is the compromisation of tight junctions. During the 24 hours before treatment with ethanol, Caco-2 cells are placed in wells with media where they are to form a monolayer of cells held together by tight junctions. Tight junctions are barriers between epithelial cells that eliminate space between cells, helping to form a sturdy layer of cells that prevent unwanted molecules from passing through. However, based on the results attained, it is likely that ethanol compromises these barriers. In the experiment where ethanol was present during infections, a decreasing trend of susceptibility was displayed at 1-hour post infection. In these ethanol treatment conditions, either the Caco-2 cells are more resistant to infection or the infectivity of Lm was compromised. These results agree with the data attained from the hemolytic assay. Under aerobic conditions, toxin production of Lm is reduced, thus lessening Lm burden on the host.
Does Ethanol Increase HAP2 Mice Susceptibility to Listeria monocytogenes infection? When HAP2 mice were orally infected with Lm, we observed that there was no significant difference in bacterial burden between ethanol supplemented mice and controls. However, we observed that these mice are particularly vulnerable to Lm infection. When infected with 108 CFU, average infection rates were around 104 CFU per organ. One explanation of this phenomenon is that because these mice are bred to prefer ethanol rather than water, their genetic predisposition to prefer alcohol, and thus their 74
tolerance may have made them more susceptible to infection. Furthermore, when plates were counted for CFU of Lm, there were many contaminations present, signaling that these mice were not only susceptible to Lm infection, but to other pathogens as well. Although there were no significant differences in infection of Lm found between the HAP2 mice tested, they seem to be more vulnerable to infections than other laboratory mice typically used for as host models of infections.
Future Research Caco-2 Epithelial Cell Infections Using Transwell Inserts Caco-2 cells grown in flat well plates similar to the type that was used in the infection procedure have been shown to not form the type of monolayer that is found in the human intestinal tract. Cells in vivo display a polarization of the monolayer, meaning that the two sides of the cell have different functions and chemical makeups to adjust for differing chemical environments on either side of the human intestinal epithelium. On the apical side (direct exposure to gut contents), cells are adapted to acquire nutrients from freshly digested stomach contents, resist the acidity of residual stomach acid, and prevent the diffusion of harmful pathogens across the intestinal epithelial barrier. By contrast, on the apical side of the Caco-2 cell displays functions that include oxygen uptake from the blood stream and releasing absorbed nutrients into the blood stream. Both sides of the cell need to balance these functions in order to sustain proper operation of the intestinal monolayer. In response to grow a Caco-2 cell layer similar to those in vivo, I propose to use transwell inserts. These inserts are defined by their semipermeable membrane that allow the Caco-2 cells to absorb nutrients from either side of their membrane. This allows the Caco-2 cells to polarize the way that would be commonly found in humans. Similar infection procedures to those done on flat well plates will be performed to observe if the effects of ethanol on Listeria monocytogenes infection are consistent between nonpolarized and polarized Caco-2 cells.
Proceedings 2017
LIFE AND PHYSICAL SCIENCES Determining the Effect of Ethanol on the Effectiveness of Common Antibiotics Antibiotics are frequently used to provide relief from symptoms associated with bacterial infections. Although the toxin production is not directly impacted by ethanol, I plan to investigate the impact of ethanol on Listeria monocytogenes susceptibility to common antibiotics. I will introduce ethanol and antibiotics to Listeria monocytogenes cultures to examine if the presence of ethanol reduces or changes the efficacy of antibiotics in controlling Listeria growth.
Acknowledgements I would like to extend my deepest thanks to all that made my research possible. Especially the University Honors Program and the Berry Family Foundation for making the Berry Summer Thesis Institute possible, my advisor Dr. Yvonne Sun for helping me plan and execute my research, my colleagues in the anaerobic microbiology lab for their unfailing help in assisting my research, and IUPUI for supplying us the HAP2 mice that proved to be invaluable in my thesis project.
Proceedings 2017
References Berkes, J, V K Viswanathan, S D Savkovic, G Hecht “Intestinal Epithelial Responses to Enteric Pathogens: Effects on the Tight Junction Barrier, Ion Transport, and Inflammation.” Gut, vol. 52, no. 3, Mar. 2003, pp. 439–451., www.ncbi.nlm.nih. gov/pmc/articles/PMC1773546/pdf/gut05200439.pdf. Cederbaum, Arthur. “Alcohol Metabolism.” Clinical Liver Disease, vol 16, no. 4, Nov. 2012, pp. 667-685., doi: 10.1016/j. cld.2012.08.002 "College Drinking." National Institute on Alcohol Abuse and Alcoholism. Department of Health and Human Services, Dec. 2015. Web. 5 Feb. 2017. Elamin, Elhaseen E, et al. “Ethanol Metabolism and Its Effects on the Intestinal Epithelial Barrier.” Nutrition Reviews, vol. 71, no. 7, June 2013, pp. 483–499., doi:10.1111/ nure.12027. Draper, L R, et al. “Effect of Alcohol on the Integrity of the Intestinal Epithelium.” Gut, vol. 24, no. 5, 1983, pp. 399–404., doi:10.1136/gut.24.5.399. Matson, L. et al. Chronic Free-Choice Drinking in Crossed High Alcohol Preferring Mice Leads to Sustained Blood Ethanol Levels and Metabolic Tolerance Without Evidence of Liver Damage. Alcoholism: Clinical and Experimental Research 37, 194–201 (2013). Portnoy, Daniel A., et al. “The Cell Biology Of Listeria Monocytogenes infection.” The Journal of Cell Biology, vol. 158, no. 3, August 2002, pp. 409–414., doi:10.1083/ jcb.200205009. Saad, Assad Joe, et al. “Ethanol Ingestion Increases Susceptibility of Mice to Listeria Monocytogenes.” Alcoholism: Clinical and Experimental Research, vol. 17, no. 1, Feb. 1993, pp. 75–85., doi:10.1111/j.1530-0277.1993. tb00729.x.
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Investigating Cell to Cell Interactions and Molecular Signals in Drosophila Glioma Models Logan J. Roebke1,2,4,5, Kirti Snigdha3, Indrayani Waghmare3 and Madhuri Kango-Singh1,3,4 University of Dayton, 300 College Park, Dayton, OH, 45469 1. Department of Pre-medicine 2. Department of Religion 3. Department of Biology 4. Berry Summer Thesis Institute 5. University Honors Program
Advisor: Madhuri Kango-Singh, Ph.D. Biology Department Corresponding Author: Logan J. Roebke, Madhuri Kango-Singh Email: roebkel1@udayton.edu, mkangosingh1@udayton.edu
Abstract Glioblastoma multiforme (GBM) is a devastating form of primary brain cancer with poor prognosis. Capitalizing on the mutations found in GBM patients and the similarities between mammalian and Drosophila genes involved in glial cell biology, Drosophila glioblastoma models have been established that show similarities to anaplastic glia from high-grade human glioma. Key aspects of disease presentation of high grade glioma include rapid growth, therapy resistance and recurrent phenotype. Here we present a review of current Drosophila glioma models focusing on the findings from these models and the advantages of Drosophila models compared to other systems.
Introduction Gliomas are glia-derived, primary brain tumors with poor prognosis (Modrek et al., 2014). Each year approximately 13,000 people die due to brain cancers, of which more than 70% die from high grade glioblastoma, the most aggressive form of this cancer (Furnari et al., 2007). Profiling "omics" analyses have generated a plethora of information on cancer genome mutations and have helped characterize glioblastoma molecularly (e.g., Proneural- or Mesenchymal-types) and have identified multiple molecular targets (Cancer Genome Atlas Research, 2008). The 76
current standard of care involves surgery followed by a combination radiation-, chemoor targeted-therapies (e.g., with cetuximab or gefitinib or rindopepimut). However, these approaches are largely ineffective as the complex treatments extend life by only an average of months (Brennan et al., 2013). One reason for the limited success of current approaches is the therapy resistance of the glioma stem cells (GSCs), which retain remarkable abilities to initiate tumors, undergo aberrant differentiation and use oncogenic pathways to maintain their stemness (Balasubramaniyan et al., 2015; Yamada and Nakano, 2012). Thus, there is a need to explore if targeting different molecules or pathways to cover non-overlapping mechanisms (molecular networks) will provide better tools for glioma therapy. Further, understanding the genetic, cell-biological properties of glioma will provide insights on the key changes that promote glioma growth.
Human Glioma: the Connection of EGFR and PI3K Pathways to Glia The human brain contains many glia types and neural stem cells that carry out diverse functions in the brain (Capdevila et al., 2017; Suh et al., 2009). Because neural stem cells are found in glioma and GBM specimens from patients, it is Proceedings 2017
LIFE AND PHYSICAL SCIENCES thought that glioma and GBM arise from neural stem cells (Singh et al., 2004b). However, other studies showed that glioma can also arise from glial populations due to accumulation of genetic mutations (Marumoto et al., 2009; Uhrbom et al., 2002). It is now believed that both glia and neural stem cells may be transformed by gliomagenic mutations to give rise to aggressive glioma. What are gliomagenic mutations? Glioma are associated with mutations in many genes and recent analysis has associated several genetic alterations to glioma sub-types (Phillips et al., 2006; Verhaak et al., 2010). For example, the pro-neural (PN) glioma are associated with mutations in either metabolic enzyme IDH1, and the onco-metabolite 2-hydroxyglutarate; or overexpression and/or amplification PDGFFRa gene (Nakano, 2014). The Mesenchymal (MES) glioma often show mutations in the NF1 gene and dysregulation of its associated pathway (Nakano, 2014). Interestingly, increased Epidermal growth factor (EGF) expression is seen in high grade glioma of all type. EGF functions to promote proliferation and survival of neural stem cells, and activation of this receptor tyrosine kinase pathway in glioma suggests that these pathways are critical targets in gliomagenesis (Huang et al., 2007). Other mutations associated with highgrade glioma include mutations in TP53, ATRX or CDK4, PDGFRA amplification or EGFR (amplification or EGFRvIII mutation), or CDKN2A deletion (Huang et al., 2007; Nakano, 2014). GBMs with EGFR amplification often harbor mutant forms of EGFR, such as EGFRvIII, resulting in constitutive kinase activity that stimulates Ras signaling to drive proliferation, promote migration and block apoptosis (Boockvar et al., 2003). Other common mutations in GBM/high-grade glioma are found in the phosphatidyl-inositol-3 kinase (PI3K) signaling pathway, another pathway involved in regulation of cell proliferation, growth and migration in various neural cell types (Endersby and Baker, 2008). Commonly-found mutations include activating mutations in the AKT kinase, or loss of PTEN through deletion, point mutations, and epigenetic silencing in approximately 70â&#x20AC;&#x201C;80% of GBMs (Eyler et al., 2008). These genetic alterations ultimately result in constitutive activation of the PI3K-AKT pathway (Sulis and Parsons, 2003). In addition, cell cycle regulators from the Rb-E2F pathway are also frequently mutated in high-grade glioma (Parsons et al., 2008). Although genetic alterations in EGFR-Ras, Proceedings 2017
PI3K-AKT and Rb-E2F pathways are often found in glioma, activation of any one of these pathways alone is not sufficient to cause glioma, suggesting that accumulation of mutations that ultimately result in co-activation of Epidermal Growth Factor Receptor (EGFR)-Ras and phosphoinositide 3-kinase pathway (PI3K), are the most commonly associated genetic lesions with human glioma. It is notable that treatment of glioma with pharmacological and molecular inhibitors of EGFR-Ras or PI3K pathways did not cause effective reduction in glioma growth (Raizer, 2011). These findings thus highlight the need to study glioma at the molecular level, to find key downstream targets and identify the oncogenic pathways that may be activated in glioma to develop better therapeutic strategies for glioma.
Modelling Glioma: Mammalian Models The need to understand the molecular underpinnings of glioma has led to the establishment of a plethora of glioma models. One system was the establishment of serum-dependent glioma cell lines, which would allow studies on the molecular and biochemical interactions important in glioma development (Lee et al., 2006). However, a key limitation of this experimental model was that the cell lines did not fully recapitulate the characteristics of the original glioma from which they were generated, thus limiting the experimental approaches as well as conclusions that could be drawn from these studies. In response to the challenges presented by glioma cell lines, glioma stem cell (GSC) lines were a second tissue culture model that was generated (Singh et al., 2004a). This model better recapitulates the genetic and phenotypic characteristics of glioma, however, it still presents experimental limitations as these cultures could be established only from a subset of glioma tumors (Singh et al., 2004b). Overall, the cell culture models do not behave like glioma as these cells no longer are interacting with neighboring cells (neurons, astrocytes, microglia, vasculature) in the central nervous system or brain (Pong and Gutmann, 2011). Another complication that emerged from these experimental approaches was the genetic and cellular heterogeneity found in different parts of a tumor, something that could not be mimicked in 77
LIFE AND PHYSICAL SCIENCES cell culture (Inda et al., 2010). These challenges led to development of in-vivo mouse models where the growth and interaction of glioma can be studied in a cellular environment in-vivo (Sarkaria et al., 2006). In these models, glioma cells from surgical samples or tissue culture are transplanted in immunocompromised mouse brains to generate tumors in mouse brains that retain characteristics from the original tumors (Sarkaria et al., 2006; Singh et al., 2004b). These xenograft in-vivo mouse models of glioma have proven useful in studying effects of known genetic lesions, the interactions of genetic mutations and the pathways affected (Fomchenko and Holland, 2006). However, these models cannot be used to identify new genes, signaling pathways and key unknown interactions that promote glioma growth downstream of the PI3K, EGFR, Rb-E2F pathways. Further, these models are costly and require complicated manipulations of cells in culture and during transplantation making these techniques very arduous for researchers.
of genetic tools available in Drosophila that provide the ability to dissect multigene interactions, identify new gene functions and cell-type specific manipulation of gene expression with single cell precision in vivo. Drosophila is now a model of choice for studying several human diseases (e.g., Alzheimerâ&#x20AC;&#x2122;s, MEN) (Bernards and Hariharan, 2001; Bier and Bodmer, 2004; Bonner and Boulianne, 2011; Miles et al., 2011; Qian and Bodmer, 2012). These studies have generated insights about the disease biology. Findings from fly research have been confirmed in mammalian models, and in some cases drugs identified in Drosophila genetic studies have proven effective in clinical trials (Dar et al., 2012; Das and Cagan, 2010; Read et al., 2005).
Overall, although mammalian glioma models are an important and useful system, they present several limitations in experimental approaches. Thus, new model systems are needed that will allow expanding the repertoire of experimental approaches and data from these new complementary systems can help improve our understanding of gliomagenesis.
Drosophila Models to Study Glioma The common fruitfly, Drosophila melanogaster, is a genetically-tractable whole animal model that provides distinct advantages because of its long history as a genetic model organism that can be used to study human diseases (Bier, 2005). Not only are 70% of human disease-linked genes functionally conserved in Drosophila (Chien et al., 2002; Hirth, 2010; Pandey and Nichols, 2011; Read, 2011), but all major signaling pathways (e.g., PI3K, EGFR, TGFb, Wnt, Hh, Notch, Hippo, etc.) that are involved in normal development and in cancer are conserved in Drosophila as well (Read, 2011; Tickoo and Russell, 2002). In fact, most signaling pathways were initially identified in genetic screens in Drosophila (Hartenstein et al., 2010; KangoSingh and Singh, 2009; Read, 2011), and several mutants that showed malignant tumors in tissues including the brain were identified in Drosophila (Gateff, 1978). Drosophila genetic models are especially advantageous due to the plethora 78
Figure 1. Drosophila brain and the distribution of neurons and glia. (A) Cartoon illustrates the major cell-types in the Drosophila brain â&#x20AC;&#x201C; neuroblasts, neurons and glia. (B) A confocal image of a Drosophila brain from mature third instar larva showing glioma caused by coactivation of PI3K and EGFR pathway. The repoGAL4>GFP, RasV12, ptenRNAi brain is stained for the neuroblast marker Prospero (red, grey), neuronal marker ELAV (blue, grey), and the glia are marked by GFP (green). Image courtesy of Indrayani Waghmare and Madhuri Kango-Singh, 2017.
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LIFE AND PHYSICAL SCIENCES Drosophila glioma models were developed given the remarkable evolutionary conservation of neural development between flies and humans. The Drosophila brain is a bilaterally symmetrical structure with two dorsal lobes and a ventral nerve cord (Figure 1) (Spindler and Hartenstein, 2010), and contains neurons and glia which arise from neuroectodermal stem-like cells called neuroblasts (Figure 1) (Apitz and Salecker, 2014). The developmental and functional conservation in glial development and the availability of cell-type specific markers that help distinguish neuroblasts, neurons and glia in the brain (Figure 1) make flies a great model for glioma studies. Further, analysis of fly genome revealed that Drosophila has single orthologs each for EGFR (dEGFR), and other pathway components Raf (dRaf), PIK3CA (dp110), PTEN (dPTEN), and Akt (dAkt), and one relevant ortholog for Ras (dRas1) and Rb (Rbf1) (Read, 2011).
PI3K-EFGR Glioma Models The first reported Drosophila glioma models were published by Read et al (2009) and Witte et al. (2009) using slightly different experimental set-ups. Read et al. (2009) used the glial cell specific driver repo-GAL4 to drive the expression of the constitutive active Drosophila EGF Receptor (dEGFRλ) with the activated form of Drosophila p110 (dp110CAAX) in the larval CNS (Read et al., 2009). Read et al. observed a post-embryonic increase in glia (50-100-fold) number that caused neoplastic overgrowth by disrupting the normal cellular architecture of the larval brain due to increased proliferation, loss of normal stellate glial morphology and formation of multilayered aggregations of abnormal glia throughout the brain. Further, akin to mouse xenograft transplantation models, when Drosophila dEGFRλ ;dp110CAAX glioma were transplanted in Drosophila female abdomens, these cells continued to proliferate into massive and invasive tumors that filled hosts’ abdomens, causing premature death (Read et al., 2009). Witte et al (2009) used the migration of glia into the ‘optic stalk’ as a model for Drosophila glioma (Witte et al., 2009). The optic stalk connects the Drosophila eye disc to the larval brain and is the structure through which the photoreceptor neurons in the developing eye imaginal disc project their axons into the dorsal optic brain lobes. In mature larvae, Repo-positive glial cells migrate from the optic stalk into the eye disc in Proceedings 2017
a stereotypical manner, and changes of proliferation or migration are easily detectable both in the optic stalk and in the eye disc (Witte et al., 2009). Using this developmental property of glial migration in optic stalk Witte et al., showed that overexpression of EGFR and/or PI3K, or other tyrosine kinase receptors like PDGFR/VEGFR, fibroblast growth factor receptor (FGFR) and insulin receptor homologs, show increased proliferation and/or overmigration of glial cells in larval eye structures, recapitulating histologic key features of human gliomas (Witte et al., 2009). Read et al. also showed that neoplastic overgrowth and inappropriate migration also occurred upon co-overexpression of other core pathway components, such as activated dRas1 (dRas1V12), dAkt or dPTENRNAi (Read et al., 2009). In addition, loss of dRbf1 enhanced the glioma caused by EGFR and PI3K co-activation. They also showed that using the ability to generate mosaic clones in Drosophila, glioma could be induced late in development that could be examined in young adult flies demonstrating that invasive neoplastic tumors can be examined in fully developed adults in flies. Since the development of the dEGFRλ ;dp110CAAX glioma model, Read et al. carried out a highthroughput genetic-modifier screen using a library of fly lines that downregulate expression of all Drosophila kinase by RNAi mechanisms (Read et al., 2013). These RNAi lines were co-expressed with dEGFRλ ;dp110CAAX in the glia and led to the identification of RIO kinases as downstream signals activated in glioma but not in normal glia in the CNS (Read et al., 2013). These studies have shown how Drosophila glioma model can be used for high-throughput screens for genetic modifiers that lead to identification of additional molecular components, or for therapeutic targets that could ultimately lead to development of better treatment strategies for glioma.
Drosophila Repo MARCM-induced Glioma About four years ago, we developed a clonal model of glioma in our lab where the coactivation of the PI3K and EGFR-Ras pathway occurred in glial clones rather than in all glia throughout the brain. This system took advantage of the ability to target glia (repo-GAL4) (Ni et al., 2009) and 79
LIFE AND PHYSICAL SCIENCES to make FLP-FRT-based MARCM mosaic clones in flies (Lee and Luo, 1999, 2001). We used a repo-FLP (Witte et al., 2009; Yuva-Aydemir and Klambt, 2011), where the flippase is linked to the repo gene restricting its expression to the glia to make GFP-tagged MARCM clones that overexpressed RasV12 and PtenRNAi in the larval brains (Figure 2). A clone refers to a small group of cells derived from a single somatic cell. The Flippase enzyme induces homologous recombination between the FRT sequences resulting in the generation of two genetically distinct daughter cells. In one daughter cell, the GAL4 is active and it drives expression of transgenes (RasV12, PtenRNAi) to form a positively marked (expression of GFP) clone. In the other daughter cell, the GAL4 repressor GAL80 is active, which does not allow any transgene expression. Thus, MARCM system allows generation of positivelymarked somatic clone, which can be characterized for expression of other signals and to study intercellular interactions between tumor cells (GFP marked) and their neighboring normal cells. The repo-MARCM system is especially advantageous as effects of gene expression can be studied in glial clones. In addition, to understand the lineage relationships the interactions of glia, neurons and neuroblasts can be explored. This system can also be used to identify new genes or pathways involved in gliomagenesis, or to identify small molecular inhibitors of glioma growth. We have used the repoMARCM glioma model to study the roles of signaling molecules identified in a tumor cell specific network in imaginal discs and to study mechanisms of gene regulation in normal glia and glioma (Roebke et al., manuscript in preparation).
Conclusion In conclusion, understanding the molecular underpinnings of glioma is of utmost importance as glioma are a devastating form of brain tumor with no cure to date. Studies from patient samples have shown the complexities of the molecular defects associated with glioma. Targeting individual pathways has shown the tremendous plasticity of glioma stem cells which circumvent these interventions by activating alternative oncogenic pathways. Thus, to find better therapeutic targets a deeper understanding of glioma biology is required which has led to the development of glioma models. In this context, Drosophila glioma models are a great complementary system for glioma studies given the genetic toolkit available in flies, and the whole animal in-vivo environment in which not only basic glioma biology can be studied but key points of intervention in oncogenic pathways can be explored using genetic and pharmacological approaches.
Acknowledgement We thank the members of the Kango-Singh lab for their help throughout the course of this work. Roebke and Kango-Singh would like to thank the Singh lab for the use of the microscope, the NEST facility at the University of Dayton for access to the Olympus Fluoview 1000 Laser Scanning Confocal Microscope. Roebke is supported by a Stander Fellowship, a Szabo grant from Premed program and a Forever Flyer grant. In addition, Roebke would like to thank the Berry family for the funding of Berry Summer Thesis Institute. Waghmare and Snigdha would like to acknowledge the GSSF awards from the Graduate Program at University of Dayton. Kango-Singh is funded by start-up support from the University of Dayton and a subaward from NIH grant R01CA183991 (PI Nakano) to Kango-Singh.
Figure 2. Repo MARCM clones in Drosophila brain. (A) Panel shows confocal image of repoGAL4>GFP, RasV12, ptenRNAi glioma brain showing the distribution of glia (marked by GFP, green). (B) Panel shows Repo MARCM clones of RasV12, PtenRNAi marked by GFP induced by the activity of repo-FLP. Note the multiple clones, the outline of the brain is marked by white line. Image courtesy of Kirti Snigdha and Logan Roebke, 2017.
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LIFE AND PHYSICAL SCIENCES References
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SERCA2: A Gatekeeper of Calcium Homeostasis in the Brain Joseph E. Saurine1,2,3,4 and Pothitos M. Pitychoutis1,2,3 University of Dayton, 300 College Park, Dayton, OH, 45469 1. Department of Biology 2. Center for Tissue Regeneration and Engineering at Dayton (TREND) 3. Berry Summer Thesis Institute 4. University Honors Program
Advisor: Pothitos M. Pitychoutis, Ph.D. Department of Biology Corresponding Author: Pothitos M. Pitychoutis Email: ppitychoutis1@udayton.edu
Summary Calcium (Ca ) ions serve as global signaling regulators with a vast array of crucial cellular and molecular functions. In most cells, a complex Ca2+-handling machinery ensures normal intracellular Ca2+ levels by distributing ions across subcellular membranes, including the endoplasmic reticulum (ER). When cytosolic Ca2+ concentrations are altered, many signaling pathways are disrupted, often leading to pathology. Herein, we focus on the sarco-endoplasmic reticulum (SER) Ca2+ ATPase (SERCA) pump and its role in maintaining cellular Ca2+ homeostasis. Despite being present in all cell types, SERCAs are typically known for regulating the distribution of Ca2+ ions across the SER membrane of myocardial cells. Therefore, dysfunction of this pump can have major physiological implications leading to cardiovascular events, such as heart failure. The role that SERCA plays in the heart is largely understood, but its implications in the brain are elusive. Even though disruption of Ca2+ homeostasis in the brain leads to debilitating neurological disorders, the role that SERCA plays in these processes is still elusive. 2+
SERCA Structure and Function Ca2+ storage is one of the functions commonly attributed to the SER in mammalian cells. Indeed, when Ca2+ ions are needed, specialized SER channels release Ca2+ from the lumen of the SER to the cytosol. When the concentration of Ca2+ ions in the cytosol needs to be reduced, Ca2+ 84
ions are pumped back into the SER to store for later use; prime examples of this are the striated muscle fibers. When the muscles need to contract, Ca2+ is released from the SER. The muscles can then contract due to the release of Ca2+ ions into the cytosol but then Ca2+ is pumped back into the SER to induce relaxation. This process is specifically mediated by the SERCA pump that resides in the SER membrane. Notably, up- or downregulating SERCA2 levels has significant cellular implications; for instance, decreased expression and/or increased inhibition of cardiac SERCA2 has been linked to heart failure in humans (Arai, Alpert, MacLennan, Barton, & Periasamy, 1993; de la Bastie et al., 1990). SERCAs are considered to be P-type ATPases, as they pump cations across membranes at the expense of an ATP molecule. SERCA is a 110-kDa protein that consists of a single polypeptide chain about a thousand amino acids long (MacLennan, Brandl, Korczak, & Green, 1985; Toyoshima & Inesi, 2004). After translation, the peptide chain ends up folded across the SER/ER membrane with ten transmembrane α-helices and short lumenal loops (Figure 1). Four of these α-helices extend beyond the SR/ER membrane to form three different domains critical to the function of SERCA (Toyoshima & Inesi, 2004). The A domain, or the actuator domain, assists in regulating the bonding of Ca2+ ions. The P domain, is where the γ-phosphate binds with Asp351. Lastly, the N domain is the nucleotide binding site (Møller et al., 2005; Periasamy & Kalyanasundaram, 2007; Toyoshima & Inesi, 2004). Proceedings 2017
LIFE AND PHYSICAL SCIENCES
Figure 1. Structural representation of SERCA. Ten É&#x2018;-helices rest across the ER membrane and three lumenal loops protrude into the cytosol to create the A, N, and P domains. J. Saurine and P. Pitychoutis, 2017.
It is well established that SERCA serves to pump Ca2+ ions from the cytoplasm of the cell into the lumen of the SER/ER, as shown in Figure 2. Throughout its conformational changes SERCA delivers two Ca2+ ions from the cytosol into the lumen of the ER per molecule of ATP as seen in Figure 3 (Periasamy & Kalyanasundaram, 2007). Initially, SERCA's first conformation (E1), exposes the Ca2+ binding sites to the cytosol where Ca2+ ions bind. Upon ATP hydrolysis SERCA becomes phosphorylated; this causes another conformational change (E2) which
Figure 2. Function of SERCA in the cell. Ca2+ ions are pumped from the cytosol of the cell to the lumen of the ER across the ER membrane. J. Saurine and P. Pitychoutis, 2017.
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guides the two Ca2+ ions to face the lumen of the ER where they are then ejected (Periasamy & Kalyanasundaram, 2007). For normal Ca2+ binding to SERCA, the E1 conformation must have a high affinity for Ca2+ ions and the E2 conformation a low affinity (Vandecaetsbeek et al., 2009). This affinity can be affected by many factors; for example, internal mutations in the SERCA gene and functional differences among the different SERCA isoforms play a role in the levels of Ca2+ affinity (Vandecaetsbeek et al., 2009). Additional factors include Ca2+ ion concentrations, pH, and abundance of ATP, ADP and inorganic phosphate (Periasamy & Kalyanasundaram, 2007). To date, at least ten different SERCA isoforms have been identified to be expressed in mammalian tissues (Baba-AÄąssa, Den Bosch, Wuytack, Raeymaekers, & Casteels, 1998; Periasamy & Kalyanasundaram, 2007). The different isoforms of SERCA are expressed in all mammalian tissue types(Periasamy & Kalyanasundaram, 2007) (Table 1). Three main SERCA genes; SERCA1, SERCA2, and SERCA3, are known to be expressed in vertebrates (Arai et al., 1993; Brandl, Green, Korczak, & MacLennan, 1986; Lyttons & Maclennanq, 1988). Although there are differences in these three genes they have all been largely conserved with none being more than 30% different than the others (Periasamy & Kalyanasundaram, 2007). In the central nervous system (CNS) it has been shown the SERCA2b isoform is ubiquitously expressed in nerve cells (Gunteski-Hamblin, Greeb, & Shull, 1988; Lyttons & Maclennanq, 1988).
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Figure 3. Conformational changes of SERCA. SERCA begins in the E1 conformation, then two Ca2+ ions bind. An ATP molecule is consumed to change to the E2 conformation. After Ca2+ is released into the lumen of the ER due to the decreased affinity of SERCA to Ca2+, SERCA moves back into the E1 conformation. J. Saurine and P. Pitychoutis, 2017.
SERCA2: A Ca2+-handling Player in the Brain Ca2+ ions are considered global signaling regulators as they are involved in cell growth, apoptosis, gene expression and development in all cell types (Mccue, Haynes, & Burgoyne, 2010). Specifically, in neurons Ca2+ ions regulate neuronal activity and neurotransmitter release, while they also help modulate synaptic activity and axonal growth (Brini, Calì, Ottolini, & Carafoli, 2014; Mccue et al., 2010; Scullin & Partridge, 2010). Moreover, Ca2+ ions comprise a prominent second messenger that influences a constellation of intracellular molecular cascades in nerve cells (Brini & Calì, 2017; Scullin & Partridge, 2010). Notably, disruption of normal Ca2+ cycling in the brain has been implicated in neuronal inflammation and oxidative stress that in turn leads to severe neuropsychiatric disorders, including Alzheimer's disease, Parkinson’s disease, dementia, bipolar disorder, schizophrenia, autism spectrum disorders and intellectual disabilities (Bergantin & Caricati-Neto, 2016; Charlene & Bezprozvanny, 2010; Dahl, 2017; Green et al., 2008; Heyes et al., 2015; LaFerla, 2002). Notably, it has been shown that Ca2+ concentrations in neurons can impact important
neurobiological processes such as learning, memory and motor function (Baker, Edwards, & Rickard, 2008; Cassidy, Dlugos, & Dlugos, 2013; Salinska, Bourne, & Rose, 2001; Simonyi et al., 2005). For instance, local increases in Ca2+ levels have been shown to drive long term potentiation (LTP) in the hippocampus, a brain region vastly implicated to learning and memory (Artola & Singer, 1993; Mulkey & Malenka, 1992). Depletion of ER Ca2+ stores in purkinje neurons of the cerebellar cortex has been shown to negatively impact motor function, specifically balance, in rats (Cassidy et al., 2013). This further demonstrates the important role Ca2+ ions play in major neurobiological processes. There is scarce evidence on the specific functions of SERCA2 in the brain. Specifically SERCA2b, the isoform that is ubiquitously expressed in the CNS, has been shown to play a key-role in regulating conditioned taste aversion in the rat insular cortex (Miranda, González-Cedillo, & Díaz-Muñoz, 2011). Localized infusion of the SERCA2-inhibitor thapsigargin showed that blocking SERCA2b only at certain time-points caused disruption of short-term memory to an aversive taste stimulus (Miranda et al., 2011). Indeed, Ca2+ ion concentration in the cytosol is typically increased to activate protein kinase C (PKC) and Ca2+-Calmodulin kinase II (CaMKII) that in turn drive proper memory formation (Dash, Moore, Kobori, & Runyan, 2007; Roth & Sweatt, 2008; Runyan, Moore, & Dash, 2005). Therefore, when SERCA is inhibited at points where Ca2+ stores must be relied on, short-term memory is impaired (Miranda et al., 2011). Taken together, this evidence suggests that SERCA2 impairment plays a critical role in memory formation. Interestingly, expression levels of SERCA2b are altered following distinct pathophysiological events, such as ischemia and ER stress
Table 1. Expression of SERCA isoforms in different adult tissues in vertebrates. *Expressed only in fetal tissue. J. Saurine and P. Pitychoutis, 2017.
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LIFE AND PHYSICAL SCIENCES (Degracia, Kumar, Owen, Krause, & White, n.d.; Kopach et al., 2016) For instance, re-oxygenation after prolonged oxygen deprivation caused time specific upregulation of SERCA2b in the CA3 region of the hippocampus. Upregulation of SERCA2b showed to relieve Ca2+ store dysfunction caused by the ischemia, suggesting that SERCA2b may play a role in neuroprotection (Kopach et al., 2016). Additionally, ischemia has been shown to cause the unfolded protein response (UPR) following ER stress. The molecular mechanisms that govern the UPR are complex but are known to involve pro-apoptotic pathways and emptying of the ER Ca2+ stores. However, cells can upregulate SERCA2b in order to promote survival and restore normal Ca2+ handling (Degracia et al., n.d.; Xia, Simonyi, & Sun, 1998). Intriguingly, the regulation of SERCA2 in the brain extends beyond neurons to include astrocytes. It has been demonstrated that astrocytes present impaired calcium cycling when regulation of SERCA is disrupted by thapsigargin (Komin, Moein, Ellisman, & Skupin, 2015; Morita & Kudo, 2010). However, upregulation of SERCA2b alone increases the oscillation of intracellular Ca2+ concentrations in astrocytes needed for their normal function. Therefore, SERCA demonstrates significant roles not only in neurons but also in glial cells as well. Overall, SERCA2 appears to be an important Ca2+ handling player in the brain. Thus, SERCA2 dysfunction in nerve cells, and subsequent deregulation of cellular Ca2+ homeostasis, could have significant pathophysiological implications and result in debilitating brain disorders. The innate complexity of neural circuits and SERCA2 regulation by other proteins leaves many unexplored research questions that need to be addressed through future research.
Acknowledgments The authors would like to thank the Berry Family for their contributions and generous support and all the members of the Pitychoutis lab, including Connor Thelen, Katerina Britzolaki and Emily Flaherty. Mr. Saurine would like to thank the University of Dayton Honors program for putting together the Berry Summer Thesis Institute and making it one of the most rewarding experiences in his undergraduate career. All images used were created in 2017 and belong to Joseph Saurine and Pothitos Pitychoutis. Proceedings 2017
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CREDITS PHOTOGRAPH AND ILLUSTRATION CREDITS Cover Row 1, left to right Repo MARCM Clones in Drosophila Brain. 2017. Image courtesy of Kirti Snigdha and Logan Roebke. Preliminary LOS Assessment. 2017. Graphic courtesy of Paige Ingram. Memorial. 2017. Photograph courtesy of Claire Bowman. Row 2, left to right Structural Representation of SERCA. 2017. Graphic courtesy of Joseph Saurine. GHG Emissions. 2017. Graphic courtesy of Lauren Murray. Hope. 2017. Photograph courtesy of Rose Rucoba. AcrAB-TolC efflux pump. 2009. Graphic courtesy of M. F. Symmons, 2009, PNAS, vol. 106, pg. 7173. Colony-forming Units. 2017. Graphic courtesy of Ryan Restrepo. Row 3, left to right The Imd Immune Response Pathway. 2017. Graphic courtesy of Steve Borchers. Black and White. 2017. Graphic illustration courtesy of Ramona Speranza. New Hope, Twin Towers Art Studio. 2017. Photograph courtesy of Mary A. Brinkman.
Articles Page 3: Mental Corset (Lauren). 2017. Photograph courtesy of C. Bowman. Page 3: Mental Corset (Diane). 2017. Photograph courtesy of C. Bowman. Page 4: Involuntary Rose-Coloured. 2017. Image courtesy of C. Bowman. Page 4-5: Infestation. 2017. Photographs courtesy of C. Bowman. Page 6-7: Memorial. 2017. Photographs courtesy of C. Bowman. Page 8-9: The Baggage of Feminism. 2017. Images and photographs courtesy of C. Bowman. Page 11: Twin Towers Neighborhood. 2017. Photograph courtesy of A. Brinkman. Page 14: Historic Buildings in the Twin Towers Neighborhood. 2017. Photograph courtesy of A. Brinkman. Page 17: Homes on Xenia Avenue. 2017. Photograph courtesy of A. Brinkman. Page 19: New Hope, Twin Tower's Art Studio. 2017. Photograph courtesy of A. Brinkman. Page 38: Hope. 2017. Photograph courtesy of Rose Rucoba. Page 57: A Mechanism of Antibiotic Resistance That Prevents the Accumulation of Antibiotics within the Cell. 2017. Graphic courtesy of S. Baxter and M. Lopper. Page 59: The Structure of the AcrAB-TolC Efflux Pump Along the Membrane of the Bacteria Cell. 2009. Graphic courtesy of M.F. Symmons, PNAS, vol. 106, pg. 7173. Page 64: GMR-Gal4/UAS System for Targeted Misexpression of Aβ42 in Drosophila melanogaster Eye. Image courtesy of A. Singh. Page 65: The Toll Immune Response Pathway. 2017. Image courtesy of S. Borchers. Page 66: The Imd Immune Response Pathway. 2017. Image courtesy of S. Borchers. Page 78: Drosophila Brain and the Distribution of Neurons and Glia. 2017. Image courtesy of I. Waghmare and M. Kango-Singh. 90
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CREDITS Page 80: Repo MARCM Clones in Drosophila Brain. 2017. Image courtesy of K. Snigdha and L. Roebke. Page 85: Structural Representation of SERCA. 2017. Graphic courtesy of J. Saurine and P. Pitychoutis. Page 85: Function of SERCA in the Cell. 2017. Graphic courtesy of J. Saurine and P. Pitychoutis. Page 86: Conformational Changes of SERCA. 2017. Graphic courtesy of J. Saurine and P. Pitychoutis.
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