Berry Summer Thesis Institute Proceedings 2016 by University of Dayton Honors Program

Berry Summer Thesis Institute

Proceedings 2016

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 Page 94 for credits Copyright ÂŠ 2016 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:

I am pleased to present the fourth volume of the Proceedings of the Berry Summer Thesis Institute.

This summer I assumed my new role as Director of the University Honors Program. Attending the Berry Summer Thesis Symposium confirmed, once again, the remarkable talents of our Honors students. This collection represents the Honors Program’s goal of introducing our top undergraduate researchers to peer review and publication and to the process of contributing to knowledge in a wide variety of disciplines. As a new director, I am especially grateful for the friends and alumni that allow programs such as the Berry Summer Thesis Institute to flourish. So let me take this opportunity to thank Mr. John W. Berry, Jr. — as well as his entire family — for his generous and continued support of the program. I also want to extend my thanks to the faculty mentors for guiding the students’ research and to the students for their extensive research and community engagement this summer. Enjoy! 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 2016 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 Process of Branding by Lucy E. Bratton . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

Catholicism, Gender, and Politics in the Music and Performance of the Nueva Canción Song Movement by Elizabeth M. Turnwald . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

SOCIAL SCIENCE The Effects of Adolescent Housing Condition and Voluntary Exercise on Alcohol Intake and Stress Response in Male Long-Evans Rats by Caroline A. Lynch and Tracy R. Butler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

A Confirmatory Factor Analysis of Normative and Pathological Personality Traits in a University Student Sample by Lisa E. Stone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

NATURAL SCIENCES The Impact of the 515nm Effect on Singlet Oxygen Quenching in Photosynthesis: Model System Studies Using β-Carotene-Acid Complexes Lauren A. Hoody and Mark Masthay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

Drosophila Eye Model to Investigate the Role of Mitochondrial Dysfunction as a Trigger for Neurodegeneration in Alzheimer’s Disease Lydia C. Payton and Amit Singh . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

A Review of the Glycosylation of Aquaporin-3 in Mammalian and Anuran Species Christopher J. Turley and Carissa M. Krane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56

Resolving the Molecular Mechanisms by Which DNA Mutations Alter the Function of a Cis-Regulatory Element Emily E. Wey and Thomas M. Williams . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

HEALTH SCIENCE Development of an Evidence-Based Strength Training Program for Individuals with Dementia Participating in Adult Day Services Jaclyn H. Franz and Kurt Jackson . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74

ENGINEERING Design and Protocol for the Utilization and Setup of a Low-Cost Slip Trainer for Fall Prevention Stephen T. McFadden and Kimberly Bigelow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80

Influence of Reverse Shoulder Implant Positioning on Patient-Specific Muscle Forces: A Simulation Study Kayla M. Pariser and Allison L. Kinney . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88

Photograph and Illustration Credits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94

ARTS AND HUMANITIES

The Process of Branding Lucy E. Bratton1,2,3 University of Dayton, 300 College Park, Dayton, OH 45469 1. Berry Summer Thesis Institute 2. University Honors Program 3. Department of Art and Design

Thesis Mentor: Roger J. Crum, Ph.D. Department of Art and Design Corresponding Author: Lucy Bratton Email: brattonl1@udayton.edu

Abstract Understanding process enhances understanding of innovation. Creation rarely occurs in a single moment; normally, creative efforts must be carried from ideation to completion by way of process. Appreciating the mechanics of process is especially necessary in the creation of brands. Branding is one of the most efficient ways to communicate the identity and even the spirit of an entity. There are many steps in the process of creating an informed, successful and memorable brand. These steps range from the active collection of data about the culture in which the brand will operate to informed speculations about the effect the brand will have. This research examines as a case study the role of process in the creation of a prospective brand for the Berry Summer Thesis Institute at the University of Dayton.

Project Description In the field of graphic design branding means visually communicating the identity of something [1]. Coca-Cola, Apple and Starbucks are instantly recognizable brands (Figure 1). As is known from these examples, branding can convey efficiently and effectively not only matters of identity but of spirit or essence. Through my research I have examined the process by which brands such as these are created. I have done this in order to inform my own process of exploring the branding of the Berry Summer Thesis Institute. Notably, I have made this work and continue its refinement as a Berry student myself.

Figure 2. I Love New York (1977). Image courtesy of iloveny.com.

Examining the process behind branding provides a deeper understanding Figure 1. Coca-Cola (2009), Apple (2014), and Starbucks of how brands are able (2011). Images courtesy of brandsoftheworld.com. to communicate so effectively. The â&#x20AC;&#x153;I Love New Yorkâ&#x20AC;? brand is a classic example of the importance of process (Figure 2). This brand emerged out of a desperate time in the history of New York State. In the 1970s crime rates in the state were skyrocketing, particularly in the City of New York. Among other crimes, looting and acts of arson were commonplace [2]. The result was that public opinion of New York, both within and beyond the city, was at an all-time Proceedings 2016

ARTS AND HUMANITIES low. Negative headlines were common and pamphlets were even given to tourists as “survival guides” for navigating what had become an economically-depressed and dangerous city (Figure 3). In response to this predicament, The New York State Department of Commerce initiated a campaign to promote tourism and uplift morale, particularly in the City of New York. Graphic designer Milton Glaser was contacted and engaged to give visual form to this campaign (Figure 4). Born and raised in New York City, Glaser was intimately familiar with the city’s Figure 3. Blackout (1977), Fear City: A Survival Guide for Visitors to the City of New environment [3]. This knowledge played York (1975). Photographs courtesy of NYC Council for Public Safety, council.nyc.gov. a major role in his design process. Glaser saw the devastation that was occurring in his city and sought to create a brand that would reflect the fondness he and other citizens felt for their hometown, despite its current state of depression and danger [4]. Originally, Glaser considered expressing his fondness for New York through the straightforward, written statement “I love New York.” However, he developed a new perspective for his emerging design while riding to his studio one day in the back of a New York City taxicab. As the cab traveled through the city, Glaser pulled a red crayon from his pocket and scribbled a new idea in both textual and coloristic form onto half of an old envelope [3] (Figure 5). Inspired by what was still the irrepressible vibrancy and energy of the city itself, this sketch served to direct Glaser’s design from that point onward. As he began to develop his sketch further, Glaser chose to set the black I, N and Y in a modified version of the slab serif typeface, Figure 4. Milton Glaser (2006). American Typewriter. He made Photograph courtesy of Sam Haskins, Sam Haskins Estate. this typographic decision so as to maintain the bold attitude of his original sketch. Glaser then chose to stack the letters in what was a clear reference to artist Robert Indiana’s Figure 5. I (Heart) NY Concept Sketch (1976) by Milton “LOVE” sculpture that had recently made its debut in the Glaser, I (Heart) NY Concept Layout (1976) by Milton city [4] (Figure 6). Finally, Glaser changed the heart back Glaser. Photograph courtesy of moma.org. to the crayon red of the initial envelope sketch. Glaser only planned for his design to serve its immediate purpose in the New York branding campaign [5]. However, the brand continued to evolve beyond Glaser’s influence. It quickly grew in popularity as its reception began to promote positivity and attract tourists to the city. To this day the “I Love New York” brand is one of the most recognized, celebrated and imitated brands in the world [5]. In fact, its influence has been so broadly felt that it can even be seen in the University of Dayton’s own “I Love UD” brand (Figure 7). The wide influence and longevity of this design was the direct product of Glaser’s extended creative process. Through Glaser’s familiarity with New York City, his ability to engage with and draw inspiration Proceedings 2016

Figure 6. LOVE (1966) by Robert Indiana. Photograph courtesy of pixabay.com.

ARTS AND HUMANITIES from his surroundings, and by editing and refining his design, Glaser produced through process the “I Love New York” brand.

Figure 7. I Love UD (2016). Image courtesy of the University of Dayton.

Learning from this understanding of Glaser’s branding process, I embarked on my own process of branding the Berry Summer Thesis Institute (Institute) I began with and held to the conviction that a future brand would need to reflect the kind of creative intensity that characterizes the Institute in which high-quality undergraduate research is designed and developed in such a short period of time [6]. My aim throughout the process was that branding the Berry Summer Thesis Institute would enhance its continuation and growth in the same way that Glaser’s brand brought new life and energy to New York. Following Glaser’s lead, my process was to engage in an intensive creative process in order to acquire a deeper understanding of the culture, mission and vision of the Institute.

I began my research by examining an existing Institute brand, as well as the design of the annual Berry Summer Thesis Institute Proceedings publication [7] (Figure 8). While these are good visual representations, I do believe that they, like all work, can be improved upon. For example, New York itself had good visual representation in the images of the Empire State building and the Statue of Liberty [8]. These were established visual “brands” well before Glaser scribbled his brand idea onto the back of an envelope. In my process I started off by following in Glaser’s bold example of brand introduction in conscious relationship with other successful brands. An important step in my branding process was researching 30 top graphic designers who, like Glaser, have been influential in the modern era. I examined the books these designers wrote, the interviews and talks they gave, and of course the design work they produced [9]. Through this research I focused on exploring the similarities and differences that characterize the unique creative processes of these designers. To continue my branding process, I collected experiential data by actively participating in and observing the operation of the Figure 8. Berry Summer Thesis Institute Proceedings 2015 and Berry Summer Thesis Institute Institute (Figure 9). I experienced logo. Images courtesy of the University Honors Program, University of Dayton. events, activities and outings with my group of fellow Institute students and collected objects from these experiences. Furthermore, I documented these experiences through extensive note taking and photograph making and filled an entire sketchbook with drawings and sketches — even, on occasion, doodles — of my observations and inspirations from the Institute experience. Perhaps most importantly, I immersed myself in following the creative processes of my fellow students. I talked with them about their work and I visited their working spaces. I lived alongside them and learned from them about the creative processes of student psychologists, engineers, musicians and biologists. Through this gathering of information I sought to capture a comprehensive understanding of the Institute experience as the basis of my developing design ideas for a new brand. Finally, for my preliminary branding processes, I examined other entities within the University of Dayton that have brands. One example that I found especially informative is the branding 4

Proceedings 2016

ARTS AND HUMANITIES

Figure 9. Photographs courtesy of Lucy Bratton, June 2016.

of the student-run business, Flyer Enterprises. The branding of Flyer Enterprises reflects the larger University of Dayton brand through its color pallet and use of purposefully joined and linked letters [10]. Furthermore, individual subdivisions of Flyer Enterprises (such as the Blend) reflect in their brands the larger Enterprise’s use of lettering and shapes [11]. Using this understanding of how brands fit within larger, pre-existing brands, I noted and examined how the Institute is housed within the University Honors Program and, more broadly, within the University of Dayton (Figure 10). This nesting relationship is reminiscent of the way in which Glaser’s “I Love New York” brand was created in relation to Robert Indiana’s “LOVE” sculpture. In addition to paying close attention to this nesting phenomenon, my process of branding the Institute has been influenced by Glaser’s process of immersion in New York City, acquiring inspiration and information from his surroundings and refining his design. I, too, have immersed myself in the Berry Summer Thesis Institute experience, gaining inspiration and information from my surroundings, and intend to refine my ideas as I continue my investigation of branding the Institute. Proceedings 2016

Figure 10. Images courtesy of the University of Dayton, University Honors Program and Flyer Enterprises.

ARTS AND HUMANITIES

Figure 11. Photographs courtesy of Lucy Bratton, June 2016.

Through my investigation of creativity, I have acquired an understanding of the importance of process in the creation of a successful brand. Taking all this research into account, I created thumbnail sketches for the Institute brand and intend to refine these ideas into a finished brand as my thesis progresses (Figure 11). When creating these sketches I sought to capture my experience of the Institute by highlighting what I have concluded to be its most central attributes. As is evident in the middle image, I kept the goal of creating a bright, optimistic, clean and versatile design at the forefront of my process. These thumbnails have an intentionally loose structure so as to convey most freely my interpretation of the Institute's spirit. In my visual process shown here, I have explored form, color and typography through sketching, note taking and visual ideation so as to more comprehensively explore potential brands. My work this summer has been a theoretical and preliminary exercise devoted largely to process. I believe that the branding of the Berry Summer Thesis Institute could play a key role in the continuation of this valuable program. Accordingly, it is my vision that the future implementation of a new Institute brand would serve to further the positive energy and sustain the great work that has always characterized the Berry Summer Thesis Institute.

Acknowledgments I would like to thank The Berry Family Foundation for supporting the Berry Summer Thesis Institute and the University Honors Program for facilitating this valuable program. I also thank my fellow "BSWI" students for their immense kindness, encouragement and inspiration. Finally, I thank my advisor, Dr. Roger Crum, for his guidance and my friends and family for their support.

Proceedings 2016

ARTS AND HUMANITIES Resource Thought of You — Behind the Scenes. Dir. Ryan Woodward. YouTube. RyanWoodwardart, 27 Aug. 2014. Web. 20 June 2016.

References 1.

Brintazzoli, Gigliola et al. “Conscious, but Not Unconscious, Logo Priming of Brands and Related Words.” Consciousness and Cognition 21.2 (2012): 824-834. Web. 11 June 2016. Standing on the Verge: Lessons and Limits from the Empirical Study of Consciousness.

Cush, Andy. “Fear City: The Insane Pamphlet the NYPD Used to Terrorize 1970s New York.” Gawker. N.p., n.d. Web. 31 July 2016.

“A Brief History of the ‘I Love New York’ Logo.” Logoworks Blog. N.p., n.d. Web. 24 July 2016.

“The Art of Milton Glaser: To Inform and Delight | The Artifice.” N.p., n.d. Web. 24 July 2016.

“Milton Glaser: From His Iconic ‘I Love New York’ Logo to His New Mad.” The Independent. N.p., 26 Apr. 2014. Web. 31 July 2016.

“Milton Glaser: His Heart Was in the Right Place.” 2-7 2011. Web. 25 June 2016.

“Berry Summer Thesis Institute : University of Dayton, Ohio.” N.p., n.d. Web. 25 June 2016.

“The New York Icons - New York - NewYork.com.” N.p., n.d. Web. 15 July 2016.

Millman, Debbie. How to Think like a Great Graphic Designer. New York: Allworth, 2007. Print.

10. "University of Dayton Brand Guidelines." Office of University Marketing (n.d.): n. pag. Web. 14 June 2016. 11. “The Blend.” Flyer Enterprises. N.p., 29 Jan. 2016. Web. 7 July 2016.

Proceedings 2016

ARTS AND HUMANITIES

Catholicism, Gender, and Politics in the Music and Performance of the Nueva Canción Song Movement Elizabeth M. Turnwald1,2,3,4 University of Dayton, 300 College Park, Dayton, OH, 45469 1. Department of Music 2. Department of Global Languages and Cultures 3. University Honors Program 4. Berry Summer Thesis Institute

Thesis Mentor: Samuel N. Dorf, Ph.D. Department of Music Corresponding Author: Elizabeth M. Turnwald Email: turnwalde1@udayton.edu

Abstract During the social and political turmoil of the 1960s and ‘70s, folk music rose to popularity throughout both North and South America. Singers such as Violet Parra of Chile, Mercedes Sosa of Argentina, and Joan Baez of the United States were viewed as hopeful and resilient voices of the people. Although much has been done to examine the political impact of their work, my aim is to study their legacies through an interdisciplinary approach. By analyzing the music of these folkloristas through musical analysis, women/gender studies, performance studies and religious studies, I seek to connect the impact of their works with Catholic liturgical practice.

Description Throughout the second half of the twentieth century, Latin American singers used music as a way to respond to the violence and tyranny devastating their respective countries. Although their songs and lyrics incorporated religious elements, the musical style, political message and commercial success were more comparable to U.S. folk singers like Bob Dylan and Peter, Paul and Mary than to Catholic liturgical music of the early twentieth century. Combining their religious undertones with the progressive messages and indigenous influences of their music, this wave of Latin American protest song appears to fall into a category entirely of its own. Coming from the Greek leitourgia, the very word “liturgy” means “public duty” or “work of the people”; therefore, at what point is control transferred back into the hands of the general public? Within the Mass we see the renewal of this mission of the people, through both the equalizing reforms of the Second Vatican Council and in the hymns of hope, struggle, and triumph sung by the congregation. Where do songs like this — songs that have seemingly transcended some imagined line between sacred and secular, songs that carry a religious power, which was not originally intended — fit into a sacred setting? Although many scholars have examined these Latin American folksingers and the political impact of their work, the topic lacks an interdisciplinary study of their legacies. By analyzing the music of these “protest singers,” or folkloristas, through musical and lyrical analysis, women/gender studies, performance studies and religious studies, I seek to connect the impact of their works with Catholic liturgical imagery and practice. Through examining four interpretations of the renowned protest 8

Proceedings 2016

ARTS AND HUMANITIES song, “Gracias a la vida,” I will show how the lyrics, poetry, music and performance share similarities with liturgical practice. Each respective discipline thoroughly investigates areas pertaining to its own subject matter. Generally, though, by focusing solely on a topic from only one perspective, there arises a certain amount of impartiality toward, or even disinterest in, other disciplines, which could potentially add insight to the study. For example, performance studies often demonstrate a bias against the Catholic Church and its institutions, while religious studies takes on a supportive, and at times defensive, view of Catholicism, yet neglects crucial aspects of performance. Throughout her work, performance studies scholar Diana Taylor misses an opportunity to explore the role of the Catholic Church in the cultural and political atmosphere of Argentina’s “Dirty War” (1976-1983). Instead of delving into the connections between Catholicism and its role in the establishment of feminist theology in Argentina, she quickly dismisses the Catholic Church, focusing solely on its connection to the country’s purist, antirevolutionary movement of nacionalismo.1 The question that begs to be answered, then, is whether or not these biases and intentional omissions within each academic discipline affect the outcomes of their respective studies. If so, in what ways can using an interdisciplinary approach bridge the gap between these distinct fields? At the root of this question is the Latin American musical genre known as nueva canción [new song]. This style came to represent more than just music but a humanitarian ideology centered on the concept of social justice. Historically, Central and South America have undergone a seemingly endless onslaught of violence, tyranny and oppression, either through imperializing forces or a selfinflicted suffering.2 As a conglomeration of countries with distinct origins, histories and cultures, the aim of the nueva canción was to seek a regional identity in hopes of freeing these countries from imperial influence and commercialization and ultimately to bring each nation together as one, united Latin America. Indeed, this hopeful, optimistic mentality is at the center of the movement. From a Northern hemispheric perspective, this genre would typically be referred to as “protest music,” yet there is an important distinction between North and South American folksong. Whereas most North American singers like Joan Baez (b. 1941), Woody Guthrie (1912-1967), and Bob Dylan (b. 1941) celebrate the term’s staunch political implications, many Latin American singers have directly objected to its application in regards to their music. Most feel that the word “protest” holds an antipathetic connotation that uncharacteristically represents their motivations. Argentine singer Mercedes Sosa (1935-2009) has described the music as “honest songs about the way things really are” and has stated that she feels the label “protest song” implies a restrictive, hostile approach.3 Similarly, Uruguayan folksinger Daniel Vigletti prefers “propose” rather than “protest,” as it better represents the genre’s constructive and unifying nature.4 In fact, the only destructive quality of the nueva canción can be found in one of its rallying cries, “¡a desalambrar!” meaning “to tear down fences.”5 Indeed, the aim is not to diminish the oppressors, but rather to motivate and empower the oppressed. It is in this spirit in which these Latin American artists wrote and performed their music — songs by the people, for the people. To the musicians, the origins of the nueva canción may have as much emotional and historical value as the actual songs themselves. In the late 1950s, as Latin America continued to be invaded by U.S. commercialization, certain artists realized that they had had enough

Diana Taylor, Disappearing Acts: Spectacles of Gender and Nationalism in Argentina’s “Dirty War” (Durham, North Carolina: Duke University Press, 1997), 34.

For more on the history of Latin America, see John Charles Chasteen’s Born in Blood and Fire: A Concise History of Latin America (New York: W.W. Norton, 2011).

Jane A. Bernstein, “Thanks for My Weapons in Battle - My Voice and the Desire to Use It: Women and Protest Music in the Americas,” in Women’s Voices Across Musical Worlds, ed. Jane A. Bernstein (Boston: Northeastern University Press, 2003), 169.

Bernstein, “Women and Protest Music in the Americas,” 169.

Robert Pring-Mill, Gracias a La Vida: The Power and Poetry of Song (London: Queen Mary and Westfield College, Department of Hispanic Studies, 1990), 9.

Proceedings 2016

ARTS AND HUMANITIES of the constant barrage of imperialism. A young folksinger named Violeta Parra (1917-1967) traveled with her children, Angel and Isabel, throughout her native Chile to study and collect indigenous and folk music of each region of the vast country. She then composed and performed her own songs over the radio and in folk cafés known as peñas. Although her music initially centered on the typical, daily lives of rural people and the working-class, as the political tension in Chile began to grow, her songs mirrored the country’s uneasy atmosphere and took on a more militant subject matter.

The Life and Legacy of “Gracias a la Vida” Parra’s most famous work, “Gracias a la Vida” [“Thanks to Life”] would later become an anthem for the nueva canción throughout all of Latin America.6 Released in 1967, just a year before her suicide, the singer thanks life for the various gifts it has given her. In the first stanza, she thanks it for granting her sight; in the second, for hearing; the third, for language; the fourth for strength; and finally, in the fifth, for her heart. Frequently described as a love song, the symbolism and depth behind the words highlight the humanistic nature of the nueva canción genre. Often dealing with the human condition, the lyrics are filled with examples of twin and opposite reactions — of good and evil, dark versus light, and of laughter and tears.7 While the lines portray a tormented woman attempting to focus on the beauty in life, the simple musical accompaniment is reminiscent of a lullaby, gently soothing the listener as if to assure him or her that all will be well. The melody (Figure 1) is in 3/8 time, and the thin, metallic sound of the solo guitar accompaniment lends a waltzlike quality, while the emphasis on the second and third beats propels the floating melody forward throughout the song.

Figure 1. Opening stanza of Parra’s “Gracias a la Vida,” 1966. Image and transcription courtesy of Elizabeth Turnwald, 2016.

Hear original audio recording of Parra’s “Gracias a la Vida” here: https://www.youtube.com/watch?v=UW3IgDs-NnA.

See Pring-Mill, Gracias a La Vida: The Power and Poetry of Song, 45.

Proceedings 2016

ARTS AND HUMANITIES “Gracias a la Vida” has become a familiar song throughout Central and South America, much like “Amazing Grace” or “Swing Low, Sweet Chariot” in the United States. And much like these two English examples, the prolific song has transcended the realm of mere popular music for Spanishspeaking populations. Composed with political intent, it has been performed in secular, popular and sacred settings, while its countless performances by artists of every genre have made the work a song of love, of thanksgiving, of strife, of mourning and of reverence. In its “afterlife,” it has become more than just a song, but an anthem of both the nueva canción and of the universal nature of the human condition.

Joan Baez Creating a Communal Experience: Protest Song and Liturgical Practice It is through the covers of “Gracias a la Vida” that we see how songs of this genre continue to evolve through repeated performance. As a song is taken out of its original setting and performed by a different artist, the audience, purpose and overall meaning change with each reproduction. Singers like Joan Baez and Mercedes Sosa have performed Parra’s famous piece both solo and together, and in various settings ranging from massive outdoor concerts in Europe, to intimate, indoor venues.8 In one particular performance in Bretagne, France, (Figure 2), as Baez sings a song with Spanish lyrics to a French-speaking audience, how does the message of the performance change? Does the audience understand the words, and if so, how are they interpreted by a European audience in the twenty-first century versus the audience Parra had intended — one set in South America in the late 1960s?

Figure 2. Joan Baez performs “Gracias a la Vida” in Bretagne, France, 2000. Photographs courtesy of https://www.youtube.com/watch?v=DFZxBvUMlG0.

In both of her live performances listed below; Baez incorporates an original vocal line (Figure 3), which acts as both a transition between stanzas and a means of increasing audience participation. The repetitive melody, sung on the syllable “la,” is quickly echoed by the entire crowd as they clap along in unison.

Figure 3. Baez original melody in “Gracias a la Vida.” 1966. Image and transcription courtesy of Elizabeth Turnwald, 2016.

See Baez and Sosa 1988 performance in Germany https://www.youtube.com/watch?v=rMuTXcf3-6A; and Baez 2000 performance in France https://www.youtube.com/watch?v=DFZxBvUMlG0.

Proceedings 2016

ARTS AND HUMANITIES From a performance studies perspective, transforming the song into an upbeat “sing-a-long” completely changes the meaning of the entire act. In The Archive and the Repertoire, Diana Taylor suggests that rather than revealing a culture’s truest essence, performances can allow further insight into our own “desire for access” and act as a reflection of “the politics of our interpretations.”9 By including a simple chorus and encouraging participation, is Baez simply tapping in to this innate “desire for access” so present in the nueva canción? Indeed, the longing for inclusivity and community in this genre mirrors the shift seen in the Catholic Church after the Second Vatican Council. With the Mass now celebrated in the vernacular, the musical repertoire expanding far beyond classical styles and Gregorian chant sung in Latin, and the priest finally addressing the people directly, a new feeling of unity grew among the congregation. Full and active participation became the end goal for the assembly and the concept of fostering a communal worship experience became increasingly more vital. In a self-conscious society, simple and repetitive melodies gave the congregation a sense of familiarity and encouraged the people to join together in song. Within Catholic practice, this idea of repetition is present in not only the hymns used during the liturgy, but in the recitation of the Rosary. When said by numbers of people, the effect of the recurrent verses has been likened to that of a sea shanty, as the rhythmic pattern of call and response “induces a sense of being caught up in the flow of something greater” and “thus [liberating] the mediator to throw himor herself completely into the task.”10 By including a repetitive melody purely to garner audience involvement, Baez creates an environment conducive to the full and active participation laid out in Vatican II, while effortlessly touching upon something so innately human: the desire to belong.

Mercedes Sosa as Pachamama: Liturgical Practice through Native Performance Mercedes Sosa’s cover of “Gracias a la Vida” has risen to international recognition well beyond its composer’s initial recording. Considered the most poignant and moving of its many interpretations, Sosa initially recorded the song in 1971 for her album entitled Homenaje a Violeta Parra [Homage to Violeta Parra]11. In the video of this live recording, Sosa is seated on a low stage, level with her select audience. The small, intimate venue is dark save for two spotlights on Sosa and the single guitarist accompanying her (Figure 4). Whereas Parra’s original version was relatively consistent in tone, vocal timbre and tempo, Sosa sings in short bursts as if overcome by emotion halfway between each line. Her swaying rubato and quasi-improvised style captivate the listener, while her frequent and meaningful pauses hold as much power over the audience as her rich alto voice. The guitar accompaniment varies in style and pattern from each stanza to the next, whereas Parra’s version had a constant, lighthearted quality throughout. Ranging from verses of pensive reservation to fervent intensity, Sosa thoroughly employs the full range of her powerful voice (Figure 5). Within not only her specific performance but in her overall image do we see evidence of the shared mentality of Argentina and of a culture heavily influenced by the Roman Catholic Church. Indeed, her very nickname, Pachamama, is an indigenous Andean term meaning “Earth Mother,” commonly used to refer to the Virgin Mary.12 This comparison to the Holy Virgin offers a clear connection between the Argentine and South American perceptions of Sosa, as well as the immeasurable impact of Catholicism in Latin America. Yet by Sosa’s likening to Mary, the question of origin begs to be

Diana Taylor, The Archive and the Repertoire: Performing Cultural Memory in the Americas, 6.

10. Sarah Jane Boss, “Telling the Beads: The Practice and Symbolism of the Rosary,” in Mary: The Complete Resource, Sarah Jane Boss, ed. (London. Continuum International Publishing Group, 2007), 386. 11. See Sosa’s 1971 live recording here: https://www.youtube.com/watch?v=cIrGQD84F1g. 12. Emilce Cuda, “Missing is the Woman/Hace Falta la Mujer,” in Linda Hogan and A. E. Orobator, eds. Feminist Catholic Theological Ethics: Conversations in the World Church (Maryknoll, New York: Orbis Books, 2014), 151.

Proceedings 2016

ARTS AND HUMANITIES

Figure 4. Mercedes Sosa’s live performance of “Gracias a la Vida,” 1971. Photograph courtesy of https://www.youtube.com/watch?v=cIrGQD84F1g.

Figure 5. Opening stanza of Sosa’s 1971 performance. Image courtesy of Elizabeth Turnwald, 2016.

answered: Do Sosa’s image and music actually hold religious meaning, or is the influence of the Catholic Church so powerful in Latin America that they simply appear that way to the public? Prevalent in virtually every aspect of daily life, Catholicism holds particular influence in the roles of women and the gender norms of the region. Specifically in the role of the wife and of the mother do we see how the importance of motherhood and virginity are embedded in an androcentric culture.13 Dually a religious and societal ideal, this concept of marianismo, or “Cult of Mary,” centers on the Virgin Mary as the ultimate example of womanhood. Both mother and virgin, she represents the infallible pillar of spirituality, humility, sacrifice and purity idealized throughout much of Latin

13. Sylvia Chant with Nikki Craske, Gender in Latin America (New Jersey: Rutgers University Press, 2003), 135.

Proceedings 2016

ARTS AND HUMANITIES America.14 We find evidence of the region’s fascination with Mary in countless representations of her, ranging from reverent hymns and intricate paintings, to ornate statues and grand monuments (Figure 6).

Figure 6. (Left to right.) Figure of Nuestra Señora de Luján (Our Lady of Luján), Buenos Aires, Argentina; photograph courtesy of paraclito.org. Statue of the Virgin Mary on top of Cerro San Cristóbal (St. Christopher’s Hill), Santiago, Chile; photograph courtesy of wikipedia.org. Painting of Our Lady of Guadalupe in Mexico City, Mexico; photograph courtesy of thecatholiccatalogue.com.

However, this reverence toward the Virgin Mary holds different meaning for Latin America’s women than for its men. Marianismo has been referred to as the equal and opposite reaction of machismo — the social construct of aggressive, dominating, and at times violent, male pride — yet this “compensatory complex” of intense admiration of Mary has been accused of “presenting major obstacles to women’s progress” and has even been described as “one of the pegs on which the ills of Latin American women’s subordination were hung.”15 Generally speaking, in this culture of machismo and marianismo, the woman’s duty is to be a wife and mother, a selfless pillar of spiritual strength. We can see references to the Virgin Mary within Sosa’s public image and personal sacrifices during Argentina’s “Dirty War.” Silenced by her own government, she refused to stop performing publicly until she was finally arrested while singing a land reform song, “Cuando Tenga la Tierra” (When They Have the Land”) at a concert in 1978.16 After enduring death threats, bomb scares and a government ban on public performances, Sosa was forced to flee her homeland and seek exile in Spain. Her audience, interpreting her actions as self-sacrificing, heralded her as a fervent and unwavering advocate of the people. Aligning with the tenets of feminist theology and the doctrines of the Medellín bishop’s conference of Vatican Council II, Sosa’s activism and passion for social justice yielded her all of the positive characteristics of marianismo, while her humbleness and personal sacrifices allowed her to remain an accessible, Latin American woman in the eyes of the public.17

Voces Unidas por Chile: A Unifying Call to Action After the massive earthquake that devastated Chile in 2010, a group of famous Latin American artists came together to produce a charity song for the victims and survivors of the disaster. The group, entitled “Voces Unidas por Chile” [Voices United for Chile], produced a cover of “Gracias a la Vida”

14. Chant with Craske, Gender in Latin America, 9. 15. Chant with Craske, Gender in Latin America, 9. 16. Bernstein, “Women and Protest Music in the Americas,” 173. 17. Bernstein, “Women and Protest Music in the Americas,” 171.

Proceedings 2016

ARTS AND HUMANITIES to raise awareness and funds for those affected (Figure 7).18 The high production value and professional lighting and camera angles result in a work utterly removed from Parra’s original and even from Sosa’s interpretation or Baez’s concert version. From a performance studies perspective, the digital instrumentation, four-part harmonies and overlapping images of weeping Chilean families and Grammy-winning popstars create a distinct message entirely divorced from those of its predecessors. No longer simply thanking life for its gifts, nor comforting a nation torn apart by violence, this modern cover is a call to the middle-classes to empower the victims through financial support.

Figure 7. Music video of “Gracias a la Vida” performed by “Voces Unidas por Chile,” 2010. Photograph courtesy of https://www.youtube.com/watch?v=_7-vTDV_aSA.

From the lens of religious studies, though, the group’s cover reflects numerous principles found within the Catholic Church and the Second Vatican Council. The song is a call to action, imploring the people to reach out and help one another in their time of need. The Catholic ministries within South America typically received orders and edicts from the European sectors of the Catholic Church, but during Vatican Council II, and specifically the Medellín Council held in Colombia, the Latin American clergy began to speak up for their constituencies and take action. Rather than simply receiving dictates and input from other continents, the Latin American church elders called for a “new era” for their nations — one of growth and change through peaceful, nonviolent means.19 Taking long-awaited initiative, this historically overlooked region of the Church came together to cry out for justice, change and liberation. The documents were said to have been written for the people with their real concerns and views taken into account and, although a secular group, in this same manner do we see “Voces Unidas por Chile” produce and perform their cover of “Gracias a la Vida.” The singers’ countries of origin range from Spain, Chile and Panama; to Mexico, Colombia and the Dominican Republic; and even to Canada (Michael Bublé) and Italy (Laura Pausini). The efforts to

18. See cover of “Gracias a la vida” by “Voces Unidas por Chile” here: https://www.youtube.com/watch?v=_7-vTDV_aSA. 19. Marcos McGrath, C.S.C. “The Impact of Gaudium et Spes: Medellín, Puebla, and Pastoral Creativity” in Sacred Music and Liturgy Reform After Vatican II, Johannes Overath, ed. (Saint Paul: North Central Publishing Company, 1969), 68.

Proceedings 2016

ARTS AND HUMANITIES bring together musicians from across Latin America — as well as those from Europe and North America — demonstrate the objective of unification prioritized throughout the recording. In the years of the Second Vatican Council during the Latin American Episcopal Conference [CELAM Consejo Episcopal Latinoamericano] in Mar del Plata, Argentina, Pope Paul VI spoke on “The Church in the Development and Integration of Latin America,” saying that the Catholic Church, “which was present at the birth and throughout the history of these nations, had to be present in these crucial moments of their history.”20 Indeed, “Voces Unidas por Chile” emphasizes this idea of a supportive presence with a subtle adjustment to the final lyrics, from “thanks to life, which has given me so much,” to “thanks to life, which has given us so much.” This intentional change, combined with the collaboration of artists from all corners of the vast continent, alters the message of the song in order to express that all of these nations are standing by the victims and survivors of the earthquake and that, together, they are ready to face whatever lies behind and ahead as a united Latin America.

Conclusion By examining these diverse covers of “Gracias a la Vida” from a musicological perspective, we see the subtle, yet impressive ability of music to inspire and promote social action. By studying the works through the lenses of performance studies and religious studies, we also see how the themes of community, inclusivity and audience participation play various roles within both the reforms of the Second Vatican Council as well as the music of the nueva canción. From the viewpoint of women/ gender studies, it is evident that there are subtle connections between the images of singers such as Sosa and Parra and the overpowering influence of marianismo within Latin America. As the Cult of Mary is such an essential part of the region’s culture, religion and gender structure, how did the female “mother” persona of these women affect their professional and musical careers? What part did the surrounding culture of marianismo play in the response to the genre and, therefore, in the context in which the songs were received? Furthermore, in examining each distinct cover we see how that context can alter the impact and message of the same work. By changing its venue, performer(s), audience and/or musical style, the meaning changes as well. Considering the countless renditions and reenactments of simply one of these folksongs, can the meaning of a song ever be transferred verbatim from one performance to another? Although Parra, Sosa, Baez and Voces Unidas por Chile were performing “Gracias a la Vida” in secular settings, protest songs were often sung in progressive Catholic churches, notably in Nicaragua, during the 1970s in defiance of the oppressive governments and dictatorial regimes. To better understand the historical role of the genre beyond the secular realm, future work may include archival research into the changing liturgical repertoires in progressive Latin American churches. As music from the nueva canción seems to fit into a category unique to a society heavily influenced by indigenous culture and the Catholic Church, in which settings can these “protest” songs be used within a sacred setting to give the underrepresented a voice? Whether intentionally religious or not, these songs of hope, struggle and triumph hold an undeniable sense of spirituality, of faith and of a unified call for change.

20. Joseph Gremillion, The Church and Culture since Vatican II: The Experience of North and Latin America (Notre Dame, Indiana: University of Notre Dame Press. 1985), 65-66. 21. Manzar Foroohar, The Catholic Church and Social Change in Nicaragua, (New York: SUNY Press, 1989), 120.

Proceedings 2016

ARTS AND HUMANITIES Acknowledgments I would like to thank the Berry Family and its foundation for this incredible opportunity to engage in undergraduate research through the Berry Summer Thesis Institute, as well as the University Honors Program and the dedicated individuals who made this summer possible: Dr. John McCombe, Dr. David Darrow, Dr. Nancy Miller, Maria Burkett, Ramona Speranza, Laura Cotten and Jill Talley. Thank you to Dr. Tracey Jaffe, Dr. Neomi DeAnda, Dr. Sandra Yocum and Dr. Marc Gidal of Ramapo College whose insights and expertise made the interdisciplinary aspects of this research a reality. Finally, I would like to thank my phenomenal advisor, Dr. Sam Dorf, for his selfless dedication, seemingly infinite wisdom and overwhelming support.

Works Cited Bernstein, Jane A., Ed. “Thanks for my Weapons in Battle - My Voice and the Desire to Use It” from Women’s Voices Across Musical Worlds. Boston: Northeastern University Press, 2003. Boss, Sarah Jane. Mary: The Complete Resource. London. Continuum International Publishing Group, 2007. Chant, Sylvia, with Nikki Craske. Gender in Latin America. New Jersey: Rutgers University Press, 2003. Chasteen, John Charles. Born in Blood and Fire: A Concise History of Latin America. New York: W.W. Norton, 2011. Fisher, Terence James. The Catholic Counterculture in America, 1933-1962. Chapel Hill, North Carolina: The University of North Carolina Press. 1989. Foroohar, Manzar. The Catholic Church and Social Change in Nicaragua. New York: SUNY Press, 1989. Gidal, Marc. “Contemporary ‘Latin American’ Composers of Art Music in the United States: Cosmopolitans Navigating Multiculturalism and Universalism.” Latin American Music Review / Revista de Música Latinoamericana 31 no. 1 (2010): 40-78. Hogan, Linda, and A. E. Orobator. Eds. Feminist Catholic Theological Ethics: Conversations in the World Church. Catholic Theological Ethics in the World Church. Maryknoll, New York: Orbis Books, 2014. Luft, Murray. “Latin American Protest Music: What Happened to The New Songs?” Canadian Folk Music Bulletin/Bulletin de Musique Folklorique Canadienne 30, no. 3 (1996): 10-18. Melhuus, Marit, and Kristi Anne Stølen, eds. Machos, Mistresses, Madonnas: Contesting the Power of Latin American Gender Imagery. London: Verso, 1996. McGrath, Marcos, C.S.C. “The Impact of Gaudium et Spes: Medellín, Puebla, and Pastoral Creativity” from Gremillion, Joseph. The Church and Culture since Vatican II: The Experience of North and Latin America. Notre Dame, Indiana: University of Notre Dame Press. 1985. Montoya, Rosario, Lessie Jo Frazier, and Janise Hurtig. Gender’s Place: Feminist Anthropologies of Latin America. London. Palgrave Macmillan, 2002. Overath, Johannes, ed. Sacred Music and Liturgy Reform After Vatican II. Saint Paul, MN. North Central Publishing Company, 1969. (68). Pring-Mill, Robert. Gracias a La Vida: The Power and Poetry of Song. London: Queen Mary and Westfield College (Department of Hispanic Studies), 1990. Roy, William G. “How Social Movements Do Culture.” International Journal of Politics, Culture, and Society 23 (2/3): 85-98, 2010. Taylor, Diana. Disappearing Acts: Spectacles of Gender and Nationalism in Argentina’s “Dirty War.” Durham, North Carolina. Duke University Press, 1997. Taylor, Diana. The Archive and the Repertoire: Performing Cultural Memory in the Americas. Durham, North Carolina. Duke University Press, 2003. Taylor, Diana and Roselyn Costantino, ed. Holy Terrors: Latin American Women Perform. Durham, North Carolina. Duke University Press, 2003.

Proceedings 2016

SOCIAL SCIENCE

The Effects of Adolescent Housing Condition and Voluntary Exercise on Alcohol Intake and Stress Response in Male Long-Evans Rats Caroline A. Lynch1,2,3 and Tracy R. Butler4 University of Dayton, 300 College Park, Dayton, OH, 45469 1. Premedical Programs 2. University Honors Program 3. Berry Summer Thesis Institute 4. Department of Psychology

Thesis Mentor: Tracy R. Butler, Ph.D. Department of Psychology Corresponding Author: Tracy R. Butler, Ph.D. Email: tbutler2@udayton.edu

Abstract Due to the remarkable genetic similarity between rats and humans, experiments with rats as subjects have long served as corollaries to human research. Examples include research on the relationships between alcohol intake and exercise, stress levels and eating behavior, and exercise and adult neurogenesis (Leasure and Nixon, 2010). The main question this research aims to answer is whether or not regular exercise during adolescence combined with living in a social environment can lead to lower stress levels and alcohol intake later in life. To gain some preliminary answers to this question, protocols have been employed to combine the variables of adolescent housing condition and voluntary exercise in the form of wheel running to discover the impact on ratsâ&#x20AC;&#x2122; subsequent response to a stressor and alcohol intake/preference. The housing conditions used in this study include Group Housed (GH; 4/cage, n=8) and Socially Isolated (SI; 1/cage, n=8). Socially Isolated housing is used in this study due to its nature as a chronic stressor, as opposed to Group Housing, which allows for natural social interaction. Behavioral tests such as an Elevated Plus Maze (EPM) and Swim Stressor are utilized to examine anxiety-like behavior and plasma corticosterone (CORT) levels. Alcohol intake/preference is measured using an intermittent access homecage drinking paradigm during which each rat receives access to a 20% alcohol solution and water (Mondays, Wednesdays, Fridays). We hypothesized that the rats with access to voluntary exercise will have less anxietylike behavior and an intact stress response and will not drink as much ethanol as the rats raised in standard laboratory housing (1/cage). Also, living in a social environment during adolescence has been shown to lower male ratsâ&#x20AC;&#x2122; ethanol intake compared to living in isolation during adolescence (Chappell et al., 2013). Gaining an understanding of how housing condition and exercise can play a role in subsequent alcohol intake and stress hormone levels will be beneficial to understanding the physiological effects of chronic stress and may be useful for the advent of new pharmacotherapies for individuals with an alcohol use disorder.

Introduction The prevalence of alcohol-use disorders among adults in the United States today remains an everpresent public health concern. To illustrate the gravity of this problem, data suggests that 30.3% of Americans will likely face an alcohol-use disorder at some point in their lifetime (Hasin et al., 2007). Many genetic, biological and environmental factors have been identified that contribute to the onset and maintenance of alcohol use disorders. Two such vulnerability factors are anxiety disorders and 18

Proceedings 2016

SOCIAL SCIENCE chronic stress. Previous studies with rats have shown that exposure to the stress hormone corticosterone (similar to cortisol in humans) increases ratsâ&#x20AC;&#x2122; ethanol intake (Besheer et al., 2013). In addition, positive correlations between displayed anxiety-like behavior and ethanol drinking have been documented in rats, meaning that the more anxiety-like behavior a rat displays, the more ethanol that rat will drink (Chappell et al., 2013). It should be noted that ethanol is the form of alcohol that is drinkable, and the terms alcohol and ethanol will be used interchangeably throughout this paper. Also, rats that spend their adolescence in a Socially Isolated (SI) condition, which is a chronic stressor, have been observed to display more anxiety-like behavior than their Group Housed (GH) counterparts. Many previous studies have used the SI/GH housing paradigm, and results from these studies have highlighted many behavioral and neurobiological deficits in SI rats compared to GH rats. For example, SI rats have exhibited deficits in neural baseline dopamine levels (Karkhanis et al., 2015), cognitive function (Hedges and Woon, 2011), deficits in sensorimotor gating (Ko and Liu, 2015), and impaired fear extinction (Skelly et al., 2015). This study represents a first attempt to lessen behavioral deficits in SI rats using voluntary exercise. Chronic exercise is commonly regarded as a means to release stress and improve overall health in humans, and a previous study has shown that it may have a similar effect on rats by lessening their anxiety-like behavior (Fulk et al., 2004). Therefore, this study represents a model where exercise, a behavioral intervention, may counteract the negative effects of a chronic stressor and lower susceptibility to alcohol dependency later in life. Also, this study may allow for the study of the biological differences in SI rats that received the behavioral intervention and those that did not. Thus, the hypothesis of this study is that the rats with access to voluntary exercise (of both housing conditions) will exhibit less anxiety-like behavior and less subsequent alcohol consumption than the rats without access to exercise. For the rats that do not have access to exercise, it has been hypothesized that GH rats will exhibit less anxiety-like behavior and alcohol intake than SI rats, who were subject to the chronic stressor of an isolated housing condition during adolescence.

Materials and Methods Animals and Housing The subjects in this study were 16 male Long Evans rats sourced from Envigo (Indianapolis, IN). The rats were received on Postnatal Day (PND) 21 and were all Group Housed (GH; 4 rats/cage) in 55 centimeters x 38 centimeters polycarbonate cages until PND 27. It is critical to note that PND 21 is immediately post-weaning, and the developmental period during which this experiment begins is equivalent to adolescence in humans. Beginning on PND 27, half of the cohort remained Group Housed while the other half became Socially Isolated (SI; 1 rat/cage) in 42 centimeters x 21 centimeters polycarbonate cages. This housing arrangement lasted for six weeks, during which time half of all GH and SI rats were allowed access to voluntary exercise (detailed below). The purpose for using this GH/SI model was to discern if Socially Isolated rats would display greater anxiety-like behavior than Group-Housed rats as has been repeatedly seen in previous studies, (Chappell et al., 2013) or if voluntary exercise could prevent greater anxiety-like behavior in SI rats. Also of interest was how housing condition could affect exercise activity, and more specifically which housing condition would result in the greatest amount of running. This housing and exercise schematic is displayed in Table 1. On PND 69, all rats became SI in order to prepare for the behavioral tests and Table 1. An Outline of Housing Condition Paired with Exercise Regimen. Courtesy of Caroline Lynch, 2016.

Proceedings 2016

SOCIAL SCIENCE drinking procedure. Food and water were available ad libitum for the entirety of the experiment. The rats were housed in an environment with a 12-hour light/dark cycle (lights on 7:00-19:00), and all experiments were performed during the light portion of the cycle. All procedures were run in accord with guidelines provided by the National Institutes of Health (NIH) and were approved by the University of Dayton Institutional Animal Care and Use Committee (IACUC).

Voluntary Exercise From PND 28 to PND 69, a subset of GH (n = 4) and SI (n = 4) rats were given access to a running wheel for 30 minutes per day for five days per week. The wheels were eight inches in diameter and made of wire mesh (Figure 2b). One BoGeer YT-813 Bicycle Odometer (Figure 2c) was attached to each wheel to record distance run (in km), in accordance with a similar technique from a previous study (Sasse et al., 2008). Each wheel was contained in a clear plastic tub with sawdust bedding so that each rat could exercise separately and had room to move around the wheel if he chose to not run. Included in Table 1 is a visual representation of which rats exercised and which rats did not according to their housing condition.

Behavioral Testing On PND 70, all 16 rats were individually given five minutes of exposure time on an Elevated Plus Maze (EPM) to assess anxiety-like behavior. The Elevated Plus Maze (Figure 3c) consists of two open arms and two closed arms. The measures we were interested in were open arm time (Figure 3a) and closed arm frequency (Figure 3b). Open arm time is the amount of time in seconds that a rat spends in the open arms of the plus maze. Generally, it is a rat’s instinct to avoid open, well-lit spaces, so the more time a rat spends in the open arms, the less anxiety-like behavior it exhibits (Chappell et al., 2013). Closed arm frequency is used as a measure of general locomotor activity (Holmes and Rodgers, 1999). On PND 71 and 72, all 16 rats underwent a swim stressor procedure in order to observe their subsequent CORT levels compared to baseline levels. Rats were individually placed in a cylindrical bucket (15” tall and 9.5” wide) that was filled with 9” of water kept at 25° C. Rats were given five minutes to swim in the bucket, after which they were removed and placed in a cage with no bedding to dry in. One pre-stressor blood sample was taken from each rat via a tail-snip technique approximately one hour before the swim stressor. Two post-stressor blood samples were taken from each rat approximately 5 and 30 minutes after rats were removed from the water. The procedure used in this study was formulated with the guidance of previous studies that used acute swimming stress (Zarein et al. 2011) and took 5 minute post-stressor blood samples (Wulsin et al., 2016), and 30 minute post-stressor blood samples (Rabasa et al., 2013) to measure the hormonal stress response reflected by concentration of rats’ primary stress hormone, corticosterone. The stress response was measured because it has been observed that individuals with alcohol-use disorders (AUDs) often show a blunted stress response, and SI rats have shown a failure to suppress plasma CORT levels when challenged with dexamethasone compared to GH rats (Butler et al., 2014).

Plasma Corticosterone Determination Pre-stressor and post-stressor blood samples were kept on ice after being acquired and were spun in a centrifuge for 10 minutes to allow for separation of the plasma. The plasma from each sample was removed and kept frozen at -80° C until plasma CORT concentration was determined using a 96-well plate ELISA method. Plasma concentrations were diluted to a 1:20 ratio before being placed into the appropriate wells. The Corticosterone enzymeimmunoassay ELISA kit used in this experiment utilized a polyclonal CORT antibody coated onto the inner surface of each of the 96 wells that attracted any CORT present in the diluted plasma samples to it. Then, the wells were washed and color was developed by using a chromogenic substrate. Finally, absorbance of the samples at 450 nm was read in a plate reader, and CORT concentration of each sample was determined using a standard curve and through a series of equations and interpolations (Immunodiagnostic Systems Inc, Gaithersburg, MD). 20

Proceedings 2016

SOCIAL SCIENCE Drinking Procedure Beginning on PND 76, following the completion of behavioral testing, ethanol consumption was assessed using an intermittent access two-bottle choice homecage drinking paradigm during which each rat received access to a 20% ethanol solution and water (in separate bottles) on Mondays, Wednesdays, and Fridays in 50ml conical bottles. On Tuesdays, Thursdays, Saturdays, and Sundays each rat was given two bottles of water in 50ml conical bottles. Due to the nature of the paradigm, all rats were singly housed for the entirety of the drinking period (PND 76-100). Ethanol consumption and preference was measured at 30 minute and 24 hour timepoints. This model was borrowed from a previous study described in an article covering Alcohol Use Disorders (Butler et al., 2016).

Data Analysis Body weight, exercise, plasma CORT concentration and ethanol intake and preference data were all analyzed separately using repeated-measures two-way ANOVA (group x timepoint of measurement). Anxiety-like behavior on the EPM was assessed by analyzing open arm time and closed arm frequency data using a one-way ANOVA. All analyses were done and graphs were created using GraphPad Prism 7.0 software. Data are expressed as mean +/- SEM.

Results Body weight Body weight data taken once weekly from PND 21-69 were analyzed using a repeated-measures two-way ANOVA (group x day of measurement), with Tukeyâ&#x20AC;&#x2122;s post hoc test. The purpose of measuring body weights was to discern if housing condition or exercise were creating significant weight differences among the groups of rats, since chronically stressed rats sometimes show less weight gain than their non-stressed counterparts (Retana-MĂĄrquez et al., 2003). A statistically significant interaction was found between group and day of measurement, [F(21, 84)= 1.829, p<0.05]. There were no significant differences in body weights from PND 21 to the start of the running protocol on PND 35. On PND 55 (beginning of week four of the running protocol) and PND 69 (shortly after the end of the running protocol), GH runners weighed significantly more than SI runners (p values <0.05) (Figure 1). It can be noted that all 16 rats consistently gained weight throughout the entirety of the experiment.

Figure 1. Body weight (g). Rats were weighed upon arrival to the lab (PND 21) and then weekly thereafter. These data show only slight differences in body weight on PNDs 55 and 69 between GH Runners and SI Runners (*p < 0.05). Courtesy of Caroline Lynch, 2016.

Proceedings 2016

SOCIAL SCIENCE Exercise Exercise data were analyzed using a repeated-measures two-way ANOVA (group x day of measurement), with Sidakâ&#x20AC;&#x2122;s post hoc test. A statistically significant interaction was found between group and day of measurement, [F(23, 138)= 2.407, p<0.05]. GH rats ran significantly more than SI rats on PNDs 35, 37, and 38 (Figure 2a). It can be noted that all four GH runners exhibited running behavior after four days of exposure to the wheels, while it took nine days of exposure for three out of the four SI runners to begin running (the fourth SI runner never ran). We also observed that running distance dropped considerably after PND 43 for both GH and SI runners. The total distance run for each rat over the five week period of the protocol can be found in Table 2.

Figure 2. Distance Run (km). (A) Daily measurements of distance run were taken of all GH runners and SI runners from PND 35 to 66. Significant differences in running distance were found on PNDs 35, 37, and 38 between GH runners and SI runners (*p<0.05). (B) Each rat was individually placed in a plastic bin with sawdust bedding and a wire mesh running wheel to run on. (C) Distance run was measured using BoGeer YT 813 Bicycle Odometers that were attached to each wheel. Courtesy of Caroline Lynch, 2016.

Table 2. Total Distance Run (km) by All GH Runners and SI Runners. Courtesy of Caroline Lynch, 2016.

EPM Measures of open arm time and closed arm frequency were analyzed using a one-way ANOVA with Tukeyâ&#x20AC;&#x2122;s post hoc test. No significant differences in open arm time were found [F(3, 12)= 2.682, p= 0.09, n.s.] (Figure 3a). However, a significant difference in closed arm frequency was found between GH runners and SI runners [F(3, 12)= 3.540, p<0.05] (Figure 3b) such that GH runners exhibited the highest number of closed arm entries on average compared to all other groups.

Proceedings 2016

SOCIAL SCIENCE A

Figure 3. Open Arm Time and Closed Arm Frequency from the Elevated Plus Maze. (A) Open arm time in seconds was measured for all groups of rats. SI runners displayed the most open arm time of all groups. (B) Closed Arm Frequency in number of entries was measured for all groups of rats. GH runners displayed the highest number of closed arm entries of all groups and significantly more closed arm entries than SI runners (*p<0.05). (C) The Elevated Plus Maze used in this experiment contains two open arms and two closed arms. Courtesy of Caroline Lynch, 2016.

C CORT Assay Plasma corticosterone (CORT) concentration in ng/ml was determined by finding the B/Bo% of each plasma sample, and then interpolating the CORT concentration from a standard curve of CORT concentration vs. B/Bo% (Figure 4). Then, CORT concentrations were analyzed using a repeated-measures two-way ANOVA (group x timepoint) with Tukeyâ&#x20AC;&#x2122;s post hoc test. No significant interaction was found between group and timepoint [F(6,24)=2.129, p=0.0870, n.s.], but there was a main effect of timepoint [F(2,24)=93.71, p<0.0001]. CORT levels for all rats were significantly higher at the five minute and 30 minute post-stressor timepoints than at the baseline timepoint. The purpose of analyzing CORT concentrations was to observe how the post-stressor samples related to the baseline samples to gain an understanding the hormonal response to the stressor. A rise in CORT levels after exposure to the stressor was expected, but it could have occurred that the chronically stressed SI rats would show a blunted CORT response as has been found in previous literature (Adinoff et al., 1998).

Figure 4. Plasma CORT Concentrations (ng/ml). CORT concentration was determined using blood samples taken via tail-snip technique at three different timepoints. Baseline samples were taken 1 hour before the stressor, and then post-stressor samples were taken at 5 and 30 minute timepoints after the stressor. Courtesy of Caroline Lynch, 2016.

Proceedings 2016

SOCIAL SCIENCE Ethanol Intake Ethanol intake (g of ethanol/kg of body weight) and preference ratio (ethanol solution consumed out of total liquid consumed) data from both 30 minute and 24 hour timepoints were analyzed using repeated-measures two-way ANOVA (group x PND) with Tukeyâ&#x20AC;&#x2122;s post hoc test interpreted when appropriate. A significant interaction was found between group and PND in 24 hour g/kg consumption [F(33, 132)=1.777, p<0.05] (Figure 5b). Post hoc tests revealed a significant difference in 24 hour g/ kg ethanol intake on PND 83 between GH runners and SI runners in that SI runners drank more ethanol (p<0.05). No significant interaction was found between group and PND in 30 minute g/kg consumption (Figure 5a), but there was a main effect of PND [F(11, 132)=3.1, p<0.05]. This means that ethanol intake changed over time (increasing in this case). No significant interaction was found between group and PND in 30 minute preference ratio (Figure 5c), but there was a main effect of PND [F(11, 132)=2.795, p<0.05] such that 30 minute preference ratio increased over time. No significant interaction was found between group and PND in 24 hour preference ratio (Figure 5d). No significant differences in 24 hour preference ratio were found between groups, but there was a main effect of PND [F(11, 132)=9.985, p<0.05] such that 24 hour preference ratio decreased over time. It is important to note that there were several occasions of bottle leakage that resulted in inaccurate data values. To correct for this, values from the day before and the day after of the same measure from the same rat were averaged. Errors occurred at the 30 minute timepoint for rats 3, 14, and 15 on PND 97 and for rat 15 on PND 87. Errors occurred at the 24 hour timepoint for rats 3, 14, and 15 on PND 97 and for rats 12 and 14 on PND 85.

Figure 5. Ethanol Consumption and Preference. (A) 30 minute ethanol consumption in g/kg. (B) 24 hour ethanol consumption in g/kg. SI runners drank significantly more than GH runners on PND 83 (*p<0.05). (C) 30 minute ethanol preference ratio. A significant difference was found in 30 minute preference ratio between GH non-runners and SI non-runners on PND 90. The solid line at 0.5 indicates the point of equal preference for water and the ethanol solution (*p<0.05). (D) 24 hour ethanol preference ratio. The solid line at 0.5 indicates the point of equal preference for water and the ethanol solution. Courtesy of Caroline Lynch, 2016.

Proceedings 2016

SOCIAL SCIENCE Discussion Effects of Adolescent Housing Condition on Voluntary Exercise It was observed that the GH rats began running earlier and more eagerly than the SI rats. This could be due to a greater response to novelty in GH rats at the time of the exercise protocol. However, the drop-off in running distance after PND 43 remains puzzling. Perhaps due to the ratsâ&#x20AC;&#x2122; repeated exposure to the wheels, they began to lose interest in running on them after this point in time. In future studies, the duration of the exercise protocol may be reduced by a few weeks, or measures will be taken to increase the novelty of the exercise environment to keep the rats running. As can be seen in Table 2, GH rats ran longer distances overall than the SI rats, with the exception of rat 12. On average, GH rats ran 1.028 +/- 0.012 km and SI rats ran 0.3225 +/- 0.0048 km. Rat 9 never ran on its wheel for the entirety of the protocol for reasons unbeknownst to the researchers in this study.

Effects of Adolescent Housing Condition and Voluntary Exercise on Anxiety-Like Behavior As can be seen in Figure 3a, SI runners spent the most time on the open arms of the Elevated Plus Maze. This means that these rats displayed the least anxiety-like behavior on the EPM according to this measure. Although this data was not statistically significant, the trend is encouraging and will require a greater sample size to determine a true effect of group on open arm time. Therefore, it is a possibility that voluntary exercise during adolescence can lessen anxiety-like behavior in Socially Isolated rats. SI non-runners spent the least amount of time on the open arms, thus displaying the highest amount of anxiety-like behavior. This falls in line with the hypothesis of this study in that we expected the SI non-runners to show more anxiety-like behavior than the SI runners. GH runners and GH non-runners showed similar amounts of open-arm time, therefore the voluntary exercise did not affect this measure too much in GH rats. Also, as Figure 3b displays, GH runners performed a significantly greater amount of closed arm entries than SI runners, thus displaying greater locomotor activity than the SI runners (p<0.05). SI non-runners performed the second highest amount of closed arm entries, followed by GH non-runners and SI runners. In the future, other tests to assess for a true effect of group on hypo- or hyperlocomotion could be conducted in an open field.

Effects of Adolescent Housing Condition and Voluntary Exercise on Plasma CORT Concentrations GH non-runners and SI non-runners had higher baseline plasma CORT concentrations on average than the GH and SI runners, as shown in Figure 4. This could mean that the rats that did not run were generally more stressed than the rats that ran (although this cannot yet be statistically confirmed). Thus, it is possible that voluntary exercise played a role in lowering the runnersâ&#x20AC;&#x2122; baseline plasma CORT levels. This CORT-lowering effect of exercise has been found in a previous study, in which rats that underwent six weeks of voluntary wheel running had significantly lower corticosterone responses to repeated audiogenic stressor exposures (Sasse et al., 2008). It was found that CORT levels for all rats were significantly higher at the 5 minute and 30 minute post-stressor timepoints than at the baseline timepoint. At the 30 minute post-stressor time point, all rats had similar plasma CORT concentrations at around 200 ng/ml. Therefore, the data from this project suggest that there may be variances in baseline CORT levels between runners and non-runners, and also that there was no blunted CORT response in SI non-runners as was hypothesized.

Effects of Adolescent Housing Condition and Voluntary Exercise on Ethanol Intake and Preference It was hypothesized by the authors of this study that GH runners would display the lowest average alcohol intake and that SI non-runners would display the greatest alcohol intake. The reasoning for this hypothesis was that both grouped housing during adolescence as well as voluntary exercise have been found to reduce anxiety-like behavior in rats (Chappell et al., 2013) (Fulk et al., 2004). Thus, a combination of these factors could reasonably result in very low anxiety-like behavior. On Proceedings 2016

SOCIAL SCIENCE the other hand, social isolation during adolescence along with no access to exercise could reasonably result in higher anxiety-like behavior. It has been found that greater anxiety-like behavior can lead to greater alcohol intake in rats and a heightened vulnerability to alcohol-use disorders in humans (reviewed in Butler et al., 2016). Thus, it was suspected that the GH runners would drink the least amount of alcohol and display the least anxiety-like behavior, and that SI non-runners would drink the most alcohol and display the most anxiety-like behavior. Although GH runners displayed less anxiety-like behavior than SI non-runners, consistent differences in alcohol intake between groups were not found.

Study Limitations and Future Directions The major limitation in this study was N value, or how many subjects were involved. Involving more subjects would allow for greater statistical power and thus more conclusive results in the future. The lack of significant differences in alcohol intake found between groups could be due to many factors. For example, the sharp decline in running activity after PND 43 could have caused a decrease in the reducing effects of exercise on alcohol intake due to the long period of time between when the rats stopped running and when they started drinking. In future studies, the ethanol protocol may be employed immediately after the conclusion of the exercise protocol to allow for the capture of an effect that was not seen in the current design. The future direction for this study is replication because the results obtained in this study are very encouraging considering this was the first use of the exercise protocol and that the sample size was relatively small.

Acknowledgements I would first and foremost like to thank my mentor, Dr. Tracy Butler, for her support and guidance throughout this project. Her organization, patience, and encouragement have been very helpful to me throughout my journey as a researcher. I would like to thank my fellow lab member, Morgan Pair, for helping me with some protocols. I would also like to thank the University Honors Program and the Berry Family for making the Berry Summer Thesis Institute a possibility, as well as my fellow 2016 cohort members for making this summer a truly memorable experience.

References Adinoff, Bryon et al. “Disturbances of the Stress Response: the Role of the HPA Axis During Alcohol Withdrawal and Abstinence.” Alcohol Health and Research World 22.1 (1998): 67-72. PubMed. Web. Besheer, Joyce et al. “Transient Increase in Alcohol Self-Administration Following a Period of Chronic Exposure to Corticosterone.” Neuropharmacology 0 (2013): 139-147. PMC. Web. Butler, Tracy R., A. N. Karkhanis, S. R. Jones, and J. L. Weiner. "Adolescent Social Isolation as a Model of Heightened Vulnerability to Comorbid Alcoholism and Anxiety Disorders." Alcoholism, Clinical and Experimental Research 40.6 (2016): n. pag. PubMed. Web. Butler, Tracy R., Olusegun J. Ariwodola, and Jeffrey L. Weiner. “The Impact of Social Isolation on HPA Axis Function, Anxiety-like Behaviors, and Ethanol Drinking.” Frontiers in Integrative Neuroscience 7 (2014): 102. PMC. Web. Chappell, A.M. et al. “Adolescent Rearing Conditions Influence the Relationship Between Initial Anxiety-Like Behavior and Ethanol Drinking In Male Long Evans Rats.” Alcoholism, Clinical and Experimental Research 37.Suppl 1 (2013): E394-E403. PMC. Web. Fulk, L. J. et al. "Chronic Physical Exercise Reduces Anxiety-Like Behavior in Rats." International Journal of Sports Medicine 25.1 (2004): 78-82. PubMed. Web. Hasin, Deborah et al. "Prevalence, Correlates, Disability, and Comorbidity of DSM-IV Alcohol Abuse and Dependence in the United States: Results from the National Epidemiologic Survey on Alcohol and Related Conditions." Archives of General Psychiatry 64.7 (2007): 830-42. National Center for Biotechnology Information. Web. Hedges, Dawson W., and Fu Lye Woon. “Early-life Stress and Cognitive Outcome.” Psychopharmacology 214.1 (2011): 121-130. PubMed. Web.

Proceedings 2016

SOCIAL SCIENCE Holmes, A., and R.J. Rodgers. “Influence of Spatial and Temporal Manipulations on the Anxiolytic Efficacy of Chlordiazepoxide in Mice Previously Exposed to the Elevated Plus-Maze.” Neuroscience and Biobehavioral Reviews 23 (1999): 971-980. ScienceDirect. Web. Karkhanis, Anushree N. et al. “Chronic Social Isolation During Adolescence Augments Catecholamine Response to Acute Ethanol in the Basolateral Amygdala.” Synapse (New York, N.Y.) 69.8 (2015): 385-395. PMC. Web. Ko, CY, and YP Liu. “Isolation Rearing Impaired Sensorimotor Gating but Increased Pro-Inflammatory Cytokines and Disrupted Metabolic Parameters in both Sexes of Rats.” Psychoneuroendocrinology 55 (2015): 173-183. PubMed. Web. Leasure, J. Leigh, and Kimberly Nixon. “Exercise Neuroprotection in a Rat Model of Binge Alcohol Consumption.” Alcoholism, Clinical and Experimental Research 34.3 (2010): 404-414. PMC. Web. Lopez, Marcelo F., and Kathy Laber. "Impact of Social Isolation and Enriched Environment duringAdolescence on Voluntary Ethanol Intake and Anxiety in C57BL/6J Mice." Physiology & Behavior 148 (2015): 151-56. PMC. Web. National Research Council. Guide for the Care and Use of Laboratory Animals: Eighth Edition. Washington, DC: The National Academies Press, 2011. PubMed. Web. Rabasa, Cristina, et al. "Adaptation Of The Pituitary-Adrenal Axis To Daily Repeated Forced Swim Exposure In Rats Is Dependent On The Temperature Of Water." Stress (Amsterdam, Netherlands) 16.6 (2013): 698-705. MEDLINE with Full Text. Web. Retana-Márquez, S et al. "Body Weight Gain and Diurnal Differences of Corticosterone Changes in Response to Acute and Chronic Stress in Rats." Psychoneuroendocrinology 28.2 (2003): 207-27. ScienceDirect. Web. Sasse, Sarah K. et al. “Chronic Voluntary Wheel Running Facilitates Corticosterone Response Habituation to Repeated Audiogenic Stress Exposure in Male Rats.” Stress (Amsterdam, Netherlands) 11.6 (2008): 425-437. PMC. Web. Solomon, Matia B., Aynara C. Wulsin, Taylor Rice, Dayna Wick, Brent Myers, Jessica McKlveen, Jonathan N. Flak, Yvonne Ulrich-Lai, and James P. Herman. "The Selective Glucocorticoid Receptor Antagonist CORT 108297 Decreases Neuroendocrine Stress Responses and Immobility in the Forced Swim Test." Hormones and Behavior 65.4 (2014): 363-71. ScienceDirect. Web. Wulsin, Aynara C., Dayna Wick-Carlson, Benjamin A. Packard, Rachel Morano, and James P. Herman. "Adolescent Chronic Stress Causes Hypothalamo-pituitary-adrenocortical Hypo-responsiveness and Depression-like Behavior in Adult Female Rats." Psychoneuroendocrinology 65 (2016): 109-17. ScienceDirect. Web. Zareian, Parvin, Mohammad Vahid Karimi, & Gita Dorneyani. "The Comparison of the Effects of Acute Swimming Stress on Plasma Corticosterone and Leptin Concentration in Male and Female Rats." Acta Medica Iranica [Online], 49.5 (2011): 284-287. PubMed. Web.

Proceedings 2016

SOCIAL SCIENCE

A Confirmatory Factor Analysis of Normative and Pathological Personality Traits in a University Student Sample Lisa E. Stone1,2,3 University of Dayton, 300 College Park, Dayton, OH, 45469 1. Department of Psychology 2. Berry Summer Thesis Institute 3. University Honors Program

Thesis Mentor: Julie Walsh-Messinger, Ph.D. Department of Psychology Corresponding Author: Lisa E. Stone Email: stonel2@udayton.edu

Abstract Personality disorders are characterized by persistent maladaptive ways of thinking about and acting in the world. Historically, personality disorders have been conceptualized as qualitatively distinct clinical syndromes based on operational criteria, and the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5; American Psychiatric Association, 2013) outlines ten distinct personality disorders. However, debate persists about whether it is more clinically useful to categorize personality disorders this way or to use a dimensional model that instead focuses on pathological levels of normative personality traits. A recent exploratory factor analysis of the NEO Personality Inventory-3 (NEO-PI-3; Costa & McCrae, 2010) and The Personality Inventory for DSM-5 (PID-5; Krueger, Derringer, Markon, Watson, & Skodol, 2012), two personality inventories designed to measure normative and pathological personality traits, respectively, indicate both normative and pathological personality traits may fall under the same common set of domains: neuroticismnegative affectivity, extraversion-detachment, openness-psychoticism, agreeableness-antagonism and conscientiousness-disinhibition (De Fruyt et al., 2013). However, a confirmatory factor analysis of this model has yet to be conducted. The purpose of this thesis is to further explore the relationship between normative and pathological personality traits and to test the De Fruyt, et al., model with a confirmatory factor analysis. It is hypothesized that pathological personality traits, measured by the PID-5, share the same underlying factor structure as normative traits, measured by the NEO-PI-3. A total of 200 undergraduate students at a private Midwestern university will participate in this study by completing both the PID-5 and the NEO-PI-3. Following completion of data collection, the psychometric properties of these measures will be examined and a conjoint confirmatory factor analysis will be conducted using mPlus software. Understanding the relationship between these two measures is important as they are consistently used to diagnose and aid in treatment of individuals with personality disorders.

Introduction Personality disorders (PD) are a type of psychiatric disorder characterized by consistent maladaptive ways of behaving, thinking and experiencing the world. Approximately nine percent of the population are diagnosed with a personality disorder (Lenzenweger, Lane, Loranger, & Kessler, 2007). According to the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5; American Psychiatric Association, 2013), there are currently 10 PDs: paranoid, schizoid, schizotypal, antisocial, borderline, histrionic, narcissistic, avoidant, dependent and obsessive-compulsive. 28

Proceedings 2016

SOCIAL SCIENCE Common symptoms include an aversion to relationships, perceptual distortions, paranoia, manipulation of others and extreme emotional instability. Individuals diagnosed with a PD often have severe difficulties with properly functioning in life, such as in a work-place environment or maintaining healthy social relationships. If the PD is left untreated, these individuals have distressing lives and are often not able to operate as healthy adults. The current PD model is ineffective, causing diagnosis and treatment to become arduous and problematic. Therefore, continued research is needed in order to fully understand PDs, more clearly diagnose them and develop more effective treatment approaches.

History of Personality Research The study of personality in its relation to psychology has been occurring since the mid to late 1800s. Empirical research, however, began to accelerate in the 1930s through the present, with a large influx in the 1980s and 2000s (Wiggins & Trapnell, 1997). Early on, research focused on the whole person, their motivations and the individual differences between people (McAdams, 1997). In the 1930s, Thurstone (1934) conducted a study in which raters were asked to analyze a list of 60 trait adjectives and choose which words they would use to describe a close friend. He ultimately found that there were five common factors that accounted for all of the correlations between words. This is possibly the earliest suggestion of the Big Five or Five Factor Model (FFM; Wiggins & Trapnell). Cattell (1943) conducted a peer-rating study that allowed him to condense 4,500 adjectives to only 171 synonym groups. Thirty-five of these word groups were determined to be a part of the normal personality domain. He ultimately found 12 factors underlying the 35 word groups, suggesting that personality traits could be grouped into 12 larger, common domains. Fiske (1949) collected data from veterans using a bipolar rating scale that used Cattell’s 35 word groups. Each participant was assessed by a self-rating, a teammate’s rating and a staff member’s rating. This allowed Fiske to analyze the consistency of factor structures among the different ratings from various sources. He found that a 5-factor solution best fit the data, not a 12-factor solution, as suggested by Cattell. To advocate the FFM even further, Tupes and Christal (1958) examined the factor structure of Fiske’s bipolar rating scale. They also identified a clear 5-factor solution and classified them as Surgency/ Extraversion, Agreeableness, Conscientiousness, Emotional Stability and Culture. Later, Tupes and Christal (1961) applied their 5-factor solution to eight diverse sample populations and found that the model was stable across all samples.

Refining the Five Factor Model Although some have argued against the comprehensiveness of the FFM (Cattell, 1973), it has been almost universally accepted as the most extensive model for normative personality (Wiggins & Trapnell, 1997). In the 1970s, Costa and McCrae investigated the possibility of a 3-factor solution for normative personality but discovered an issue with having only three domains. They proposed the NEO model, a 3-factor model, that only accounted for Neuroticism, Extraversion and Openness (Costa & McCrae, 1976). The researchers decided to expand the original 3-factor model after conducting correlational analysis between the 3-factor model and the Big Five markers set by Goldberg (1983). They found that the Neuroticism, Extraversion and Openness domains converged on the corresponding markers set by Goldberg but did not correlate with the remaining two domains, thus causing them to believe that there were additional personality traits that existed that were not being measured by only three factors (Costa & McCrae, 1985). Subsequently, they developed two more domains: Agreeableness and Conscientiousness and conceived The NEO Personality Inventory (NEO-PI; Costa & McCrae) to comprehensively measure their theoretical FFM. The NEO-PI and successive revisions of it will be further explored later in this paper.

Personality Pathology When certain personality traits lead to difficulty in cognition, affectivity, interpersonal function and impulse control, personality pathology develops and negatively affects a person’s ability to adapt Proceedings 2016

SOCIAL SCIENCE and function in life. The study of personality pathology has existed almost as long as the study of personality itself. Originally, maladaptive personality traits were simply thought of as moral deficiencies and not a psychological issue (Morey, 1997). Towards the conclusion of the 19th century, Koch (1891) used the term “psychopathic” to describe a person’s personality and used it to reflect antisocial or criminal behavior. His research led others to study and treat abnormal personality as a serious disorder and not a failure of morals. Kurt Schneider (1958) was one of the first researchers to identify that maladaptive personality traits could lead to a diagnosable disorder. Schneider’s term “psychopathic personality” is what is now known as a personality disorder. He conceptualized 10 psychopathic personality types, which were the first widely accepted proposals for personality pathology. Schneider’s 10 types have been found to significantly correlate to the DSM’s current PDs (Standage, 1986).

Categorical Model vs. Dimensional Model The Diagnostic and Statistical Manual of Mental Disorders, 3rd Edition (DSM-III; American Psychiatric Association, 1980) aimed to improve the process of psychiatric diagnosis to make it much more reliable and valid. Subsequently, a system was created in which a person must meet certain criteria for a particular disorder in order to receive a diagnosis. This system was also carried over to the DSM-IV (American Psychiatric Association, 1994) and became known as the categorical model. In this model, for example, borderline personality disorder has nine diagnostic features, and at least five of the features must be displayed by the patient in order to receive a borderline personality disorder diagnosis (DSM-IV). This system allows for the natural diversity among individuals, but it also causes issues in regards to comorbidity, consistency and the development of adequate instrumentation. One of the major issues with the categorical model is comorbidity, which essentially means that most patients do not fit nicely into one exact diagnosis and instead often display the criteria of many PDs (Krueger, 2013). Gunderson (1996) indicated that the average number of PD diagnoses per patient has ranged from 2.8 to 4.6. This means that on average, if a person is diagnosed with one PD, they are also likely to be diagnosed with another two to five disorders (Krueger, Hopwood, Wright, & Markon, 2014). Comorbidity causes a great issue when a clinician attempts to treat the individual, due to the fact that his or her diagnoses is so complex. Additionally, heterogeneity is a large issue with the categorical model. Although there is natural diversity among individuals, heterogeneity occurs when patients within the same diagnosis significantly differ from each other. This is often because they do not have one diagnoses and instead have three or four, so naturally, they will look unique and display contrasting behavior, despite sharing the same categorical diagnosis (Krueger et al.). It is immensely difficult to treat individuals with borderline PD, for example, when each person with the disorder is completely different from one another. This problem also occurs with the other nine PDs. These issues have led to further difficulties in developing an adequate instrument to assess PDs, which makes it that much harder for clinicians to efficiently diagnose. In theory, the main advantage of the categorical model is that it can provide a simple and efficient way for clinicians to communicate with each other about patient symptoms and diagnoses. However, this approach to clinical communication often does not provide complete or accurate information due to heterogeneity and comorbidity (Krueger et al.). Recent research has shown that the categorical model of PDs is fundamentally broken and an alternative model is becoming increasingly necessary. The DSM-5 (American Psychiatric Association, 2013) included two different models of PDs. In Section II, the traditional categorical model was reported as the official model. In Section III, however, an alternative model was proposed and is known as the dimensional model. The DSM-5 called for additional research on the dimensional model in order for it to become more accepted and empirically endorsed in the field so that it may be considered as a possible future replacement for the outdated categorical model. In this new model, PDs are abnormal or maladaptive extensions of normative traits. For example, the abnormal trait of “detachment” is just an extreme extension of the normal trait “introversion.” In this system, a patient is no longer simply given a diagnosis, category or label. Instead, they are described as ranging somewhere on a set of trait continuums, 30

Proceedings 2016

SOCIAL SCIENCE which will be explored later in this paper. Although this can decrease the simplicity of diagnosis, the model has quite a few advantages. The model has been shown to have more reliable scores, both across time and across scorers (Heumann & Morey, 1990). It also helps to clarify the lack of boundaries between the categorical diagnoses. There is an extreme amount of overlap between the criteria for PDs and the dimensional model allows and accounts for this. Additionally, since the abnormal traits would be put on the same scale as normal traits, the dimensional model links PDs to the already immense literature on normal personality. Since the publication of the DSM-5, an impressive amount of research has been done on the dimensional model and almost all of the studies have shown not only its validity but also its clinical utility (De Fruyt et al., 2013; Griffin & Samuel, 2014; Suzuki, Samuel, Pahlen, & Krueger, 2015; Thomas et al., 2012; Wright et al., 2012; Wright & Simms, 2014). More research is needed, however, in order for it to become the dominant model and this study aims to add to that body of evidence.

How to Measure Personality There are a number of different ways psychologists assess personality, and personality measures, or questionnaires, are one of the most common methods. In a self-report questionnaire, the participant fills out the questionnaire by himself, while in a clinician-reported inventory the clinician fills out the questionnaire on behalf of the patient. Personality measures can tell the psychologist a great deal about the individual, such as how the person will behave in social situations, how he is feeling and how he thinks he interacts with others. In psychology, it can be difficult to operationalize and define variables because psychologists often work with variables that are difficult to quantify, such as human emotion and thought. Therefore, it is essential to have measures that actually assess what they claim to do. The quality of a questionnaire can be determined through multiple types of statistical analyses, with reliability and validity being the largest indicators of a measureâ&#x20AC;&#x2122;s capabilities. Reliability is consistency and high reliability means that the same person will get a similar score every time they take the measure. It is most commonly measured through a statistic called Cronbachâ&#x20AC;&#x2122;s Îą, which ranges from zero to one. A higher score indicates that the measure is reliable. Validity is the extent to which the questionnaire actually measures what it claims to be doing. It is assessed through various methods, such as criterion-related validity, content-related validity and construct validity. Criterion-related validity is the degree to which a measure is related to another outcome, such as a questionnaire that assesses similar qualities. If the new measure is similar to previous ones, it likely measures the intended variables. Content-related validity is how well the questionnaire measures the attributes that it intends to measure. Construct validity is a type of content-related validity and essentially asks if the measure relates to the underlying theoretical concepts behind the measure. All of these aspects of validity are calculated for each measure in order to ensure that the questionnaire is credible and legitimate. Psychometrics is the field of study concerned with the theory and technique of psychological measurement. It essentially studies how psychologists measure and study psychological constructs, such as personality traits. Factor analyses are common methods of psychometrics, including exploratory factor analysis (EFA) and confirmatory factor analysis (CFA). The purpose of an EFA is to discover the underlying structure or relationship within a large set of variables. This is done with the use of statistical software that will produce a numerical score for each variable, called its loading. A higher loading indicates that the variable belongs to that factor, and the researcher is essentially trying to find out how many overarching factors fit the large set of variables. A CFA is used to verify and confirm a hypothetical structure within a set of variables. Typically, a model is created by a researcher based on the results of a previous EFA or factor analysis and then runs new data through the hypothetical model using statistical software. The software will produce a numerical score for each variable, with a larger number indicating a strong relationship between the variable and proposed factor. Factor analyses are typical methods among personality assessment, since it can tell psychologists how personality traits, or facets, can be group together to form a larger domain or how domains are related to one another.

Proceedings 2016

SOCIAL SCIENCE Neo-pi In the mid-1980s, Costa and McCrae (1985, 1989, 1992) developed The NEO Personality Inventory (NEO-PI), which was designed to measure the FFM, or normative personality traits. Costa and McCrae found convergence, or overlap, between their NEO-PI and seven other commonly used personality assessments, including the Myers-Briggs Type Indicator, Personality Research Form, Eysenck Personality Test, Minnesota Multiphasic Personality Inventory, Institute of Personality Assessment and Research, California Q-Set, Interpersonal Adjective Scales, and Self-Directed Search. The fact that they found convergence across a wide variety of personality scales supports that the validity of the NEO-PI and suggests that it may be the most comprehensive model of personality to date (Wiggins & Trapnell, 1997). In the 1990s, Costa and McCrae released a revised version of the NEO-PI (NEO-PI-R; Costa & McCrae, 1992), which added six facets for each of the five main domains. These additions made the questionnaire much more detailed and exhaustive. In 2010, Costa and McCrae released the NEO Personality Inventory-3 (NEO-PI-3), an updated version of the NEO-PI-R that was the first rendition to include normative data for adolescents. The NEO-PI-3 contains 240 questions, with Cronbach’s α for the domains ranging from .87 to .95, suggesting that it has high reliability (Costa, McCrae, & Martin, 2010). However, a few facets obtained relatively low Cronbach’s α scores, meaning a value of .70 or lower: Impulsiveness, Activity, Excitement Seeking, Actions, Values, Straightforwardness, Compliance, Tender-Mindedness and Dutifulness. This suggests that these facets have poor reliability, and additional research on them is needed. Countless studies have been conducted on Table 1. Domains and Facets within the PID-5 and NEO-PI-3.

Proceedings 2016

SOCIAL SCIENCE the validity and reliability of the NEO-PI-R and the NEO-PI-3, and research supports its test-retest reliability, criterion validity and construct validity (DeFruyt, DeBolle, McCrae, Terracciano, & Costa, 2009; McCrae, Costa, & Martin, 2005; McCrae, Kurtz, Yamagata, & Terracciano, 2010). The domains and facets measured by the NEO-PI-3 are provided in Table 1.

Pid-5 Before a new edition of the DSM is constructed and written, the APA assembles a workgroup for each diagnostic section, gathering experts from that specific branch of psychiatry and psychology. Personality, for example, had its own dedicated workgroup. Along with revising the Personality Disorders section for the DSM-5, the personality workgroup developed the Personality Inventory for DSM-5 (PID-5; Krueger, Derringer, Markon, Watson, & Skodol, 2012). Their main aim in developing this new personality measure was to construct an empirical trait model for the DSM-5 and to provide a corresponding assessment to reflect this model (Krueger et al.). The PID-5 measures trait attributes that are typically displayed by individuals with PDs. Workgroup members and their colleagues created a preliminary list of 37 facets, or specific personality traits (i.e. anxiousness, emotional lability). They conducted two rounds of data collection and analyses in order to test the reliability of their measure and also to determine if the 37 facets could be condensed into a smaller number of groups. In the first round, the researchers conducted an EFA and used item response theory to determine that they were able to accurately measure most of the facets reliably. However, six facets were not deemed reliable: Submissiveness, Separation Insecurity, Attention Seeking, Oppositionality, Perseveration and Rigidity. In the second round of analyses, additional questions for each of the unreliable facets were added in order to improve accuracy. The results indicated that the original 37 facets could be collapsed into a more concise 25 facets (Krueger et al., 2012). Factor analysis revealed that a 5-factor solution best fit the data, meaning the 25 facets belong to five overarching domains. The researchers named these five domains Negative Affect, Detachment, Antagonism, Disinhibition and Psychoticism. Ultimately, the final version of the PID-5 contains 220 questions, measures 25 facets and five domains, and has a strong Cronbachâ&#x20AC;&#x2122;s Îą ranging from .72 to .96. The domains and facets measured by the PID-5 are provided in Table 1. The PID-5 was released relatively recently, in 2012, so studies are still being conducted to assess its validity, but early studies suggest that its validity is high. In undergraduate and inpatient samples, Zimmermann et al., (2014) found a positive correlation between PID-5 traits and the Inventory of Personality Organization - 16 Item Version, a self-report measure of personality dysfunction. The authors also found a positive correlation between the DSM-5 model, as measured by the PID-5, and general PD severity, as measured by the Patient Health Questionnaire. These results suggest that the PID-5 strongly relates to previous measures of PD, implying that it has strong criterion validity. Samuel, Hopwood, Krueger, Thomas and Ruggero (2013) conducted a study to determine if the DSM-5 dimensional model was able to reproduce the DSM-IV PD constructs. Using a sample of undergraduate students, the researchers utilized the PID-5 to measure the DSM-5 model and the Personality Diagnostic Questionnaire-Version 4 (PDQ-4) to measure the DSM-IV constructs. Correlational analysis revealed that the PID-5 was able to dependably account for the variance within the DSM-IV PDs, meaning that it was able to describe the DSM-IV constructs (Samuel et al.). This supports the criterion validity of the PID-5 because it shows that the new measure relates to and accounts for a previous one. Similarly, Fossati, Krueger, Markon, Borroni and Maffei (2013) also found correlations between the DSM-5 dimensional model, again measured by the PID-5, and DSM-IV PD constructs, measured by an Italian translation of the PDQ-4. Additionally, the researchers found that the DSM-5 model may capture personality characteristics that were not previously covered in the DSM-IV, such as psychopathy, thus increasing its clinical utility. Other studies have added to the idea that the PID-5 has strong criterion validity by comparing it to previous measures of personality pathology (Few et al., 2013; Wright et al., 2014).

Proceedings 2016

SOCIAL SCIENCE Psychometric Support for the Dimensional Model Construct validity is a type of content-related validity, and it essentially asks if the measure relates to the underlying theoretical concepts. Theoretically, the PID-5 and the FFM have the same underlying construct because both are based off of a 5-factor structure and, according to the dimensional model, the PID-5 just measures extreme extensions of the FFM. Each domain covered by the PID-5 is expected to match with a domain measured by the NEO-PI-3. Studies that support this idea increase the construct validity of the PID-5 because the underlying theoretical constructs are being reinforced. Griffin and Samuel (2014) studied the lower-order structure of the PID-5 and the NEO-PI-R. Using an undergraduate sample, they conducted a joint EFA of the 25 facets of the PID-5 and the 30 facets of the NEO-PI-R. They investigated both a 5-factor and a 6-factor solution but ultimately determined that the 5-factor solution was much simpler to interpret and it was able to strongly reproduce the FFM structure. They also found that four facets did not load as predicted: Hostility, Depressivity, Restricted Affectivity and Submissiveness. This suggests that more research is needed to fully support the dimensional model. Overall, however, 51 facets loaded as predicted, indicating that the PID-5 can be viewed as an adequate measure of the FFM. Furthermore, using a clinical sample, Wright and Simms (2014) performed an EFA using the PID-5, the NEO Personality Inventory-3 First Half and the Computerized Adaptive Test of Personality Disorder, which measures PDs. The EFA revealed that the 5-factor solution best fit the data, suggesting that the PID-5 reflects the same structure as the FFM. Their study indicated that there is a strong convergence between the PID-5 and the first half of the NEO-PI-3 (Wright & Simms). Suzuki, Samuel, Pahlen and Krueger (2015) used item-response theory to compare pathological traits as measured by the PID-5, and normative FFM traits assessed with the International Personality Items Pool-NEO PI-R (IPIP-NEO). They found that four of the five domains overlapped, with the exception of the openness-psychoticism domain, which is somewhat consistent with previous research. In general, however, the PID-5 had an advantage at the upper (abnormal) levels, while the IPIP-NEO had advantages at the lower (normative) levels of the traits, which makes sense considering what each measure was designed to capture (Suzuki et al.). Overall, these results suggest that the PID-5 and the IPIP-NEO can measure both pathological and normative domains. These results enhance the construct validity of the PID-5 as well as the validity of the dimensional model. A conjoint EFA of the PID-5 and the NEO-PI-3 would greatly support the dimensional model due to the fact that it would create a direct connection between the domains and facets of each measure; it would match together the five domains from the two measures. De Fruyt et al., 2013, analyzed the dimensional model by running a series of EFAs. The investigators first ran two EFAs on the PID-5 and the NEO-PI-3 separately in order to confirm their 5-factor structure. Then they conducted a joint exploratory factor analysis of both measures in order to determine the extent to which they share a common structure. De Fruyt and his colleagues examined the measures at the facet level and were ultimately able to find significant loadings on every facet. They found that a 5-factor solution best fit the data and suggested that the FFM is able to describe both adaptive and maladaptive personality (De Fruyt et al., 2013). The authors proposed the following joint domains: neuroticismnegative affectivity, extraversion-detachment, agreeableness-antagonism, openness-psychoticism and conscientiousness-disinhibition. Essentially, the investigators linked the domains from the NEO-PI-3 to the domains of the PID-5, thus suggesting that they lie on the same continuum, which supports the dimensional model. The current study will conduct a confirmatory factor analysis using the hypothetical joint model reported by De Fruyt et al., 2013. A conjoint CFA of the full version of the NEO-PI-3 and the PID-5 has yet to be conducted and the current study seeks to perform it. The results of De Fruyt et al.â&#x20AC;&#x2122;s study were encouraging, as significant loadings emerged for every facet on the intended theoretical domain. It is hypothesized that a CFA would produce similar results and would further strengthen the joint relationship between the two measuresâ&#x20AC;&#x2122; domains. If this hypothesis is supported, research advocating for the dimensional model would be enhanced and the validity of the PID-5 would be reinforced because it is supporting the idea that both normative and pathological personality traits lie on the same domain.

Proceedings 2016

SOCIAL SCIENCE Proposed Methods For the present study, participants will complete the NEO-PI-3, the PID-5 and a short demographic questionnaire. The order in which they complete the measures will be counterbalanced so that administration order can be eliminated as a confounding variable. Participants will be undergraduate students enrolled in psychology courses that require psychology research credit and will be recruited online using the University of Dayton's Experiment Management System (SONA). Additional participants will be recruited through word-of-mouth and voluntary participation. Students not participating in the study for research credit will be eligible to enter a raffle for a drawing of a $40 Target gift card. The present study aims to collect data from at least 200 participants; so far, 56 participants have completed the study and it is anticipated that data collection will be completed in approximately 12 to 18 months. Preliminary models for the CFA of the two measures will be created in Mplus based on De Fruyt et al.â&#x20AC;&#x2122;s (2013) EFA 5-factor model. The 25 facets that loaded significantly on more than one factor will be included in both domains for a total of 80 variables, or facets, with five proposed factors representing the joint NEO-PI-3 and PID-5 domains as previously established by De Fruyt et al.â&#x20AC;&#x2122;s model (Figures 1-6). Subsequent analyses will also test whether a model that includes each facet on only its highest loading domain better fits the data.

Figure 1. Preliminary confirmatory factor analysis model for NEO-PI-3 and PID-5. This figures illustrates the proposed model. Blue represents facets from the NEO-PI-3 and red represents facets from the PID-5. See Figures 2-6 for detailed models of each joint domain. Chart courtesy of Lisa Stone, 2016.

Proceedings 2016

SOCIAL SCIENCE

Figure 2. Preliminary confirmatory factor analysis model for NEO-PI-3 and PID-5. This figures illustrates the proposed model for the NeuroticismNegative Affectivity domain. Blue represents facets from the NEO-PI-3 and red represents facets from the PID-5. * represents facets that double loaded and do not normally belong on this domain. Chart courtesy of Lisa Stone, 2016.

Figure 3. Preliminary confirmatory factor analysis model for NEO-PI-3 and PID-5. This figures illustrates the proposed model for the Extraversion-Detachment domain. Blue represents facets from the NEO-PI-3, and red represents facets from the PID-5. * represents facets that double loaded and do not normally belong on this domain. Chart courtesy of Lisa Stone, 2016.

Proceedings 2016

SOCIAL SCIENCE

Figure 4. Preliminary confirmatory factor analysis model for NEO-PI-3 and PID-5. This figures illustrates the proposed model for the Openness-Psychoticism domain. Blue represents facets from the NEO-PI-3, and red represents facets from the PID-5. * represents facets that double loaded and do not normally belong on this domain. Chart courtesy of Lisa Stone, 2016.

Figure 5. Preliminary confirmatory factor analysis model for NEO-PI-3 and PID-5. This figures illustrates the proposed model for the Agreeableness-Antagonism domain. Blue represents facets from the NEO-PI-3, and red represents facets from the PID-5. * represents facets that double loaded and do not normally belong on this domain. Chart courtesy of Lisa Stone, 2016.

Figure 6. Preliminary confirmatory factor analysis model for NEO-PI-3 and PID-5. This figures illustrates the proposed model for the Conscientiousness-Disinhibition domain. Blue represents facets from the NEO-PI-3, and red represents facets from the PID-5. * represents facets that double loaded and do not normally belong on this domain. Chart courtesy of Lisa Stone, 2016.

Proceedings 2016

SOCIAL SCIENCE Conclusion Personality and PDs are an important focus of research, as approximately nine percent of the population suffer from at least one PD (Lenzenweger, Lane, Loranger, & Kessler 2007). Those who endure PDs are often not able to successfully function as an adult, both personally and professionally. For example, they often are not capable of maintaining a steady job, making them a financial burden on society. Since they are not able to afford it, the cost of the treatment for their disorder is often the responsibility of loved ones or the government. Additionally, relationships with family and friends is strained for these individuals because they are not mentally capable of successfully and healthily maintaining them. The categorical model, the current way practicing clinicians diagnose PDs, is fundamentally broken, and the need for an alternative model is becoming increasingly necessary. The evidence for the dimensional model, as proposed in Section III of the DSM-5, is accumulating and extremely promising, as research heavily supports the reliability and validity of the measure designed to capture the features of the model: the PID-5. The current thesis will add to this growing body of empirical evidence by conducting a CFA of De Fruyt et al.â&#x20AC;&#x2122;s (2013) model of the NEO-PI-3 and the PID-5. Confirmation of this model would further support the dimensional model in which both normative and pathological personality traits lie on the same domain. This is important as it would help to streamline and clarify the diagnosis of PDs and subsequently improve treatment efficacy. Ultimately, the goal of PD research is to effectively identify maladaptive personality traits and improve interventions to better the lives of those suffering from PDs. Adopting a new and updated model would allow clinicians and researchers to properly aid individuals who experience these issues.

Acknowledgements I would like to thank my amazing mentor and advisor Dr. Julie Walsh-Messinger for her seemingly endless guidance and support. I would also like to thank the Psychopathology, Personality and Affective Science Lab for their assistance with data collection. I express gratitude toward the Berry Family and the Berry Family Foundation for making this experience possible. I thank the entire University of Dayton Honors Program staff for their help this summer.

References American Psychiatric Association (1980). Diagnostic and Statistical Manual of Mental Disorders (3rd ed.). Washington, D.C.: American Psychiatric Association. American Psychiatric Association (1994). Diagnostic and Statistical Manual of Mental Disorders (4th ed.). Washington, D.C.: American Psychiatric Association. American Psychiatric Association (2013). Diagnostic and Statistical Manual of Mental Disorders (5th ed.). Washington D.C.: American Psychiatric Association. Cattell, R.B. (1943). The description of personality. II. Basic traits resolved into clusters. Journal of Abnormal and Social Psychology, 38, 476-507. Cattell, R.B. (1973). Personality and Mood by Questionnaire. San Francisco: Jossey-Bass. Costa, P.T. & McCrae, R.R. (1976). Age differences in personality structure: A cluster analytic approach. Journal of Gerontology, 31(5), 564-570. Costa, P.T. & McCrae, R.R. (1985). The NEO Personality Inventory Manual. Odessa, FL: Psychological Assessment Resources. Costa, P.T. & McCrae, R.R. (1989). The NEO-PI/NEO-FFI Manual Supplement. Odessa, FL: Psychological Assessment Resources. Costa, P.T. & McCrae, R.R. (1992). Revised NEO Personality Inventory (NEO PI-R) and NEO Five-Factor Inventory (NEO-FFI) Professional Manual. Odessa, FL: Psychological Assessment Resources. Costa, P. T., & McCrae, R. R. (2010). NEO Personality Inventory-3. Lutz, FL: PAR, Inc. De Fruyt, F., De Bolle, M., McCrae, R.R., Terracciano, A., & Costa, P.T. (2009). Assessing the universal structure of personality in early adolescence: The NEO-PI-R and NEO-PI-3 in 24 cultures. Assessment, 16(3), 301-311. De Fruyt, F., De Clercq, B., De Bolle, M., Wille, B., Markon, K., & Krueger, R.F. (2013). General and maladaptive traits in a five-factor framework for DSM-5 in a university student sample. Assessment, 20(3), 295-307.

Proceedings 2016

SOCIAL SCIENCE Few, L.R., Miller, J.D., Rothbaum, A.O., Meller, S., Maples, J., Terry, D.P., Collins, B., & MacKillop, J. (2013). Examination of section III DSM-5 diagnostic system for personality disorders in an outpatient clinical sample. Journal of Abnormal Psychology, 122(4), 1057-1069. Fiske, D.W. (1949). Consistency of the factorial structures of personality ratings from different sources. Journal of Abnormal and Social Psychology, 44, 329-344. Fossati, A., Krueger, R.F., Markon, K.E., Borroni, S., & Maffei, C. (2013). Reliability and validity of the Personality Inventory for DSM-5 (PID-5): Predicting DSM-IV personality disorders and psychopathy in community-dwelling Italian adults. Assessment, 20(6), 689-708. Griffin, S.A., & Samuel, D.B. (2014). A closer look at the lower-order structure of the Personality Inventory for DSM-5; Comparison with the five-factor model. Personality Disorders: Theory, Research, and Treatment, 5(4), 406-412. Goldberg, L.R. (1983, June). The magic number five, plus or minus two: Some conjectures on the dimensionality of personality descriptors. Paper presented at a research seminar, Gerontology Research Center, Baltimore. Gunderson, J. G. (1996). Introduction to section IV: Personality disorders. In T.A. Widiger, A.J. Frances, H. A. Pincus, et al. (Eds.), DSM-IV Sourcebook (Vol. 2, pp. 647-664). Washington, D.C.: American Psychiatric Association. Heumann, K.A., & Morey, L.C. (1990). Reliability of categorical and dimensional judgments of personality disorder. American Journal of Psychiatry, 147, 498-500. Koch, J.L. (1891). Die Psychopathischen Minderwertigkeiten. Ravensburg: Maier. Krueger, R.F. (2013). Personality disorders are the vanguard of the post-DSM-5.0 era. Personality Disorders, 4(4), 355-362. Krueger, R. F., Derringer, J., Markon, K. E., Watson, D., & Skodol, A. E. (2012). Initial construction of a maladaptive personality trait model and inventory for DSM-5. Psychological Medicine, 42(9), 1879-1890. Krueger, R.F., Hopwood, C.J., Wright, A.G.C., & Markon, K.E. (2014). DSM-5 and the path toward empirically based and clinically useful conceptualization of personality and psychopathology. Clinical Psychology: Science & Practice, 21(3), 245-261. Lenzenweger, M.F., Lane, M.C., Loranger, A.W., & Kessler, R.C. (2007). DSM-IV personality disorders in the national comorbidity survey replication. Biological Psychiatry, 62(6) 553-564. McAdams, D.P. (1997). A conceptual history of personality psychology. In R.H. Hogan, J. Johnson, & S. Briggs (Eds.), Handbook of Personality Psychology (pp. 3-39). San Diego: Academic Press. McCrae, R.R., Costa, P.T., & Martin, T.A. (2005). The NEO-PI-3: A more readable revised NEO personality inventory. Journal of Personality Assessment, 84(3), 261-270. McCrae, R.R., Kurtz, J.E., Yamagata, S., & Terracciano, A. (2010). Internal consistency, retest reliability, and their implications for personality scale validity. Personality and Social Psychology Review, 15(1), 28-50. Morey, L.C. (1997). Personality Diagnosis and Personality Disorders. In R.H. Hogan, J. Johnson, & S. Briggs (Eds.), Handbook of Personality Psychology (pp. 919-946). San Diego: Academic Press. Samuel, D.B., Hopwood, C.J., Krueger, R.F., Thomas, K.M., & Ruggero, C.J. (2013). Comparing methods for scoring personality disorder types using maladaptive traits in DSM-5. Assessment, 20(3), 353-361. Schneider, K. (1958). Psychopathic Personalities. London: Cassell. (Original work published 1923). Standage, K. (1986). A clinical and psychometric investigation comparing Schneiderâ&#x20AC;&#x2122;s and the DSM-III typologies of personality disorders. Psychiatry, 36, 815-817. Suzuki, R., Samuel, D.B., Pahlen, S., & Krueger, R.F. (2015). DSM-5 alternative personality disorder model traits as maladaptive extreme variants of the Five-Factor Model: An item-response theory analysis. Journal of Abnormal Psychology, 124(2), 343-354. Thomas, K.M., Yalch, M.M., Krueger, R.F., Wright, G.C., Markon, K.E., Hopwood, C.J. (2012). The convergent structure of DSM-5 personality trait facets and Five-Factor Model trait domains. Assessment, 20(10), 1-4. Thurstone, L.L. (1934). The vectors of mind. Psychological Review, 41, 1-32. Tupes, E.C. & Christal, R.E. (1958). Stability of personality trait ratings factors obtained under diverse conditions USAF WADC Technical Note, No. 58-61. Tupes, E.C. & Christal, R.E. (1961). Recurrent personality factors based on trait ratings. USAF ASD Technical Report, No. 61-97. Wiggins, J.S. & Trapnell, P.D. (1997). Personality structure: The return to the Big Five. In R.H. Hogan, J. Johnson, & S. Briggs (Eds.), Handbook of Personality Psychology (pp. 737-765). San Diego: Academic Press. Wright, G.C., Calabrese, W.R., Rudick, M.M., Yam, W.H., Zelazny, K., Williams, T.F., Rotterman, J.H., Simms, L.J. (2014). Stability of the DSM-5 section III pathological personality traits and their longitudinal associations with psychosocial functioning in personality disordered individuals. Journal of Abnormal Psychology, 124(1), 199-207. Wright, G.C. & Simms, L.J. (2014). On the structure of personality disorder traits: Conjoint analyses of the CAT-PD, PID-5, and NEO-PI-3 trait models. Personality Disorders: Theory, Research, and Treatment, 5(1), 43-54. Wright, G.C., Thomas, K.M., Hopwood, C.J., Markon, K.E., Pincus, A.L., & Krueger, R.F. (2012). The hierarchical structure of DSM-5 pathological personality traits. Journal of Abnormal Psychology, 121(4), 951-957. Zimmermann, J., Altenstein, D., Krieger, T., Holtforth, M.G., Pretsch, J., Alexopoulos, J., Spitzer, C., Benecke, C., Krueger, R.F., Markon, K.E., & Leising, D. (2014). The structure and correlates of self-reported DSM-5 maladaptive personality traits: finding from two German-speaking samples. Journal of Personality Disorders, 28(4), 518-540.

Proceedings 2016

NATURAL SCIENCES

The Impact of the 515nm Effect on Singlet Oxygen Quenching in Photosynthesis: Model System Studies Using β-Carotene-Acid Complexes Lauren A. Hoody1,2,3 and Mark Masthay1 University of Dayton, 300 College Park, Dayton, OH, 45469 1. Department of Chemistry 2. Berry Summer Thesis Institute 3. University Honors Program

Thesis Mentor: Mark Masthay, Ph.D. Department of Chemistry Corresponding Author: Mark Masthay, Ph.D. Email: mmasthay1@udayton.edu

Abstract β-carotene (βC) is an orange pigment present in the photosynthetic reaction center (PRC) of green plants, where it plays a vital role in photosynthesis: it quenches singlet oxygen (1O2), a toxic oxidizing species generated during photosynthesis) before the 1O2 damages chlorophyll and other components of the PRCs. During photosynthesis, βC temporarily converts from its native orange-450 state to a pink-515 state via the so-called 515nm Effect. Because of the differences between the electronic structures of orange-450 and pink-515, I hypothesize that pink-515 will quench 1O2 less efficiently than orange-450. This hypothesis has not been tested to date because orange-450 and pink-515 states are both inherently present during photosynthesis, making deconvolution of their relative 1O2-quenching efficiencies effectively impossible. In this work, I detail (1) the properties of βC and its role in photosynthesis, (2) the 515nm Effect, and (3) the properties of βC-Acid Complexes (βC-ACs, which are structurally similar to the pink-515 species and formed upon reaction of βC with acids). In addition, I detail (4) my attempts to model the pink-515 state using blue βC-acid complexes in which the native orange-450 state has been converted to a blue-900 state with trifluoroacetic acid (TFA). Lastly, I detail (5) future plans to obtain the 1O2-quenching efficiencies 40

of the orange-450 and blue-900 states that will be characterized using a state-of-the-art NIRQuest spectrophotometer which monitors the intensity of the 1,270nm near infrared (NIR) emission of 1O2.

Introduction β-carotene (C40H56, Figure 1; βC hereafter) is a naturally-occurring biological pigment responsible for the orange color of “yellow” fruits and vegetables such as carrots, squash, and oranges. [1] It is also present in the photosynthetic reaction centers (PRCs) of green plants where it plays an essential photoprotective role: βC rapidly “quenches” singlet oxygen (1O2, a toxic oxidizing species which is generated as a by-product of photosynthesis), thereby preventing oxidative damage to the PRCs. [1] In its native orange-450 state, βC has a wavelength absorption maximum (λmax) of 450 nm. The λmax increases by 65nm as βC converts from the orange-450 state to a transient pink-515 state (λmax = 515nm) for approximately 0.01 seconds during the photosynthetic cycle. [2-4] This so-called 515nm Effect is expected

Figure 1. β-Carotene (βC; C40H56). Figure courtesy of Lauren Hoody, 2016.

Proceedings 2016

NATURAL SCIENCES to decrease the 1O2-quenching efficiency ΦQ of βC during the brief periods in the photosynthetic cycle when βC is in its pink-515 state; consequently, ΦQpink-515 < ΦQorange-450. Since the 515nm Effect is inherent to photosynthesis, both the native orange-450 and pink-515 states of βC contribute to 1O2-quenching in PRCs; in studies of green plants, then, it is not possible to deconvolute ΦQpink-515 from ΦQorange-450. Even so, by using model systems which — like the pink-515 state — are red-shifted with respect to the native orange-450 state, the impact of the 515nm Effect on 1O2-quenching can be inferred. In this research, the pink-515 state will be modeled using Blue βC-Acid Complexes (βC-ACs) — in which the native orange-450 state of βC is converted to a blue-900 state (Figure 2 and Figure 3) by various acids. [5-12] βC-ACs are particularly good models for the pink-515 state, for two reasons: (1) the red shift for the blue-900 state is greater than that for the pink-515 state; and (2) the efficiency of 1O2-quenching is expected to vary inversely with the λmax of the quencher (i.e., ΦQcolor-λmax oc λmax). Hence, the blue-900 state is expected to be a poorer 1O2-quencher than the pink-515 state; the quenching efficiencies are thus expected to follow the trend ΦQorange-450 > ΦQpink-515 > ΦQblue-900.

of our working hypothesis will strongly suggest that the 515 Effect decreases the efficiency of 1O2-quenching in photosynthesis. Competitive kinetic quenching studies have long been used to characterize 1O2-quenching efficiencies. However, in recent years, optical quantitation of the 1270nm, near infrared (NIR) 1 O2 phosphorescence using (custom-built) transient NIR spectrometers, similar to the (commercial) Ocean Optics NIRQuest NIR Spectrophotometer used in my studies, has become the method of choice for determining ΦQ. Accordingly, my original proposal emphasized the NIRQuest as a way to characterize the 1O2 phosphorescence signals resulting from the prepared solutions.

Figure 3. (Black) Absorption spectrum of 10-5M βC in benzene solvent in its native orange-450 state. (Red) The same solution in the presence of 0.5M TFA, illustrating the acid-induced +450 nm bathochromic shift as βC converts to the blue-900 species. This shift significantly exceeds the +65 nm bathochromic shift observed when photosynthetic systems undergo the 515nm Effect. The “noisiness” of the spectra at λ < 300nm is due to absorption by benzene solvent, which has a UV cutoff of 280 nm, below which %T ≤ 10%. Graph courtesy of Lauren Hoody and Ashlee Wertz, 2016.

Figure 2. (Left) A 10-5M solution of β-carotene (βC) in its native orange-450 state in benzene solvent; (right) A solution of the blue-900 βC-TFA complex in benzene solvent, with [βC] = 10-5M and [TFA] = 0.5M (TFA = trifluoroacetic acid). Photograph courtesy of Lauren Hoody, 2016.

My research was designed to test a single, central working hypothesis — namely, that the 1 O2-quenching efficiency of βC is maximized for its native orange-450 state and decreases with increasing λmax. This hypothesis would be verified if experiments indicate that ΦQblue-900 < ΦQorange-450. Because the pink-515 and acid blue-900 states have similar electronic structure, confirmation Proceedings 2016

This project required many steps to optimize the system both chemically and optically. Unfortunately, because of unexpected difficulties with the NIRQuest, we have not yet been able to characterize the 1270nm phosphorescence from our native orange-450 and acid blue-900 samples. Even so, the chemical optimization of our system is essentially complete: the system to be used in future studies will utilize acid blue-900 species comprised of 10-5M βC combined with 0.5M trifluoroacetic acid (TFA) with 10-5M C60 as the 1O2 photosensitizer, all dissolved in benzene solvent. During the course of this research, a secondary goal evolved — to generate new insights into the chemical nature of the blue-900 βC-TFA 41

NATURAL SCIENCES complexes and βC-TFA degradation products. [6-8, 12] Accordingly, I divide the Experimental Method, Results, and Discussions into “Chemical Optimization” (which are essentially complete) and “Optical Optimization” (which are only partially complete) sections.

Experimental Methods, Results and Discussion Purification of Chemicals βC (Sigma-Aldrich Type I, synthetic, ≥ 93%) was purified using standard column chromatographic protocols developed in Dr. Masthay’s laboratory. [13] All other chemicals were used as purchased.

Chemical Optimization of Sensitizer + βC + Solvent and Sensitizer + βC + Acid + Solvent Combinations As detailed below, optimal conditions for the solutions were determined for our experiments. Our solutions will be made up of 10-5M C60 (the 1 O2 sensitizer) in benzene solvent with 10-5M βC in either the absence or presence of 0.5M TFA (a 50,000-fold molar excess). [12] A 355nm pulsed laser or 365nm Hg lamp will then be used to irradiate these solutions to generate 1O2. Solvent Optimization Initially, the majority of the solutions were prepared in CH2Cl2 (CHROMASOLV® Plus, for HPLC, ≥ 99.9%) solvent because βC and C60 are both soluble in CH2Cl2. Unfortunately, however, βC photodegrades in CH2Cl2 and other

chloroalkane solvents via photoinduced electrontransfer reactions; [13-15] our βC-CH2Cl2 solutions accordingly rapidly lost color upon irradiation. Hence, benzene (Sigma-Aldrich, for HPLC ≥ 99.9%) was our ultimate solvent choice for two reasons: (1) βC and C60 are both soluble in benzene; and (2) βC is photostable in benzene. Acid Optimization A variety of solutions of 10-5M βC and 0.5M acid in solvent (CH2Cl2) were prepared. Sulfuric acid (H2SO4; Fischer Scientific, Certified ACS Plus) was problematic because two layers formed upon adding H2SO4 to the 10-5M βC solution in CH2Cl2. The formation of the two layers was not ideal for our experiments — even though both layers were blue — because it was difficult to determine the composition of each layer. Since the density of H2SO4 (1.84 g/cm3) is greater than that of CH2Cl2 (1.33 g/cm3), the bottom layer of the solution was presumably composed of H2SO4 and βC; however, it was uncertain if CH2Cl2 was also present in this bottom layer. The top layer was presumably comprised of CH2Cl2 and blue βC-H2SO4 complexes. Ultimately, because (1) βC is photolabile in CH2Cl2; and (2) we believe CH2Cl2 is present in both H2SO4 layers, it was decided that H2SO4 would not be the ideal acid for our experiments. Similar results were obtained when nitric (HNO3; Fischer Scientific, Certified ACS Plus) and methanesulfonic (CH3SO3H, Sigma-Aldrich, 99%) acids were added to 10-5M βC solutions in CH2Cl2. In the case of HNO3, the solution initially became blue, but over a short period of time formed a yellowish/brown top layer and a

Figure 4. Emission from 10-5M C60 in benzene upon irradiation with 532 nm laser pulses, illustrating the absence of 1270nm 1O2 emission (spectrum obtained with the Ocean Optics NIRQuest Transient Absorption Spectrophotometer. The spurious “spike” at 1064nm is due to a damaged photodiode in the NIRQuest, and is not indicative of true emission from the sample, as this spike was present whenever the NIRQuest was triggered—even in the absence of a sample or laser pulses. Figure courtesy of Lauren Hoody and Ashlee Wertz, 2016.

Proceedings 2016

NATURAL SCIENCES colorless bottom layer, consistent with reports in the literature. [16] Presumably, the bottom layer was primarily composed of HNO3 since the density of HNO3 is 1.5129 g/cm3 (greater than that of CH2Cl2). Apparently, βC was absent from the bottom layer (since it showed no coloration), which is possibly due to the moderate polarity of HNO3, which has a dipole moment of 2.17±0.02D. The βC-CH3SO3H solution was similar to the βC-H2SO4 solution in that it formed two blue layers.

Concentration Optimization In accord with the method of Mortensen and Skibsted [12] our optimized solutions are made up of 10-5M βC and 0.5M TFA in benzene (50,000fold molar excess of TFA). These conditions were ideal because of the far-red shift in the βC-TFA complexes (λmax ~900nm) present with these concentrations. 10-5M C60 appears optimal for our experiments because we anticipate that its concentration is high enough to generate a detectable 1O2 1270nm signal. [17-22]

Ultimately, TFA (also CF3COOH; Sigma-Aldrich, 99+% Spectrophotometric grade) became our acid of choice because it formed one, blue (λmax~900nm) uniform layer when mixed with the βC in both benzene and CH2Cl2.

Chemical Characterization of βC-TFA Complexes and βC-TFA Degradation Products

O2 Sensitizer Optimization [17-22] A solution of C60 (Sigma-Aldrich, 99.5%) in benzene produced no 1O2 signal (Figure 4) because C60 does not absorb the 532nm second harmonic pulse (ε532(C60)~0 (Figure 5) from our pulsed Nd:YAG laser (Spectra Physics INDI-40-10). Because we wanted to confirm the existence of a 1270nm 1O2 phosphorescence signal before changing the dichroic optics of the laser to the 355nm option, a temporary alternative 1O2 sensitizer (Rose Bengal, which does absorb the 532nm pulses) in ethanol solvent was used. Ultimately, however, C60 will be our 1O2 sensitizer of choice because Rose Bengal is not soluble in benzene. 1

The structures of βC-ACs [6-8] in general and of βC-TFA complexes and βC-TFA degradation products in particular [12] are poorly characterized. Accordingly, a secondary goal of our experiments was to characterize the chemical properties of the βC-TFA complexes and βC-TFA degradation products [12] using a combination of vacuum and thin layer chromatographic (TLC) methods.

βC-TFA Complexes A solution of βC-TFA in benzene was prepared and then immediately dried under vacuum (~2-3 torr). When all the solvent had evaporated, an orange oil remained in the bottom of our 25mL round bottom flask. This product was dissolved in benzene, and an absorption spectrum was obtained. The spectrum showed a peak similar — but not identical — to that of pure βC; the wavelength maxima of the vibronic peaks were shifted to slightly longer wavelengths and their relative intensities were altered, suggesting that the orange vacuum product was similar, but not identical, to βC (Figure 6).

βC-TFA Degradation Products

Figure 5. Absorption spectrum of 10-5M C60 in benzene, illustrating that the molar extinction coefficient ε532 oc A532 ~ 0 (see green line), hence accounting for the lack of 1O2 emission from these solutions. In future studies the 355 nm third harmonic line from the laser will be used; because C60 absorbs strongly at 355nm (see purple line), a 1270nm 1O2emission signal is anticipated in these future studies. Figure courtesy of Lauren Hoody and Ashlee Wertz, 2016.

Proceedings 2016

Another solution of βC-TFA in benzene was prepared and incubated for 24 hours in the dark. As βC-TFA complexes are known to degrade over time [12] an absorption spectrum was taken to insure the 900nm peak of the βC-TFA complexes had disappeared. To characterize the colorless polar products, fluorescent silica gel TLC plates were then run with benzene as the mobile phase. Residual βC was the mobile and moved with the solvent front, (this compound showed an absorption identical to that of βC when it was extracted from the TLC plate), whereas the βC-TFA degradation product was the immobile (RF=0). 43

NATURAL SCIENCES is proportional to the concentration of 1O2 in solution, this intensity will vary inversely with the 1O2-quenching efficiencies: I1270 oc ΦQ-1. Hence, if we find that the 1,270 nm emission intensities orange-450 follow the trend I1270 < I blue-450 , our working 1270 hypothesis will be verified.

Figure 6. (Black) Absorption spectrum of 10-5M βC in benzene solvent. (Red) Absorption spectrum of βC-TFA complexes ([βC] = 10-5M; [TFA] = 0.5M) following the removal of benzene solvent by vacuum. (This product was an orange oil; see Section II.C.1). The relative intensities of the vibronic peaks in the two species differ significantly, and the absorbance maxima of the peaks differ slightly. Hence, the vacuum-isolated product is similar — but not identical — to βC. Figure courtesy of Lauren Hoody and Ashlee Wertz, 2016.

To further characterize the polar degradation products, preparative TLC was used. The immobile spot on the plate was scraped off and the immobile (in benzene) products were dissolved in 90% ethyl acetate/10% methanol. A second TLC plate was run of this immobile substance, but with 90% ethyl acetate/10% methanol as the mobile phase. The βC-TFA degradation products were mobile (displaying one spot that moved with the solvent front), thus confirming that the βC-TFA degradation products are polar, unlike βC.

Optical Optimization: Attempts to Observe 1270nm 1O2 Phosphorescence To Date

O2 Phosphorescence Signal and Characterization We plan to characterize ΦQorange-450 and ΦQblue-900 using a state-of-the-art transient near infrared emission spectrophotometer (Ocean Optics NIRQuest). More specifically, the 1O2 generator buckminsterfullerene (C60; Sigma-Aldrich, 99.5%) will be added to solutions of βC and serially diluted to concentrations of 10-5M C60. 1O2 will then be generated by irradiating these βC + C60 and βC-TFA + C60 solutions with the 355nm third harmonic of a pulsed Nd:YAG laser and/ or the 365nm line of a 100 Watt Hg lamp. The intensity I1270 of the resulting 1,270nm emission of 1O2 [23] emanating from these solutions will be monitored using an Ocean Optics NIRQuest Spectrophotometer operating in Edge Trigger mode (with 355nm laser pulses) or Free Trigger mode (with the 365nm CW Hg line). Since I1270 1

NIRQuest Operation An Ocean Optics NIRQuest Spectrophotometer (a germanium photodiode array-based transient infrared spectrometer with an operating range of 900-1700nm) was selected for our experiments for three reasons. First, its operating range encompasses the anticipated 1270nm 1O2 emission signal. Second, the NIRQuest interfaces easily with our pulsed Nd:YAG laser. Third, its cost ($16,000) was fundable using Chemistry Department funds. The NIRQuest is triggered by the 20ns rising edge of a trigger pulse from the sync/out on the laser power supply, which is generated at the same time as the 6.5ns, 532nm pulses. The laser operates at a frequency of 10Hz; hence pulses come at 0.1 sec (105 μs) intervals. 1O2 has an optical lifetime of 10-5s (10μs), so that each 1270nm phosphorescence signal comes ~10μs after the trigger, but is complete long before the next laser pulse reaches the sample. The NIRQuest thus integrates the 1270nm signal on a pulse-by-pulse basis. Unfortunately, the cable that was provided from Ocean Optics to carry the trigger pulse from the laser to the NIRQuest was nonfunctional when it arrived. Five weeks of work with an expert electrical engineer/optical physicist (Dr. Perry Yaney of the UD Physics Dept. and Electrooptics Program) was required to make the cable functional, at which point we commenced with the transient emission studies detailed below. Rose Bengal A solution of Rose Bengal (Allied Chemical) in ethanol (AAPER Alcohol, 95%) produced a broad, asymmetric emission signal extending from 1100-1300nm with a peak at 1170nm and a shoulder at 1270nm (Figure 7). Though this signal encompasses the range of wavelengths expected for 1O2, the signal does not originate from 1O2—for two reasons: (1) the 1170nm signal does not match the emission spectrum of 1 O2 [17-22]; and (2) the intensity of the 1170nm signal was identical in air-saturated solutions ([O2]=1.91×10-3M at 25°C), O2-saturated solutions generated by bubbling with O2(g) for 30 minutes Proceedings 2016

NATURAL SCIENCES

Figure 7. Transient absorption spectrum obtained with NIRQuest upon exciting ~10-5M solution of Rose Bengal in ethanol solvent with 532 nm laser pulses. The broad, asymmetric emission signal ranging from 1100-1300nm with a peak at 1170nm and a shoulder at 1270nm does not originate from 1O2, and is currently of unknown origin (see Section II.D.3). Figure courtesy of Lauren Hoody and Ashlee Wertz, 2016.

prior to irradiation ([O2]=9.12×10-3M) [24], and solutions sparged with high purity (99.998%) Ar(g) [24] prior to irradiation ([O2]~0M). Thus, the 1170nm signal was independent of oxygen concentrations. Furthermore, the signal does not originate from Rose Bengal, the solvent, or impurities in either. Nor does it originate from thermal lensing of blackbody emission, for the reasons detailed below. 1. The signal clearly originates in some way from Rose Bengal, since (i) low Rose Bengal concentrations resulted in no I1270 signals, and (ii) very high Rose Bengal concentrations also produced no signals (because the laser pulse was absorbed entirely at the entry edge of the cuvette, and hence did not pass by the fiber optics portal at the middle of the cuvette). [25] 2. The signal does not originate from fluorescent or phosphorescent impurities in the solvent, since the signal was observed only in the presence of Rose Bengal; the signal was absent for neat ethanol. Even so, the signal was not due to emission from Rose Bengal [20] or Rose Bengal photoproducts (which manifest no emission from 1050-1100nm [i.e. near the short-wavelength edge of the 1170nm signal]) upon excitation with 532nm using a Varian Cary 1 Eclipse Fluorimeter (which has an upper wavelength range of 1100nm). [26] 3. The signal cannot originate from a thermal lens generated via thermal relaxation of Rose Bengal following photoexcitation for Proceedings 2016

two reasons: (1) though a thermal lens could cause the beam to expand and intersect with the entry portal of the fiber optic cable with each pulse, resulting in a transient signal, the signal would occur at the actinic wavelength (532nm) which lies outside the spectral range of the NIRQuest, and hence would not be observed; and (2) the maximum temperature rise which would be generated by our 14mJ laser pulses in the irradiated beam volume of πRlaser2 Ɩ = .636 mL would be ΔT = ΔH/Cp = Epulse / (Cpm×n) = 0.0114 K; (calculated assuming CP ethanol = 112.4 J mole-1 K-1 and dethanol = .789 g mL-1). [20] This small temperature rise would seem to make a thermal lens unlikely. 4. The signal appears to be independent of laser intensity. 5. The 1170nm signal cannot originate from blackbody emission from the Rose Bengal [20] or ethanol, since at ambient temperature (21°C in our laboratory) the blackbody emission maximum is 9,860nm, according to the Wien displacement law λmax = (2.898×106nm K) / T. The observed emission maximum of 1170nm corresponds to a temperature of ~2,500K, which is clearly unattainable in our samples (0.0114 K temperature rise, as detailed above) — even if all of the absorbed laser energy were deposited into the sample as heat [20]. Hence, at this point, the nature of the 1170nm signal remains ambiguous. We accordingly plan to begin studies using C60 in benzene with a focused 355nm Nd:YAG third harmonic beam to see if we obtain 1270nm 1O2 phosphorescence signals. [17-22] 45

NATURAL SCIENCES DPBF Experiments 2,6-Diphenylisobenzofuran (DPBF; Sigma-Aldrich, 97%) is commonly used as a singlet oxygen substrate. [27] We accordingly attempted to use DPBF to quantify [1O2] in our solution to supplement our anticipated NIRQuest I1270 results. We were surprised to find that quantification of [1O2] with DPBF may yield spuriously high estimates of [1O2]. Photolability of DPBF Effects of Ambient Light and Sunlight on DPBF When yellow-blue solutions of DPBF in benzene were placed in quartz cuvettes, the solutions turned colorless over time. In fact, upon exposure to 3 minutes of direct sunlight (mid-morning, July, Dayton, Ohio, 39.8°N, 84.2°W), the DPBF absorption peak was completely eliminated and the solutions became colorless. Similar — but slower — photodegradation resulted upon exposure of the solutions to ambient (overhead fluorescent) light. Surprisingly, the rate of photodegradation was approximately equal in both aerated and sparged solutions, indicating that the process is O2-independent. To confirm that the degradation is initiated by light, two cuvettes of DPBF in benzene were prepared; one was placed in a dark drawer and the other was placed in ambient light overnight. The next morning, the solution kept in the dark retained its original yellow-blue color, whereas the solution kept in the light had become colorless. Clearly then, DPBF is photolabile in benzene in both ambient light and sunlight. O2 Plays No Role, Eliminating 1O2 As an Intermediate Zhang and colleagues characterized the role that light and oxygen play in the photolysis of DPBF. [27] Consistent with our results — which indicate that the reaction is O2-independent — Zhang and colleagues reported that DPBF does not form a triplet state, in which case it cannot sensitize the generation of 1O2. Hence, the degradation of DPBF is not induced by 1O2 (though this result does not eliminate possible roles for other reactive oxygen species). Benzene Plays no Direct Photochemical Role in the Photodegradation of DPBF To characterize the possible role of benzene in the photodegradation of DPBF, we prepared 10-5M solutions of DBPF in benzene and in ethanol (in the latter of which it was reported to be photostable). [27] Both benzene and ethanol 46

solutions were used because benzene has a UV cutoff (%TλUVcutoff ) ≤ 10%) of 280nm, whereas the UV cutoff of ethanol is 205nm. Because solar UV light with wavelengths as short as 270nm reaches the earth’s surface, photons with wavelengths between 270 and 280nm can be absorbed by benzene, resulting in potential photoactiviation. This in not possible for ethanol, which is transparent at 270nm. Our observations that DPBF in both solutions completely degraded after 3 minutes of exposure to sunlight indicates that the benzene is not involved in the photodegradation. Further tests were accordingly performed by irradiating DPBF-ethanol solutions with the 254nm and 436nm lines of the Hg lamp. Unexpectedly, the DPBF photodegraded about three times faster when irradiated with the 436nm line. It was noted that the percentage transmittance for the 254nm filter was ~5.5% and the 436nm filter was ~17%. This may be the explanation for the unexpected results we obtained. DPBF Degrades in the Presence of TFA We found that DPBF degrades in benzene solutions in the presences of TFA. This result is consistent with reports in the literature, which indicate that, in ambient light, TFA causes the intensity of the visible absorption band of DPBF to lose its absorption peak at a rate that is proportional to [TFA]. [27] These results preclude the use of DPBF as a 1O2-substrate in βC-TFA solutions.

Conclusions and Future Studies The next steps required to reach our final goal of characterizing the 1O2-quenching efficiency of our βC and βC-TFA solutions involve optimizing the optical conditions of our experiment. More specifically, this will entail changing the actinic wavelength of the laser from 532nm (Nd:YAG 2nd harmonic wavelength) to 355nm (Nd:YAG 3rd harmonic wavelength) and focusing the narrowed beam with the use of a 150nm focal length lens. We anticipate that, with the adjustment, we will be able to generate the 1O2 in our βC-C60 and βC-C60-TFA solutions and obtain signals using the NIRQuest.

Proceedings 2016

NATURAL SCIENCES Acknowledgements First and foremost, I would like to thank my thesis advisor, Dr. Mark Masthay, for all of his help and support in guiding me in my research this summer, as well as my lab coworker, Ashlee Wertz. I would also like to thank the Berry Family, the Berry Family Foundation, and the University Honors Program, with an extra special thanks to Dr. David Darrow, Dr. Nancy Miller, Maria Burkett, Ramona Speranza, Jill Talley, Laura Cotton and Dr. John McCombe, for making this summer research experience possible. Finally, an extra special thank you to Dr. Perry Yaney, without whom the progress we made with the optics of our experiment would not have been possible.

References 1.

Krinsky, N.I., Function, in Carotenoids, O. Isler, Editor. 1971, Birkhäuser: Basel. p. 669-716.

Duysens, L.N.M., Transfer of light energy within the pigment systems present in photosynthesizing cells. Nature, 1951. 168: 548-550.

Telfer, A., What is β-carotene doing in the photosystem II reaction centre? Phil. Trans. R. Soc. Lond. B 2002. 357: 1431-1440.

Govindjee, Carotenoids in Photosynthesis: An Historical Perspective, in The Photochemistry of Carotenoids, H.A. Frank, et al., Editors. 1999, Kluwer Academic Publishers: Dordrecht. p. 1-19.

Carr, F.H. and E.A. Price, Colour reactions attributed to vitamin A. Biochem. J., 1926. 20: 497-501.

Haugan, J.A. and S. Liaaen-Jensen, Blue carotenoids. Part I. Novel oxonium ions derived from fucoxanthin. Acta Chem. Scand., 1994. 48: 68-75.

Haughan, J.A. and S. Liaaen-Jensen, Blue carotenoids. Part 2: The chemistry of the classical colour reaction of common carotenoid 5,6 epoxides with acid. Acta Chem. Scand., 1994. 48: 152-159.

Britton, G., R.J. Weesie, D. Askin, J.D. Warburton, L. Gallardo-Guerrero, F.J. Jansen, H.J.M. DeGroot, J. Lugtenburg, J.P. Cornard and J.C. Merlin, Carotenoid blues: Structural studies on carotenoproteins. Pure Appl. Chem., 1997. 69: 2075-2084.

Wassermann, A., Proton-acceptor properties of carotene. J. Chem. Soc., 1954: 4329-4336.

10. Wassermann, A., Properties of ion-pairs formed by proton transfer to conjugated polyenes. Trans. Far. Soc., 1957. 53: 1029-1030. 11. Prum, R.O., A.M. LaFountain, C.J. Berg, M.J. Tauber and H.A. Frank, Mechanism of carotenoid coloration in the brightly colored plumages of broadbills (Eurylaimidae). J. Comp. Physiol. B, 2014. 184: 651-672. 12. Mortensen, A. and L.H. Skibsted, Kinetics and mechanism of the primary steps of degradation of carotenoids by acid in homogeneous solution. J. Agric. Food Chem., 2000. 48(2): 279-286.

Proceedings 2016

13. Wang, W., Photochemical Characterization of the Intensity Dependence in Multiphoton-Reactive Systems: Application to the Photodegradation of β-Carotene, 2008M.S. Thesis, Chemistry, University of Dayton, Dayton, OHpp. 14. Gurzadyan, G.G. and S. Steenken, Photoionization of β-carotene via electron transfer from excited states to chlorinated hydrocarbon solvents. A picosecond transient absorption study. Phys. Chem. Chem. Phys., 2002. 4(13): 2983-2988. 15. Fujii, R., Y. Koyama, A. Mortensen and L.H. Skibsted, Generation of radical cation of β-carotene in chloroform via the triplet state as revealed by time-resolved absorption spectroscopy. Chem. Phys. Lett., 2000. 326: 33-38. 16. Karrer, P. and E. Jucker, Carotenoids, E.A. Braude, Editor. 1950, Elsevier Publ. Co.: New York. 17. Hare, J.P., H.W. Kroto and R. Taylor, Preparation and UV/visible spectra of fullerenes C60 and C70. Chem. Phys. Lett., 1991. 177(4,5): 394-398. 18. Snyder, J.W., E. Skovsen, J.D.C. Lambert, L. Poulsen and P.R. Peter R. Ogilby, Optical detection of singlet oxygen from single cells. Phys. Chem. Chem. Phys., 2006. 8(37): 4280. 19. Andersen, L.K. and P.R. Ogilby, A nanosecond nearinfrared step-scan Fourier transform absorption spectrometer: Monitoring singlet oxygen, organic molecule triplet states, and associated thermal effects upon pulsed-laser irradiation of a photosensitizer. Rev. Sci. Instrum., 2002. 73(12): 4313-4325. 20. Nagano, T., K. Arakane, A. Ryu, T. Masunaga, K. Shinmoto, S. Mashiko and M. Hirobe, Comparison of single oxygen production efficience of C60 with other photosensitizers, based on 1268 nm emission. Chem. Pharm. Bull., 1994. 42(11): 2291-2294. 21. Bilsky, P., B. Zhao and C.F. Chignell, Singlet oxygen phosphorescence photosensitized in nanoaggregates of C60 buckminsterfullerene is insensitive to solvent and quenchers and strongly red-shifted indicating highly polarizable interior. Chem. Phys. Lett., 2008. 458: 157-160. 22. Prat, F., C. Martí, S. Nonell, X. Zhang, C.S. Foote, R. González-Moreno, J.L. Bourdelande and J. Font, C60 fullerene-based materials as singlet oxygen O2(1Dg) photosensitizers: A time-resolved near-IR luminescence and optoacoustic study. Phys. Chem. Chem. Phys., 2001. 3: 1638-1643. 23. Krasnovsky, A.A.J., Y.V. Roumbal and A.A. Strizhakov, Rates of 1O2 (1∆g) production upon direct excitation of molecular oxygen by 1270 nm laser irradiation in air-saturated alcohols and micellar aqueous dispersions. Chem. Phys. Lett., 2008. 458: 195-199. 24. Murov, S.L., Handbook of Photochemistry. 1973, New York: Marcel-Dekker, Inc. 272. 25. Additionally, we noted that Rose Bengal degraded rapidly upon laser pulse intensities of 14mJ pulse-1. However, with pulse intensities of 1.5mJ*pulse-1, the Rose Bengal degraded more slowly. 26. While it is possible that laser-induced two-photon excitation of Rose Bengal could result in NIR emission from Rose Bengal differing from the one-photon excitation emission induced in the fluorimeter, this possibility seems unlikely. 27. Zhang, X.F. and X. Li, The photostability and fluorescence properties of diphenylisobenzofuran. J. Luminescence, 2011. 131: 2263-2266.

NATURAL SCIENCES

Drosophila Eye Model to Investigate the Role of Mitochondrial Dysfunction as a Trigger for Neurodegeneration in Alzheimer’s Disease Lydia C. Payton1,2,3 and Amit Singh1 University of Dayton, 300 College Park, Dayton, OH, 45469 1. Department of Biology 2. Berry Summer Thesis Institute 3. University Honors Program

Thesis Mentor: Amit Singh, Ph.D. Department of Biology Corresponding Author: Amit Singh, Ph.D. Email: asingh1@udayton.edu

Abstract

Introduction

Alzheimer’s disease is a progressive neurodegenerative disorder that affects cognitive function and memory of an affected individual. It results from plaques formed by the abnormal cleavage of the Amyloid Precursor Protein (APP). This abnormal cleavage creates a 42 amino acid polypeptide, also known as amyloid beta 42 (Aβ42). Accumulation of the Aβ42 peptide triggers cell death in the neuronal cell population of the central nervous system. The trigger for this abnormal cell death is unknown. Mitochondrial dysfunction is one possible explanation for the trigger of neurodegeneration. Mitochondria are involved in vital cellular functions, including ATP production, calcium ion homeostasis, reactive oxygen species production and apoptosis. In this review, possible mitochondrial gene mutations that are involved in these vital functions are discussed, along with how they can relate to Alzheimer’s mediated neurodegeneration. In addition, areas of future investigation are mentioned. In order to study these issues, a Drosophila melanogaster eye model is proposed using the Gal4/UAS system to misexpress the human Aβ42 polypeptide in the developing photoreceptor neurons of the fly eye.

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder that results in cognitive dysfunction, memory loss, and ultimately death of the patient (Sarkar et al., 2016; Drake et al., 2003; Shankar et al., 2008; Barnes and Yaffe, 2011a,b; Cavedo et al., 2014; Seifan et al., 2015). It primarily affects people aged 65 years and older by causing neuronal death in the hippocampus and cortex, creating shrinkage in brain morphology (Cavdeo et al., 2015; Sarkar et al., 2016). In 2015, this disease affected more than 5.3 million Americans, and this number is expected to rise to 10 million by 2050. It is currently the sixth leading cause of death in America and is the only leading cause of death without an effective cure, treatment or preventive method (Götz et al., 2012; Cavedo et al., 2015). From 2000 to 2013, mortality rates for diseases like prostate cancer, stroke and heart disease decreased by 11%, 23%, and 14%. However, the Alzheimer’s disease mortality rate increased by a dramatic 71% (Alzheimer’s Association, 2015). In addition, there is an enormous economic burden left by the disease, which cost the United States $217 billion dollars in 2014 (Alzheimer’s Association, 2015). These health and economic threats posed by Alzheimer’s heighten the need for continued research into the molecular mechanisms of Alzheimer’s disease.

Proceedings 2016

NATURAL SCIENCES AD neuropathology is created from two types of abnormal protein deposition within in the brain: (1) neurofibrillary tangles (NFTs), which are formed from hyper-phosphorylated forms of Tau, a microtubule protein; and (2) amyloidbeta (Aβ42) plaques, which are formed from abnormal cleavage of the Amyloid Precursor Protein (APP) (Sarkar et al., 2016). The Amyloid Hypothesis of AD states that Aβ accumulation is the primary event in Alzheimer’s neuropathology (Hardy et al., 2002). This review, which proposes directions for a new study, focuses on the role of mitochondrial dysfunction as it relates to Alzheimer’s disease and the Amyloid Hypothesis. In order to investigate mitochondria’s role, a Drosophila melanogaster model can be employed. In addition, methods of detection for reactive oxygen species and mitochondrial dynamics are mentioned.

Generation of Amyloid-beta Plaques in AD Alzheimer’s disease can be familial or sporadic. Familial forms of the disease result from abnormal mutations in the amyloid precursor protein (APP) (Eggert et al., 2004; Evin and Weidemann, 2002). The amyloid hypothesis states that the formation of amyloid-beta plaques is the primary event in AD neuropathology (Hardy et al., 2002). Normally, APP is cleaved by α and γ secretases that create an amino acid chain of 40 peptides in length. However in AD, instead of α-secreatase, a β-secretase cleaves the protein at 42 peptides. This extra cellular peptide with two extra amino acids generates a toxic, hydrophobic structure within the brain (Speretta et al., 2012). The products of this abnormal cleavage began as fibrils, but eventually conglomerate into oligomers and then plaques. The formation of Aβ42 plaques creates aberrant signaling, oxidative stress, and eventually neuronal death (Sarkar et al., 2016).

Figure 1. Alzheimer’s disease neuropathology results from abnormal cleavage of APP. The Amyloid Precursor Protein (APP) is abnormally cleaved by the β-secretase at forty-two peptides in length, creating a hydrophobic structure within the brain that results in damaged mitochondrion, increased ROS levels, aberrant signaling and ultimately neuronal death. Illustration courtesy of the National Institute on Aging through http://neurosciencenews.com/buphenylpossible-treatment-alzheimers-disease-neurotrophic-factors/.

Proceedings 2016

NATURAL SCIENCES Drosophila melanogaster as a Disease Model System Because of the enormous amount of amassed knowledge concerning its development and genetics, the Drosophila has been argued that it is the most important model organism available to the scientific community (Roberts, 2006; Bier, 2005). The amount of genetic tools and methods available to study the organism surpasses any other multi-cellular organism, and this alone has allowed for advances in molecular genetics that will continue to generate novel research (Beckingham et al., 2005). The benefits to using the organism as a model system are numerous; Drosophila is inexpensive, reproduces quickly and has a short lifespan (Mhatre et al., 2014). Most importantly, around 70% of human diseasecausing genes have Drosophila orthologs (PruBing et al., 2013; Singh and Irvine, 2012), and there is a high conservation of important biological and metabolic pathways (Bilen et al., 2005). All of these aspects make it an ideal organism to study neurodegenerative diseases. In order to study the human Alzheimer’s pathology within the model system, a two-part expression method taken from yeast was used known as the UAS/Gal4 system (Brand and Perrimon, 1993; Tare et al., 2013). The first part consists of a transcription factor known as Gal4. This element then binds to UAS, also known as the upstream activating sequence, which transcribes the downstream target sequence. In the interests of this study, the target sequence is the human Aβ42 gene. The Gal4 driver line can be expressed under a tissue specific enhancer in order to express the target gene in a variety of tissues and organs. In this study, the GMR (Glass Multimer Repeat) driver is used in order to express the human Aβ42 gene within the fly eye region (Moses, 1991). After crossing the transgenic flies harboring the Gal4 unit and the UAS element, the target gene is expressed in a spatiotemporal manner (Brand and Perrimon, 1993).

bi-layer structure in the larva stage, known as the eye-imaginal disc. These cells differentiate in the third instar larva stage from photoreceptor precursor cells to photoreceptor neurons. The eye-imaginal disc then metamorphoses into the pupal retina, which in turns develops into the adult fly eye (Ready et al., 1976). In wild-type phenotypes, the compound fly eye consists of 800 highly ordered hexagonal structures, known as ommatidia (Ready et al., 1976; Tare et al., 2013). However, when the human Aβ42 gene is expressed within the fly eye region, a strong neurodegenerative phenotype is expressed at 29°C (Tare et al., 2013). This neurodegenerative phenotype resembles Alzheimer’s like neuropathology, reducing overall fly eye size, creating a glassy appearance and producing uneven pigmentation

Figure 2. The UAS/Gal4 system is used to express the human Aβ42 gene in the Drosophila model. The binary yeast system, UAS/Gal4 (Brand and Perrimon, 1993), is used to express the Aβ42 gene within the differentiating photoreceptor neurons. The first half of the system consists of the Gal4 transcription factor. This is expressed under a tissue specific enhancer, GMR, which isolates the manifestation in the fly eye region. The second half of the system consists of a UAS element, which transcribes a target gene (Aβ42) located downstream from it. When two flies with each element are crossed, the target gene can be expressed in a spatiotemporal manner (Brand and Perrimon, 1993). Courtesy of Lydia Payton and Amit Singh, 2016.

Drosophila Eye Model The fly eye of the Drosophila can be genetically modified without affecting other major lifesustaining organs (Tare et al., 2011; PruBing et al., 2013). For this reason, it has been extensively used to study various biological processes and diseases. The fly eye develops from an epithelial 50

Figure 3. Drosophila Life Cycle. The Drosophila life cycle is approximately 12 days long and has four distinct stages: egg, larva, pupa and adult fly. The larval stage is comprised of three distinct developmental stages: first, second and third instar larvae. This short life cycle and the fly’s small size allows it to be an excellent model organism. Courtesy of Lydia Payton and Amit Singh, 2016.

Proceedings 2016

NATURAL SCIENCES and evident dark necrotic spots (Cutler et al., 2015; Moran et al., 2013; Steffensmeier et al., 2013). With this Drosophila model, mitochondrial dysfunction is investigated as it relates to Alzheimer’s disease.

Mitochondria’s Role in Neurodegeneration Mitochondria’s role in neurodegeneration from AD is ultimately unknown. The scientific community is unsure if abnormal function of the organelle triggers the Aβ42 plaque formation or if the plaques generate the organelle’s dysfunction noted in AD pathology. Mitochondria are responsible for many cellular functions, including ATP and energy production, calcium ion homeostasis, reactive oxygen species formation and apoptotic signaling (Santos et al., 2010). This review highlights mitochondrial dysfunction, possible gene targets, and tools for detection as it relates to future studies.

Figure 4. Photoreceptor degeneration in Drosophila model is mediated through human Aβ42 toxicity. (A) Wild-type Drosophila fly eyes are round and consist of 800 highly organized structures known as ommatidia. (B) As GMR> Aβ42 is expressed in the differentiating photoreceptor neurons, a decrease in eye size occurs along the anterior and posterior regions, as well as a glassy appearance with dark necrotic spots. Courtesy of Sarkar & Singh, unpublished data.

Mitochondrial Homeostasis The human brain receives 20% of total oxygen consumption resulting from a high-energy demand (Moreira et al., 2010). This is largely driven by the fact that neurons have a low glycolytic capacity, making them highly dependent on aerobic oxidative phosphorylation (OXPHOS) (Santos et al., 2010). During the aerobic energy process, electrons are transported through a series of electron carriers in the Electron Transport Chain (ETC) to generate an electromotive force that can create ATP for the cell to use these ATP as energy (Owusu-Ansah et al., 2016). However, OXPHOS is a major contributor Proceedings 2016

to the generation of reactive oxygen species (ROS) within the cell, including hydrogen peroxide (H2O2), hydroxyl (HO-), and superoxide (O2-) (Moreira et al., 2010). At low levels, ROS can often be modulators of cellular function; however, it is only when ROS reach a high level that toxic effects begin to occur (Remacle et al., 1995). While the cell employs several enzymatic defense mechanisms, such as SOD, GPx, superoxide reductase (sRed), catalase (CAT), and more, the cell often faces a challenge when the radicals produced overwhelm the defense mechanisms available to the cell (Moreira et al., 2010). The reactive species produced then target many mitochondrial components, including their DNA, lipids and proteins. The mitochondrial genome is relatively unprotected, as it lacks histones, and its DNA repair processes are inefficient when compared to the nucleus. These damaged mitochondria can then produce ten times more ROS than normal mitochondria, eventually resulting in neuronal death (Hagberg et al., 2014). The free radical theory of aging suggests that the generation of free radicals is the main cause for lifespan decrease in organisms (Liochev et al., 2013). To support this, a study done by Sanz et al. (2010), noted that there is a negative correlation between oxidative stress and life span. The study also concluded that major contributors to the generation of these free radicals included complex III and I from the ETC as well as alpha-ketoglutarate dehydrogenase in the Citric Acid Cycle (Sanz et al., 2010). While it is clear that oxidative stress is elevated in AD brains relative to age-matched brains of normal individuals (Williamson et al., 2001), one controversy within the scientific community is whether or not oxidative stress precedes or occurs after the deposition of Aβ42 plaques. In a study done by Drake et al., results suggested that oxidative stress precedes fibrillar deposition of amyloidbeta plaques in a transgenic Caenorhabditis elegans model. However, more investigation must occur before a clear distinction can be reached between the two. As stated before, a central role assumed by mitochondria is calcium homeostasis. In general, the organelle serves as a high-capacity Ca2+ sink, which allows them to either release or retain ions when there are changes in cytosolic levels (Moreira et al., 2010). However, excessive accumulation of the ion has been shown to increase ROS production, disrupt 51

NATURAL SCIENCES ATP production and induce mitochondrial permeability transition pore (PTP), and initiate apoptosis through cytochrome c and apoptosisinducing factor (AIF) (Moreira et al., 2010). After cytochrome c is released, it then binds to apoptotic protease-activating factor 1 (Apaf-1), which activates the caspase cascade, leading to neuronal death (Green et al., 2004). Dysfunction relating to either ROS levels or Ca2+ regulation is believed to relate to mitochondrial dysfunction seen in AD pathology.

Figure 5. The Electron Transport Chain (ETC) generates reactive oxygen species (ROS). The Electron Transport Chain (ETC), located within the intermembrane space of mitochondria, consists of four complexes, which use electromotive force to generate ATP for the cell to use as energy. The complexes do this through a series of oxidation-reduction reactions that inevitably generate amounts of superoxide (O2-) molecules. Courtesy of Lydia Payton and Amit Singh, 2016.

Mitochondrial Dynamics It is hypothesized that half of mitochondria are replaced through cellular quality control mechanisms every month (Lieff, 2016). Neurons, which are poorly or never replaced through division or by differentiating stem cells, become dependent upon intracellular degradation pathways (Terman et al., 2010). In terms of the mitochondrial organelle, an ongoing fusionfission cycle occurs, known as mitochondrial turnover. During this process, mitochondria undergo biogenesis, or synthesis of new mitochondria. After biogenesis, mitochondria then increase in mass through fusion with other nearby mitochondria. After the generation of these new large organelles, mitochondria then undergo fission and selective degradation of unhealthy mitochondria (Seo et al., 2010). This process has been shown to be extremely crucial in order to maintain mtDNA integrity, as well as reduce ROS damage within the cell. Additionally, studies have shown that mutations in genes involving fusion and fission result in apoptotic cell death (Seo et al., 2010). In terms of neurodegeneration, if decreased fission was occurring, 52

large senescent mitochondria could be produced, resulting in larger amounts of ROS damage. If decreased fusion was occurring, large amounts of fragmented, unusable mitochondria could be generated, resulting in apoptotic death and decreased ATP production. Possibly the most important aspect to mitochondrial turnover is the selective degradation that occurs, or mitophagy. It is essential for healthy neurons, as it removes damaged, or energy-insufficient mitochondria that would then stimulate mitochondrion-mediated cell death (Seo et al., 2010). In order for this to occur, fission must be completed beforehand. Large mitochondria that are formed from fusion, also known as giant mitochondria, are unable to be degraded through mitophagy (Seo et al., 2010). It is possible that in Alzheimer’s disease, large senescent mitochondria are accumulating because of mutations in fission related genes. Another explanation relates to a mutation in mitophagy, which could impair the degradation process. Besides mitochondrial turnover mechanisms, mitochondrial localization is crucial when investigating mitochondria’s role in Alzheimer’s disease and neurodegeneration. A study done by Iijama-Ando et al. (2009) found that the number of mitochondria in neurons of Alzheimer’s Drosophila model was not globally reduced, but the disease did induce mitochondrial mislocalizaiton. There were fewer mitochondria in axons and dendrites, and an apparent reduction in mitochondrial transport was detected (Iijima-Ando et al., 2009). The study presented possible reasons for this abnormality, some of which will be investigated in the Drosophila model presented earlier in this review. One of these explanations involves an elevated amount of Ca2+ within the cell. An altered calcium ion homeostasis has been known to affect transport of mitochondria through disruption of signaling pathways. In addition, another explanation presented involved the dynamin-like GTPase, Drp1, which is involved in fission.

Tools to Detect Mitochondrial Dysfunction After choosing mitochondrial gene targets to investigate, this study will use multiple investigative methods to display the mitochondrial health of transgenic human Aβ42 flies. One method of detecting reactive oxygen species, Proceedings 2016

NATURAL SCIENCES

Figure 6. Mitochondrial turnover as a quality control mechanism for healthy cells. The hypothesis surrounding mitochondrial turnover involves the organelle undergoing successive rounds of fusion and fission to separate functional and healthy components (green) from non-functional and unhealthy components (red). Non-functional components can then be degraded through intracellular mechanisms, such as mitophagy. This process is extremely important for post-mitotic cells. Courtesy of Lydia Payton and Amit Singh, 2016.

specifically O2 –, is a fluorescent dye called Dihydroethidium (DHE) (Hilenski, 2011). This dye is able to freely permeate cell membranes and react with superoxide anions to form a red fluorescent product, which attaches to DNA (Owusu-Ansah et al., 2016). After mild fixation, samples are able to undergo confocal microscopy for imaging. As stated before, mitochondrial quality control is crucial for post-mitotic cells with high-energy demands, such as neurons. A tool to be used that monitors mitochondrial turnover and mitophagy is a time-sensitive fluorescent protein called Mitotimer (Hernandaz et al., 2013). This protein is targeted to the mitochondrial matrix by fusion to the COX8A subunit (Ferree et al., 2013). When the protein is newly synthesized, it emits a green fluorescence. However, it irreversibly shifts to a red fluorescence after being oxidized (Laker et al., 2014). After fixation, these tissues can also be imaged through confocal microscopy.

Future Directions With this knowledge in mind, we plan to investigate genes involved in mitochondrial biogenesis, fusion and fission, and mitophagy using the tools mentioned in Aβ42 mediated neurodegeneration. One specific area of study involves investigating mutations in the Optic Atrophy 1 (OPA1) dynamin-like GTPase located within the inner mitochondrial membrane (Tang et al., 2009). Mutations in this protein Proceedings 2016

have resulted in increased ROS levels and cytochrome c mediated apoptosis by affecting mitochondrial cristae configurations (Tang et al., 2009). Furthermore, a future exploratory path involves the Krebs cycle as it modulates caspase regulation. For example, the Krebs cycle protein Succinyl-CoA synthetase β (A-Sβ) has been shown to restrict or interfere with caspase activity using a Drosophila model in the mitochondrial outer membrane (MOM) (Minis et al., 2016). If a mutation like this were to occur with Alzheimer’s pathology, greater amounts of senescent mitochondria creating ROS would be present, leading to accelerated neuronal death.

Concluding Remarks This review provides some insights into Alzheimer’s mediated neurodegeneration as it relates to mitochondrial dysfunction. Whether or not mitochondria are the trigger for this neurodegeneration is still to be investigated. However, mitochondria do assume extremely crucial functions for the neuron, including ATP production, calcium ion homeostasis, ROS generation, and apoptotic signaling. A mutation in genes involving any one of these functions could be disastrous for the cell and will be investigated as it relates to the human Aβ42 Drosophila model presented. With Alzheimer’s disease mortality rates steadily rising, it is important that the scientific community investigate all areas of cellular function, including mitochondrial dysfunction.

Acknowledgements The authors would like to thank the Berry Family and the Berry Family Foundation for the financial contribution and support. Also, many thanks are owed to the members of the Singh Lab, specifically Ankita Sarkar and Neha Gogia for their guidance and contributions. Also, thanks are due largely to Dr. Amit Singh for his assistance during the summer program and for his ideas contributing to this project.

NATURAL SCIENCES References 2015 Alzheimer's disease facts and figures. (2015). Alzheimer's & Dementia: The Journal of the Alzheimer's Association, 11(3), 332-384. doi:10.1016/j.jalz.2015.02.003 Alexei, T., Tino, K., Marian, N., Edgar A., A., & Ulf T., B. (2010). Mitochondrial Turnover and Aging of Long-Lived Postmitotic Cells: The Mitochondrial–Lysosomal Axis Theory of Aging. Antioxidants & Redox Signaling, 12(4), 503-535. Barnes, D. E. & Yaffe, K. (2011). Accuracy of summary risk score for prediction of Alzheimer disease: better than demographics alone? Archives of neurology 68, 268; author reply 268-270, doi:10.1001/archneurol.2011.4 Barnes, D. E. & Yaffe, K. (2011). The projected effect of risk factor reduction on Alzheimer's disease prevalence. The Lancet. Neurology 10, 819-828, doi:10.1016/ S1474-4422(11)70072-2 Beckingham, K. M., Armstrong, J. D., Texada, M. J., Munjaal, R., & Baker, D. A. (2005). Drosophila melanogaster--the model organism of choice for the complex biology of multi-cellular organisms. Gravitational And Space Biology Bulletin: Publication Of The American Society For Gravitational And Space Biology, 18(2), 17-29. Bier E (2005) Drosophila, the golden bug, emerges as a tool for human genetics. Nat Rev Genet 6: 9-23). Bilen, J., & Bonini, N. M. (2005). Drosophila as a Model for Human Neurodegenerative Disease. Annual Review Of Genetics, 39153-171. Brand, A. H., & Perrimon, N. (1993). Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development (09501991), 118401-415. Cavedo, E. et al. (2014). The Road Ahead to Cure Alzheimer's Disease: Development of Biological Markers and Neuroimaging Methods for Prevention Trials Across all Stages and Target Populations. J Prev Alzheimers Dis 1, 181-202, doi:10.14283/jpad.2014.32 Cutler T, Sarkar A, Moran M, Steffensmeier A, Puli OR, Mancini G, et al. (2015) Drosophila Eye Model to Study Neuroprotective Role of CREB Binding Proteing (CBP) in Alzheimer’s Disease. PLoS ONE 10(9): e0137691. doi:10.1371/ journal.pone.01237691 Drake, J., Link, C., & Butterfield, D. (n.d). Oxidative stress precedes fibrillar deposition of Alzheimer's disease amyloid beta-peptide (1-42) in a transgenic Caenorhabditis elegans model. Neurobiology Of Aging, 24(3), 415-420. Eggert, S. et al. (2004). The proteolytic processing of the amyloid precursor protein gene family members APLP-1 and APLP-2 involves alpha-, beta-, gamma-, and epsilon-like cleavages: modulation of APLP-1 processing by nglycosylation. J Biol Chem 279, 18146-18156 Evin, G. & Weidemann, A. (2002). Biogenesis and metabolism of Alzheimer's disease Abeta amyloid peptides. Peptides 23, 1285-1297 Ferree, A. W., Trudeau, K., Zik, E., Benador, I. Y., Twig, G., Gottlieb, R. A., & Shirihai, O. S. (2013). MitoTimer probe reveals the impact of autophagy, fusion, and motility on subcellular distribution of young and old mitochondrial protein and on relative mitochondrial protein age. Autophagy, 9(11), 1887-1896. doi:10.4161/auto.26503 Götz, J., Matamales, M., Götz, N. N., Ittner, L. M., & Eckert, A. (2012). Alzheimer's disease models and functional genomicsHow many needles are there in the haystack?. Frontiers In Physiology, 3320. doi:10.3389/fphys.2012.00320

Green, D. R., & Kroemer, G. (2004). The Pathophysiology of Mitochondrial Cell Death. Science, (5684). 626. Hagberg, H., Mallard, C., Rousset, C., & Thornton, C. (n.d). Mitochondria: hub of injury responses in the developing brain. Lancet Neurology, 13(2), 217-232. Hardy, J., & Selkoe, D. J. (2002). The Amyloid Hypothesis of Alzheimer's Disease: Progress and Problems on the Road to Therapeutics. Science, (5580). 353. Hilenski, L. (2011, April ). Methods Used in Internal Medicine Imaging Core. Retrieved from Emory Medicine, http:// medicine.emory.edu/cardio/ic/index.cfm Hernandez, G., Thornton, C., Stotland, A., Lui, D., Sin, J., Ramil, J., & ... Gottlieb, R. (n.d). MitoTimer A novel tool for monitoring mitochondrial turnover. Autophagy, 9(11), 1852-1861. Iijima-Ando, K., Hearn, S. A., Shenton, C., Gatt, A., LiJuan, Z., & Iijima, K. (2009). Mitochondrial Mislocalization Underlies Ab42-Induced Neuronal Dysfunction in a Drosophila Model of Alzheimer's Disease. Plos ONE, 4(12), 1-13. doi:10.1371/ journal.pone.0008310 Laker, R. C., Peng Xu1, 2., Ryall, K. A., Sujkowski, A., Kenwood, B. M., Chain, K. H., Zhang, M., Royal, M., Hoehn, K., Driscoll., Adler, P., Wessells, R., Saucerman, J., & Zhen Yan1, 2. z. (2014). A Novel MitoTimer Reporter Gene for Mitochondrial Content, Structure, Stress, and Damage in Vivo. Journal Of Biological Chemistry, 289(17), 12005-12015. Lieff, J. (2014, February 2). Dynamic relationship of Mitochondria and Neurons. Retrieved August 31, 2016, from Blog, http://jonlieffmd.com/blog/ dynamic-relationship-of-mitochondria-and-neurons Liochev, S. I. (2013). Review Article: Reactive oxygen species and the free radical theory of aging. Free Radical Biology And Medicine, 601-4. doi:10.1016/j.freeradbiomed.2013.02.011 Mhatre, S. D., Michelson, S. J., Gomes, J., Tabb, L. P., Saunders, A. J., & Marenda, D. R. (2014). Development and characterization of an aged onset model of Alzheimer's disease in Drosophila melanogaster. Experimental Neurology, 261772781. doi:10.1016/j.expneurol.2014.08.021 Minis, A., & Steller, H. (2016). Preview: Krebs Cycle Moonlights in Caspase Regulation. Developmental Cell, 371-2. doi:10.1016/j.devcel.2016.03.016 Moran, M. T., Tare, M., Kango-Singh, M., & Singh, A. (2013). Homeotic Gene Teashirt (tsh) Has a Neuroprotective Function in Amyloid-Beta 42 Mediated Neurodegeneration. eCommons Moreira, P. I., Carvalho, C., Zhu, X., Smith, M. A., & Perry, G. (2010). Mitochondrial dysfunction is a trigger of Alzheimer's disease pathophysiology. Biochimica Et Biophysica Acta, 1802(1), 2-10. doi:10.1016/j.bbadis.2009.10.006 Moses, K Rubin (GM) (1991). Glass encodes a site-specific DNA-binding protein that is regulated in response to positional signals in the developing Drosophila eye. Gnes Dev5: 583-593. Owusu-Ansah, E., Yavari, A., & Banerjee, U. (2008, February 28). Nature Protocals. Retrieved May 25, 2016, from http:// www.nature.com/protocolexchange/protocols/414 Prussing, K., Voigt, A., & Schulz, J. (n.d). Drosophila melanogaster as a model organism for Alzheimer's disease. Molecular Neurodegeneration, 8 Ready, D.F., Hanson, T., & Benzer, S. (1976) Development of the Drosophila retina, a neurocrystalline lattice. Dev Biol 53: 217-240.

Proceedings 2016

NATURAL SCIENCES Remacle, J., Raes, M., Toussaint, O., Renard, P., & Rao, G. (1995). Low levels of reactive oxygen species as modulators of cell function. Mutation Research, 316(3), 103-122. Roberts, D. B. (2006). Drosophila melanogaster: The model organism. Entomologia Experimentalis et Applicata, 121(2), 93–103. doi:10.1111/j.1570-8703.2006.00474.x Santos, R. X., Correia, S. C., Wang, X., Perry, G., Smith, M. A., Moreira, P. I., & Zhu, X. (2010). Alzheimer's disease: diverse aspects of mitochondrial malfunctioning. International Journal Of Clinical And Experimental Pathology, 3(6), 570-581. Sanz, A., Stefanatos, R., & Mellroy, G. (2010). Production of reactive oxygen species by the mitochondrial electron transport chain in Drosophila melanogaster. Springer, 42, 135-142. Sarkar, A., Irwin, M., Singh, A., Riccetti, M., & Singh, A. (2016). Alzheimer's disease: the silver tsunami of the 21(st) century. Neural Regeneration Research, 11(5), 693-697. doi:10.4103/1673-5374.182680 Seifan, A., Schelke, M., Obeng-Aduasare, Y. & Isaacson, R. (2015).Early Life Epidemiology of Alzheimer's Disease – A Critical Review. Neuroepidemiology 45, 237-254, doi:10.1159/000439568 Seo, A. Y., Joseph, A., & Dutta, D. (2010). New insights into the role of mitochondria in aging: mitochondrial dynamics and more. Journal Of Cell Science, 123(15), 2533-2542. Shankar, G. M. et al. (2008). Amyloid-beta protein dimers isolated directly from Alzheimer's brains impair synaptic plasticity and memory. Nat Med 14, 837-842 Singh, A., & Irvine, K. D. (2012). Drosophila as a Model for Understanding Development and Disease. eCommons. Speretta, E., Jahn, T. R., Tartaglia, G. G., Favrin, G., Barros, T. P., Imarisio, S., & ... Dobson, C. c. (2012). Expression in Drosophila of Tandem Amyloid β Peptides Provides Insights into Links between Aggregation and Neurotoxicity. Journal Of Biological Chemistry, 287(24), 20748-20754. Tang, S., Le, P. K., Tse, S., Wallace, D. C., & Huang, T. (2009). Heterozygous mutation of Opa1 in Drosophila shortens lifespan mediated through increased reactive oxygen species production. Plos One, 4(2), e4492. doi:10.1371/journal. pone.0004492 Tare, M., Puli, O. R., Moran, M. T., Kango-Singh, M., & Singh, A. (2013). Domain specific genetic mosaic system in the Drosophila eye. Genesis (New York, N.Y.: 2000), 51(1), 68-74. doi:10.1002/dvg.22355 Tare, M., Modi, R., Nainaparampil, J., Puli, O. R., Bedi, S., Fernandez-Funez, P., Kango-Singh, M. and Singh, A. (2011). Activation of JNK signaling mediates Amyloid-β-dependent cell death. PLoS ONE 6 (9) e24361, 1-12 PMID: 21949710 Terman, J. R., Williamson, W. R., Taehong, Y., & Hiesinger, P. R. (2010). Guidance Receptor Degradation Is Required for Neuronal Connectivity in the Drosophila Nervous System. Plos Biology, 8(12), 1. doi:10.1371/journal.pbio.1000553 Williamson, K. S., Gabbita, S. P., Mou, S., West, M., Pye, Q. N., Markesbery, W. R., Cooney, R.V., Grammas, P., ReimannPhillip, U., Floyd, R., & Hensley, K. (2002). The nitration product 5-nitro-gamma-tocopherol is increased in the Alzheimer brain. Nitric Oxide: Biology And Chemistry / Official Journal Of The Nitric Oxide Society, 6(2), 221-227.

Proceedings 2016

NATURAL SCIENCES

A Review of the Glycosylation of Aquaporin-3 in Mammalian and Anuran Species Christopher J. Turley1,2,3 and Carissa M. Krane1 University of Dayton, 300 College Park, Dayton, OH, 45469 1. Department of Biology 2. Berry Summer Thesis Program 3. University Honors Program

Thesis Mentor: Carissa M. Krane, Ph.D. Department of Biology Corresponding Author: Carissa M. Krane, Ph.D. Email: ckrane1@udayton.edu

Abbreviations AA: Amino Acid

AQP: Aquaporin

GLP: Aquaglyceroporin

ICC: Immunocytochemistry

MIP: Major Intrinsic Protein

NPS: Nonpenetrating Solutes

PS: Penetrating Solutes

RBCs: Red Blood Cells (erythrocytes)

Abstract Major Intrinsic Proteins (MIPs) are a family of channel proteins that allow for the movement of water, and sometimes organic molecules, across the cellular membrane with the osmotic gradient. The MIP family can be broken down into three functional classifications: Aquaporins (AQPs), Aquaglyceroporins (GLPs) and Super Aquaporins. GLPs are channel proteins that allow for the movement of both water and specific small organic molecules across the cellular membrane. Aquaglyceroporin 3 (AQP3), is expressed in both mammalian and anuran RBCs, kidney cells and skin cells. AQP3 protein is post-translationally modified with N-linked glycosylation, but the effect of this glycosylation is not experimentally understood. However, based on the role of glycosylation in other MIPs, it is likely that glycosylation stabilizes AQP3 in colder environments.

Principles Surrounding Osmotic Equilibrium The cellular membrane acts as a semipermeable barrier between extracellular and intracellular 56

fluid compartments. The cellular membrane is also essential in cell-cell communication and acts as a protective and selective barrier [10, 60]. In the early 1970s, Singer and Nicholson proposed the widely accepted fluid mosaic model of the cellular membrane [61]. According to this model, the cellular membrane is a selectively permeable, phospholipid bilayer that is studded with various transport and channel proteins [10, 60, 61]. The phospholipid bilayer is oriented so that hydrophobic (water insoluble) lipid tails are sandwiched between hydrophilic (water soluble) phosphate heads. This â&#x20AC;&#x153;sandwichâ&#x20AC;? orientation gives the cellular membrane its selectively permeable nature, meaning that only certain molecules are able to freely cross with the concentration gradient [10, 60, 61]. Ideally, cells exist in a state of homeostasis. In the context of nonpenetrating solutes (NPS) and ions, which cannot freely cross the cellular membrane, this is often a state of uneven distribution. This chemical and electrical disequilibrium give cells the ability to fulfill certain physiological processes, such as cellular communication [60, 61]. In addition to NPSs and ions, cells are also influenced by the presence of penetrating solutes (PS) and water, which freely cross the cellular membrane to achieve equilibrium [60]. The diffusion, or movement, of water across a cellular membrane is known as osmosis [10]. The osmotic flux of water across a cellular membrane is directly related to the concentration of NPS, or the osmolarity. If a cell, with a high osmolarity, were to be introduced to a Proceedings 2016

NATURAL SCIENCES solution that has a lower osmolarity, an osmotic gradient would form. This gradient would result in the net movement of water from outside of the cell, across the cellular membrane, into the cell. Once the extracellular fluid and the intracellular fluid have similar osmolarities, the cell would begin to approach osmotic equilibrium, where the flow of water across the cellular membrane is equal in both directions.

Major Intrinsic Proteins Major intrinsic proteins (MIPs) are a family of channel proteins that allow for the flux of water and, in some cases, small organic molecules across the cellular membrane. They were first isolated in the mid 1970s from the lens of bovine eyes [7, 16], but their function and distribution was not completely understood until the early 1990s [1, 13, 16, 56, 63]. Since this time, the MIP family in mammals has grown to include 13 individual members, which can be subdivided into three functional groups: Aquaporins, Aquaglyceroporins and Super Aquaporins [reviewed in 57]. In addition to these mammalian MIPs, there are also several orthologous AQPs, which are found in plants, insects and a variety of other organisms [reviewed in 9, 46]. Because the functions of Aquaporins and Aquaglyceroporins are well characterized, they will be the focus of this paper. As can be seen in Figure 1, members of the MIP family have six transmembrane spanning domains connected by three extracellular loops and two intracellular loops [30]. Both intracellular loop B and extracellular loop E contain a highly conserved amino acid region of Asparagine, Proline and Alanine (NPA). Within the membrane, the MIP assembles in to what is referred to as the â&#x20AC;&#x153;hour-glassâ&#x20AC;? structure [30], which positions the two NPA regions together to form the functional pore. This pore allows for the flux of water and, in some cases, small organic molecules across the cellular membrane. In addition to the NPA region, extracellular loop E also contains a conserved Cysteine amino acid, which confers to mercury inhibition of MIPâ&#x20AC;&#x2122;s function as a water/glycerol channel [65]. AQPs are often expressed in the cellular membrane as tetramers, or groupings of four monomeric units [36]. The tetramerization of AQPs was found to occur outside of the golgi apparatus and is suggested to have no effect on the rate of osmotic flux in AQPs. Tetramerization is, however, Proceedings 2016

likely to affect the insertion of AQPs into the membrane when triggered by a sudden change in osmolarity [32]. The four monomeric units of a tetramer are held together by amino acid interactions found in extracellular Loop E [36]. Because this amino acid region is not found in GLPs, it has been suggested that these MIPs are primarily inserted into the cellular membrane as monomers instead of tetramers. This is, however, conflated with more recent findings on AQP3, which show that the protein is expressed in the cellular membrane as both monomers and oligomers, or groupings of monomers [58].

Figure 1. General Structure of Major Intrinsic Protein. The red balls correspond to the cellular membrane and the black line to the MIP. Additionally, the green, orange and purple dots represent the conserved NPA region and the yellow dot represents the conserved cysteine region. Reproduced from [33]. Krane, C.M. and Goldstein, D.L. (2007) Mammalian Genome; 18:452-462.

Aquaporins Currently, five of the thirteen known mammalian MIPs (AQP0, AQP1, AQP2, AQP4 and AQP5) are classified as traditional Aquaporins. While all five of these AQPs function as water channel proteins, each has a unique physiological role. AQP1, the first member of the MIP family to be characterized as a functional water pore, was originally isolated from human erythrocytes as CHIP 28, the channel- like integral protein of 28 kDa [1, 13, 56, 63]. Since this initial isolation and characterization, AQPs have been found expressed in the cells of various mammalian tissues. Mammalian aquaporins are expressed in a wide variety of cell types where they function in diverse physiological processes including absorption and secretion mechanisms, cell volume regulation, and fluid homeostasis. For example, in the lens of the eye, AQPs allow for the transmembrane flux of water that hydrates 57

NATURAL SCIENCES the lens [34, 35]. In kidney cells, AQP expression functions in urinary concentration mechanisms [12, 21, 50]. In the brain, AQPs are expressed in astrocytes where they help maintain the osmotic pressure and communication within the cranium [20, 40, 51]. In skin cells, AQPs are expressed in sweat glands, where they function in the secretion of water for thermoregulation [48]. Finally, the transmembrane flux of water with the concentration gradient in lung cells is also maintained by AQP expression [69].

Expression of AQP3 in Mammals

Aquaglyceroporins

Kidney Cells AQP3 is expressed in the kidneys of rats, mice and humans. In rats, AQP3 is found localized in the basolateral membrane of medullary collecting duct cells and in renal connecting tubule cells [11, 14, 15]. In mice, AQP3 is also found in kidney medullary collecting duct cells [44]. In humans, AQP3 is found primarily in the basolateral membrane of kidney medulla collecting duct cells [28]. Functionally, the medullary collecting duct and renal collection tubules are responsible for the reabsorption of water from liquid waste, or urine [60]. AQP3 is suggested to take part in the urine concentrating mechanism, which requires the expression of AQPs and GLPs in order to work [42, 43].

Aquaglyceroporins (GLPs) are channel proteins that allow for the transmembrane flux of both water and small organic molecules, such as glycerol. Structurally, GLPs have a larger aromatic/arginine region (Ar/R), which is a conserved region that forms the narrowest part of the NPA pore. This larger Ar/R region most likely contributes to GLPâ&#x20AC;&#x2122;s ability to allow larger organic molecules to pass through its pore [27]. AQP3 was the first mammalian AQP to be identified as a GLP [28]. Since its initial isolation and characterization, AQP3 has been found localized in numerous tissues across a variety of organisms. As seen in Table 1, AQP3 is found in mammalian, as well as anuran, kidney cells [11, 15, 28], erythrocytes (RBCs) [58, 59, 71] and skin cells [6, 22, 45]. Like many other cellular proteins, the expression of AQP3 is dynamic and is largely dependent upon physiological conditions such as pH and temperature [73, 74].

Erythrocytes AQP3 is expressed in the membranes of rat and human erythrocytes (RBCs), but is absent in mouse RBCs [58, 71]. RBCs are responsible for bringing water and nutrients to cells and disposing of cellular waste [60]. When AQP1 is down regulated, AQP3 was found to act as a water channel for transmembrane water flux [59]. It is therefore likely that AQP3 aids in the flux of both water and glycerol across the cellular membrane of RBCs, with the concentration gradient.

Skin Cells AQP3 is expressed in the skin of rats, mice and humans. In rats and mice, AQP3 is found primarily in the basal and intermediate layers of the epidermis [22, 45]. In humans, AQP3 is found

Table 1. Summary of AQP3 Expression in Mammalian and Anuran Erythrocytes, Kidney Cells and Skin Cells

Proceedings 2016

NATURAL SCIENCES in the epidermis and in the stratum corneum layers [6, 29]. In addition to being expressed, AQP3 is upregulated in water deprived rat and human skin cells [45, 67]. Based on the localization it is likely that AQP3 plays a significant role in the hydration and healing of mammalian skin [22, 45, 64].

Mammalian GLPs- AQP7, AQP9 and AQP10 Since the isolation of AQP3, three other GLPs have been identified in mammals: AQP7, AQP9 and AQP10. While all GLPs have the same basic function, their physiological roles vary. AQP3 and AQP7 are expressed in mouse embryos during preimplantation, a major phase in the early development of mammalian embryos. While the function of these AQPs is not fully known, their down regulation results in the death of developing embryos [70]. In addition to its localization in developing embryos, AQP7 is also found in the renal proximal tubes of mouse kidneys. In the proximal tubule, AQP7 primarily functions as a channel for glycerol reabsorption [62]. Furthermore, AQP7 is found in pancreatic beta cells, where it mediates glycerol concentration. Because the concentration of glycerol is suggested to influence insulin production, AQP7 is a potential target for diabetes treatment [41]. AQP9 is the major glycerol channel in mouse RBCs [38]. In the brain, AQP9 is expressed in glial cells, astrocytes and in certain neurons. While AQP9 has not been identified as a major osmoregulator, it is likely that it acts a metabolic channel for neurons, which use glycerol and lactose as energy sources [3]. The final GLP, AQP10, is primarily expressed in the duodenum, jejunum and ileum of mammalian small intestines. Here, AQP10 absorbs glycerol and helps maintain proper osmoregulation [24]. AQP3 is also coexpressed with AQP10 in the stratum corneum (SC) layer of human skin. Together, these AQPs maintain skin hydration in varying environmental conditions [29].

AQP3 Expression Anurans Evolutionarily, amphibians were the first vertebrates to make the transition to a terrestrial habitat [10]. This transition makes amphibians exciting to study in conjunction with mammals because many of their adaptations have been Proceedings 2016

conserved within the mammalian genome. Copeâ&#x20AC;&#x2122;s Gray Treefrog (Hyla chrysoscelis) and the Japanese Treefrog (Hyla japonica) are of particular interest to this study because they not only contain AQPs homologous to AQP3, but because they have also developed an acclimation mechanism of freeze tolerance [Reviewed in 66]. Freeze tolerance, a coping mechanism for subfreezing temperatures, utilizes small organic molecules, cryoprotectants, to control freezing [66]. H. chrysoscelis likely utilize glycerol as a cryoprotectant [66] and studies on H. japonica are exploring glycerol and glucose as a potential cryoprotectants [23, 26]. When released, cryoprotectants surround the organismsâ&#x20AC;&#x2122; cells and lower the freezing point of the extracellular free water. Additionally, cryoprotectants likely utilize the osmotic gradient to cross the cellular membrane through GLPs to stabilize the internal osmolarity [66]. In H. chrysoscelis, AQP HC-3 is the only GLP that has been identified to date [75]. In H. japonica, there are multiple GLPs including AQP-h3BL and AQP-h9, which are homologous to mammalian AQP3 and AQP9 [2, 26]. Based on the results of a freeze fracture study of AQP3 in mammalian erythrocytes, the oligomerization of AQP3 in the plasma membrane is dynamic [58]. Results produced monomers, dimers, trimers and tetrameric assemblies of AQP3. Figure 2 (see page 60) depicts the predicted monomeric structure and quaternary tetrameric assembly of HC-3 within the lipid bilayer, based on high degree of amino acid identity shared between AQP3 and HC-3. Erythrocytes AQP HC-3 is expressed in the plasma membrane of erythrocytes (RBCs) in H. chrysoscelis [55]. Additionally, AQP HC-3 expression is upregulated in cold acclimated organisms, or organisms that have adjusted to winter temperatures [47]. AQP-h3BL is not expressed in the RBCs of Hyla japonica [2]. AQP HC-3 is suggested to be the channel protein responsible for the transmembrane flux of glycerol with the concentration gradient. This mechanism could aid in both cryoprotection and osmoregulation. Kidney Cells AQP HC-3 is expressed in the apical and basolateral membranes of H. chrysoscelis endothelium cells [47]. AQP-h3BL is expressed in the basolateral membrane of proximal tubule cells and in the collecting duct cells of H. japonica [2, 54]. In H. chrysoscelis, AQP HC-3 59

NATURAL SCIENCES

Figure 2. Hour Glass Figure of AQP HC-3. Homology based images of AQP HC-3 developed using SWISS IMAGE software (proteinmodelportal.org). Images A and B correspond to monomeric units of AQP HC-3 and images C and D correspond to the tetrameric assembly of the monomers in the plasma membrane. In each of the images: the blue ribbons correspond to transmembrane loop 1 (amino acid sequence 21-48); the dark green ribbons correspond to transmembrane loop 2 (AA sequence 54-74); the light green ribbons correspond to transmembrane loop 3 (AA sequence 103-123); the yellow ribbons correspond to transmembrane loop 4 (AA sequence 161-180); the orange ribbons correspond to transmembrane loop 5 (AA sequence 192-214); and the red ribbons correspond to transmembrane loop 6 (AA sequence 242-261) [4, 17, 19, 31]. Image by C. Turley, 2016.

allows for the transmembrane flux of cryoprotectant glycerol in preparation for freezing and likely contributes to the concentration mechanism of urine [75]. In H. japonica kidneys, AQP-h3BL is suggested to aid in osmoregulation and a different GLP, AQP-h9, is suggested to be responsible for cryoprotectant flux [2, 54]. Skin Cells Both H. chrysoscelis and H. japonica express AQP3 homologs in the basolateral membrane of stratum granulosum skin cells [2, 53, 55, 68]. While both anuran and mammalian species are constantly losing water due to evaporation through their skin, anurans have thinner skin and are more susceptible to dehydration [reviewed in 37]. Therefore, it is likely that both AQP HC-3 and AQP-h3BL are used to maintain skin hydration through water absorption and glycerol excretion.

Glycosylation of AQP3 A perpetuating theme in cellular biology and biochemistry states that the function of a protein is influenced by its structure [10]. Upon translation, a protein is often altered with different post-translational modifications. These modifications can range from the â&#x20AC;&#x153;foldingâ&#x20AC;? of a protein with chaperons, to attaching different moieties onto the protein [49]. Because post-translational modifications physically alter the structure of proteins, it is likely that they also influence their function in some way.

Overview of Glycosylation Glycosylation, or the attachment of a sugar moiety (oligosaccharide) onto a protein, is one of the many forms of post-translational modification. The two most common types of glycosylation are O-linked and N-linked [49]. O-linked glycosylation is the attachment of an oligosaccharide onto the hydroxyl group of either a serine or threonine amino acid. In order for O-linked glycosylation to occur, proteins must be inserted in to small vesicles located in the cytosol of the cell. Usually O-linked oligosaccharides are only 1-2 sugar residues long, and each sugar group is added to the protein one molecule at a time. N-linked glycosylation differs from O-linked insofar as it involves the attachment of an oligosaccharide onto the amide group of asparagine amino acids. Additionally, N-linked glycosylation usually includes a more sporadic attachment of larger, more branched sugar groups. Furthermore, N-linked oligosaccharides are added onto protein molecules inside of the Rough Endoplasmic Reticulum and modifications are made in the Golgi Apparatus [39, 49].

N-Linked Glycosylation of AQP3 As can be seen in Table 2, both mammalian and anuran AQP3 homologs are found post translationally modified with N-linked glycosylation. In rats and humans, AQP3 is glycosylated in RBCs [59, 63], kidney cells [14, 28] and skin cells [45, 64]. While AQP3 has potential N-linked glycosylation sites in mice (unpublished results retrieved from NetNGlyc 1.0 Server), its glycosylation has not yet been confirmed through experimental Proceedings 2016

NATURAL SCIENCES Table 2. Summery of the Glycosylation of AQP3 in Mammalian and Anuran RBCs, Kidneys and the Skin. *Results generated using NetNGlyc 1.0 Server [18]

evidence. AQP HC-3 is glycosylated in H. chrysoscelis RBCs, kidney cells, and skin cells [47, 55, 75], and AQP-h3BL is glycosylated in H. japonica skin cells and kidney cells [2, 53]. The effects of glycosylation on AQP3 function are not currently understood, so comparisons will be made between it and other MIPs that have known functions for glycosylation.

The Effects of Glycosylation on AQP3 According to Darwinian theory, organisms evolve in such a way that their traits are best suited for environmental success [10]. In order to be successful, organisms must conserve energy and resources. Because glycosylation requires cells to expend energy and use resources, it is likely to contribute to the successful expression and/or function of MIPs. In mammalian red blood cells, glycosylation has no apparent effect on the oligomerization of AQP3 [58]. These results parallel those of another mammalian MIP, AQP2 [25]. Glycosylation of AQP2 was found, however, to stabilize mutated proteins before insertion into the cellular membrane [8]. There are, however, no commonly known mutations of AQP3, which makes these findings incomparable. AQP10 and AQP HC-3 are both GLPs that are post-translationally modified with N-linked glycosylation. AQP10 is glycosylated at Asn133 [52] and AQP HC-3 is glycosylated at Asn141*. Glycosylation of mammalian AQP10 stabilizes proteins expressed in temperatures below 6 Â°C [52]. As mentioned earlier, both AQP HC-3 and AQP-h3BL are expressed in freeze tolerant organisms [2, 75]. Additionally, the expression and glycosylation of AQP HC-3 increases in cold acclimated anurans [47, 55]. Given the similarities between function as a glycerol transporter and proximity of the glycosylation sites, it is Proceedings 2016

probable that glycosylation of AQP-HC3 could have the same stabilizing effects that are seen in the glycosylation of AQP10. Therefore, glycosylation of AQP3 is most likely an evolutionary adaptation that improves the stability of its expression in cellular membranes at colder temperatures.

Original Research Results Copeâ&#x20AC;&#x2122;s Gray Treefrog (Hyla chrysoscelis) is a species of frog that is found throughout the eastern half of the United States [5]. As previously mentioned, H. chrysoscelis has evolutionarily developed a mechanism of freeze tolerance to survive harsh winter conditions. Based on living conditions, we hypothesize that RBCs from freshly caught H. chrysoscelis will have a more abundant expression and membrane localization of AQP HC-3 in the cellular membrane when compared to cells from lab-acclimated organisms. In order to better understand the differences between lab acclimated and freshly caught frog cells, protein was isolated from the erythrocytes of H. chrysoscelis. Using immunoblotting techniques, AQP HC-3 was found to be upregulated in RBCs from freshly caught H. chrysoscelis when compared to protein samples from the RBCs of lab acclimated H. chrysoscelis. Additionally, immunocytochemistry (ICC) revealed an apparent localization of HC-3 around the cellular membrane of RBCs from freshly caught H. chrysoscelis.

Western Blots RBC samples from freshly caught and lab acclimated H. chrysoscelis were collected and cultured according to the techniques established by Mutyam et al. [47]. In order to determine 61

NATURAL SCIENCES

Figure 3. Western Blot Results. Lanes 1 and 2 contain proteins isolated from the RBCs of lab acclimated frogs before and after a 24-hour culture. In addition to lanes 3 and 4, lanes 5 and 6 contain proteins isolated from the RBCs of freshly caught frogs before and after a 24-hour culture. Each column contains HC-3 immuno-reactive bands (32kDa) and Beta Actin Bands (42kDa). Additionally, high molecular weight bands that likely correspond to glycosylated HC-3 were detected (75-200kDa). Image by C. Turley, 2016.

abundance, protein was isolated from each of the RBC cultures. The protein was then loaded in to SDS- Page gels, which separates protein based on size. As seen in Figure 3, AQP HC-3 immunoblots form 31.5kDa bands. Quantitatively, AQP HC-3 is more abundant in protein samples isolated from the RBCs of freshly caught H. chrysoscelis. While there appears to be no difference in AQP HC-3 abundance after the 24hr. culture, two of the protein samples isolated from cells after the 24hr. culture period have larger immunoreactive bands around the 75-200kDa range. Based on

previous studies, these are likely glycosylated forms of AQP HC-3 [47]. Beta actin, the control protein, was present in all samples around the 42kDa range, in equal proportions.

ICC Images RBC samples collected from freshly caught and lab acclimated H. chrysoscelis were fixed and, using immunocytochemistry, AQP HC-3 subcellular localization was examined. Figure 4 corresponds to the images captured using a confocal

Figure 4. ICC Images of Erythrocytes. (A) Immunocytochemistry shows AQP HC-3 distribution in the cytosol (green) and the location of the nucleus (blue) in erythrocytes of a lab acclimated frog (60x). (B) Immunocytochemistry shows AQP HC-3 distribution in the cytosol (green) and the location of the nucleus (blue) in the erythrocytes of a freshly caught frog (60x). Images by C. Turley, 2016.

Proceedings 2016

NATURAL SCIENCES microscope. As seen in Figure 4A, RBCs from lab acclimated H. chrysoscelis have an even distribution of AQP HC-3 throughout the cell. This is contrasted by RBCs from freshly caught H. chrysoscelis, in which AQP HC-3 is localized within the cellular membrane (Figure 4B).

physiological cues, initiated during a period of cold-acclimation, will trigger cells via an intracellular signaling cascade, to synthesize, glycosylate, and insert aquaglyceroporins into the cell membrane to allow glycerol to permeate cells as a cryoprotectant. Based upon previous studies of AQP3 in human Caco 2 cells [72], epinephrine is the focus of future neurohormone studies.

Discussion Based on the Western Blot results, it appears that RBCs obtained from freshly caught H. chrysoscelis have a greater abundance of AQP HC-3, when compared to lab-acclimated animals. Additionally, the ICC images of RBCs suggest that, when compared to lab-acclimated organisms, more AQP HC-3 is inserted in the plasma membrane of freshly caught H. chrysoscelis. Together, these results suggest that an inherent difference in AQP HC-3 expression and localization exists between the RBCs of freshly caught and lab acclimated H. chrysoscelis. Such a difference is likely to be the result of the environmental conditions that each type of frog was exposed to. Unlike freshly caught frogs, lab acclimated H. chrysoscelis have been living in a controlled environment free of environmental stresses such as dehydration. Therefore, it is likely that the RBCs of these anurans have slowly adjusted their expression of AQP HC-3 to fit the needs of their controlled environment. Because freshly caught H. chrysoscelis came from an unpredictable environment, it was advantageous for their cells to be ready for droughts and sudden rain storms. It is likely that the cells of these organisms prepared for such events by expressing more AQP HC-3 in the cellular membrane. Glycosylation of AQP HC-3 was detected in two of the samples after the 24-hour culture. This means that the time in culture triggered a cellular mechanism that resulted in the glycosylation of AQP HC-3. This is interesting considering that these RBCs were separated from hormone releasing organs. Additionally, in one of the freshly caught RBC samples, there is no apparent difference in glycosylation after the 24-hour culture. This is likely to be due to inter-individual differences found in nature. With an understanding of how AQP HC-3 differs in lab acclimated and freshly caught H. chrysoscelis RBCs, the cell signaling pathway needs to be investigated. We further hypothesize that Proceedings 2016

Conclusion Based on its similarities to AQP10, the N-linked glycosylation of AQP3, and of its homologs, is suggested to stabilize proteins expressed in cold environments. This helps explain how H. japonica and H. chrysoscelis cells are able to survive the physiological stresses brought on by freezing. In a study that focused specifically on the expression of AQP HC-3 in freshly caught and lab acclimated H. chrysoscelis RBCs, cells from freshly caught organisms were found to express more AQP HC-3. Additionally, AQP HC-3 was found localized more in the cellular membrane of freshly caught treefrog RBCs. After culture, AQP HC-3 was found glycosylated in both lab-acclimated and freshly caught treefrog RBCs. Together, these results could suggest that glycosylation is elicited by stresses such as cooler temperatures or separation of the cells from the organism. GLPs maintain physiological functions that are essential for an organismâ&#x20AC;&#x2122;s survival. Because structure and the physical characteristics such as stability of proteins are so closely related, it is crucial that post-translational modifications such as N-linked glycosylation be studied in the context of alteration to physical properties. From such results, conclusions on how we can stabilize the expression of GLPs in different conditions can be made.

Acknowledgements I would like to thank the University of Dayton Honors Program, the Berry Family Foundation, the Schuellein Endowed Chair and the National Science Foundation for providing me with the funding necessary to engage in research this summer. I would also like to also thank the members of the Krane lab, Dr. Clara do Amaral, Lauren McDaniel and Raphael Crum, for all of their help and support this summer. Finally, I would like to thank Dr. Carissa Krane for all 63

NATURAL SCIENCES of the time and energy she invested in me as a student and as a researcher. Without her influence and patience this summer would not have been a success.

References 1.

Agre P, Saboori AM, Asimos A. Purification and partial characterization of the Mr 30,000 integral membrane protein associated with the erythrocyte Rh(D) antigen. Journal of Biological Chemistry. 262: 17497–17503, 1987.

Akabane G, Ogushi Y, Hasegawa T, Suzuki M, Tanaka S. Gene cloning and expression of an aquaporin (AQP-h3BL) in the basolateral membrane of waterpermeable epithelial cells in osmoregulatory organs of the tree frog. The American Journal of PhysiologyRegulatory, Integrative and Comparative Physiology. 292: R2340-R2351, 2007.

Badaut J, Petit JM, Brunet JF, Magistretti PJ, CharriautMarlangue C, Regli L. Distribution of Aquaporin 9 in the adult brain: preferential expression in catecholaminergic neurons and in glial cells. Neuroscience. 128: 27-38, 2004. Biasini M, Bienert S, Waterhouse A, Arnold K, Studer G, Schmidt T, Kiefer F, Cassarino TG, Bertoni M, Bordoli L, Schwede T. SWISS-MODEL: Modelling Protein Tertiary and Quaternary Structure using Evolutionary Information. Nucleic Acids Research. 42: W252-W258, 2014. Blackburn LM, Najappa P, Lannoo MJ. United States Amphibian District Maps. (2001) Department of Biology at Ball State University. http://home.bsu.edu/ home/00mjlannoo [12 September 2016]

Boury-Jamot M, Sougrat R, Tailhardat M, Varlet BL, Bonté F, Dumas M, Verbavatz JM. Expression and function of aquaporins in human skin: Is aquaporin-3 just a glycerol transporter? Biochimica et Biophysica Acta- Biomembranes. 1758: 1034-1042, 2006.

Broekhuyse RM, Kuhlmann ED, Stols ALH. Lens membranes II: Isolation and characterization of the main intrinsic polypeptide (MIP) of bovine lens fiber membranes. Experimental Eye Research. 23: 365-371, 1976.

Buck TM, Eledge J, Skach WR. Evidence for aquaporin-2 folding mutants by N-linked glycosylation in endoplasmic reticulum. American Journal of Physiology. 287: C1292-C1299, 2004.

Campbell EM, Ball A, Hoppler S, Bowman AS. Invertebrate aquaporins: A review. Journal of Comparative Physiology B-Biochemical Systemic and Environmental Physiology. 178: 935-955, 2008.

10. Campbell NA, Reece JB, Urry LA, Cain ML, Wasserman SA, Minorsky PV, Jackson RB. Biology. 8th Edition. San Francisco, CA: Pearson, 2008: p. 77-86, 125-135. 11. Colman RA, Wu DC, Liu J, Wade JB. Expression of aquaporins in the renal connecting tubule. The American Journal of Physiology: Renal Physiology. 279: F874-F883, 2000. 12. Deen PM, Verdijk MA, Knoers NV, Wieringa B, Monnens LA, van Os CH, van Oost BA. Requirement of human renal water channel aquaporin-2 for vasopressin-dependent concentration of urine. Science. 264: 92-95, 1994.

13. Denker BM, Smith BL, Kuhajda FP. Identification, purification, and partial characterization of a novel Mr 28,000 integral membrane protein from erythrocytes and renal tubules. Journal of Biological Chemistry. 263: 15634–15642, 1988. 14. Ecelbarger CA, Terris J, Frindt G, Echevarria M, Marples D, Nielsen S, Knepper MA. Aquaporin-3 water channel localization and regulation in rat kidney. American Journal of Physiology: Renal Physiology. 269: F663-F672, 1995. 15. Echevarria M, Windhager EE, Tate SS, Frindt G. Cloning and expression of AQP3, a water channel from medullary collecting duct of rat kidney. Proceedings of the National Academy of Sciences of the United States of America. 91: 10997-11001, 1994. 16. Gorin MB, Yancey SB, Cline J. The major intrinsic protein (MIP) of the bovine lens fiber membrane: Characterization and structure based on cDNA cloning. Cell. 39: 49–59, 1984. 17. Guex N, Peitsch MC, Schwede T. Automated Comparative Protein Structure Modeling with SWISSMODEL and Swiss-PdbViewer: A Historical Perspective. Electrophoresis. 30: S162-S173, 2009. 18. Gupta R, Jung E, Brunak S. Prediction of N-glycosylation sites in human proteins [online]. Center for Biological Sequence Analysis- Technical University of Denmark. http://www.cbs.dtu.dk/services/NetNGlyc/ [21 Sep 2016]. 19. Haas J, Roth S, Arnold K, Kiefer F, Schmidt T, Bordoli L, Schwede T. The protein model portal—a comprehensive resource for protein structure and model information. The Journal of Biological Databases and Curation. NA: 1-8, 2013. 20. Haji-Yasein NN, Vindedal GF, Eliert-Olsen M, Gundersen GA, Skare Ø, Laake P, Klungland A, Thorén AE, Burkhardt JM, Ottersen OP, Nagelhus EA. Glialconditional deletion of aquaporin-4 (Aqp4) reduces blood-brain water uptake and confers barrier function on perivascular astrocyte endfeet. Proceedings of the National Academy of Sciences of the United States of America. 108: 17815-17820, 2011. 21. Hara-Chikuma M, Verkman AS. Aquaporin-1 facilitates epithelial cell migration in kidney proximal tubule. Journal of the American Society of Nephrology. 17: 39-45, 2006. 22. Hara M, Ma T, Verkman AS. Selectively reduced glycerol in skin of aquaporin-3-deficient mice may account for impaired skin hydration, elasticity, and barrier recovery. The Journal of Biological Chemistry. 277: 46616-46621, 2002. 23. Hasegawa T, Tanii H, Suzuki M, Tanaka S. Regulation of water absorption in the frog skins by two vasoactindependent water channel aquaporins, AQP-h2 and AQP-h3. Endocrinology. 144: 4087-4096, 2003. 24. Hatakeyama S, Yoshida Y, Tani T, Koyama Y, Nihei K, Ohshiro K, Kamiie JI, Yaoita E, Suda T, Hatakeyama K, Yamamoto T. Cloning of a new Aquaporin (AQP10) abundantly expressed in duodenum and jejunum. Biochemical and Biophysical Research Communications. 287: 814-819, 2001. 25. Hendricks G, Koudijs M, van Balkom BW, Oorshot V, Klumperman J, Deen PM, van der Sluijis P. Glycosylation is important for cell surface expression of the water channel aquaporin-2 but is not essential for tetramerization in the endoplasmic reticulum. Journal of Biological Chemistry. 279: 2975-2983, 2004.

Proceedings 2016

NATURAL SCIENCES 26. Hirota A, Takiya Y, Sakamoto J, Shiojiri N, Suzuki M, Tanaka S, Okada R. Molecular cloning of cDNA encoding an Aquaglyceroporin, AQP-h9, in the Japanese Tree Frog, Hyla japonica: possible roles of AQP-h9 in freeze tolerance. Zoological Science. 32: 292-306, 2015. 27. Hub JS, de Groot BL. Mechanism of selectivity in aquaporins and aquaglyceroporins. Proceedings of the National Academy of Sciences of the United States of America. 105: 1198-1203, 2007. 28. Ishibashi K, Sasaki S, Uchida S, Kuwahara M, Saito H, Furuwaka T, Nakajima K, Yamaguchi Y, Gojobori T. Molecular cloning and expression of a member of the aquaporin family with permeability to glycerol and urea in addition to water expressed at the basolateral membrane of kidney collecting duct cells. American Journal of Physiology- Cell Physiology. 266: C189-C197, 1994. 29. Jungerstead JM, Bomholt J, Bajraktari N, Hansen JS, Klaerke DA, Penderson PA, Hedfalk K, Nielson KH, Agner T, Helix-Nielson C. In vivo studies of aquaporin 3 and 10 in human stratum corneum. Archives of Dermatological Research. 305: 699-704, 2013. 30. Jung JS, Preston GM, Sith BL, Guggino WB, Agre P. Molecular structure of the water channel through aquaporin CHIP: The hourglass model. Journal of Biological Chemistry. 269: 14648-14654, 1994. 31. Kiefer F, Arnold K, Küzli M, Bordoli L, Schwede T. The SWISS-MODEL Repository and Associated Resources. Nucleic Acids Research. 37: D387-D392, 2009. 32. Kitchen P, Conner MT, Bill RM, Conner AC. Structural determinants of oligomerization of the Aquaporin-4 channel. The Journal of Biological Chemistry. 291: 6858-6871, 2016. 33. Krane CM, Goldstein DL. Comparative functional analysis of aquaglyceroporins/ glyceroporins in mammals and anurans. Mammalian Genome. 18: 452-462, 2007. 34. Kumari SS, Gupta N, Shiels A, FitzGerald PG, Menon AG, Mathias RT, Varadaraj K. Role of aquaporin 0 in lens biomechanics. Biochemical and Biophysical Research Communications. 462: 339-345, 2015. 35. Kumari SS, Varadaraj K. Aquaporin 0 plays a pivotal role in refractive index gradient development in mammalian eye lens to prevent spherical aberration. Biochemical and Biophysical Research Communications. 452: 986-991, 2014. 36. Lagrée V, Froger A, Deschamps S, Pellerin I, Delamarche C, Bonnec G, Gouranton J, Thomas D, Hubert JF. Oligomerization state of water channels and glycerol facilitators: involvement of loop E. The Journal of Biological Chemistry. 273: 33949-33953, 1998. 37. Lillywhite HB. Water relations of tetrapod integument. Journal of Experimental Biology. 209: 202-226, 2006. 38. Liu Y, Promeneur D, Rojek A, Kumar N, Frøkiær J, Nielsen S, King LS, Agre P, Carbrey JM. Aquaporin 9 is the major pathway for glycerol uptake by mouse erythrocytes, with implications for malarial virulence. Proceedings of the National Academy of Sciences of the United States of America. 104: 12560-12564, 2007. 39. Lodish H, Berk A, Zipursky SL, Matsudaira P, Baltimor D, Darnell J. Molecular Cell Biology. 4th Edition. New York, NY: W.H. Freeman and Company, 2000, Section 17.7, Protein Glycosylation in the ER and Golgi Complex. Available from: http://www.ncbi.nlm.nih.gov/books/NBK21744.

Proceedings 2016

40. Manley GT, Binder DK, Papadopoulos MC, Verkman AS. New insights into water transport and edema in the central nervous system from phenotype analysis of aquaporin-4 null mice. Neuroscience. 129: 981-989, 2004. 41. Matsumura K, Chang BHJ, Fujimiya M, Chen W, Kulkarni RN, Eguchi Y, Kimura H, Kojima H, Chan L. Aquaporin 7 is a β-cell protein and regulator of intraislet glycerol content and glycerol kinase activity, β-cell mass, and insulin production and secretion. Molecular and Cellular Biology. 27: 6026-6037, 2007. 42. Ma T, Frigeri A, Hasegwa H, Verkman AS. Cloning of a water channel homolog expressed in brain meningeal cells and kidney collecting duct that functions as a stilbene-sensitive glycerol transporter. Journal of Biological Chemistry. 269: 21845-21849, 1994. 43. Ma T, Frigeri A, Skach W, Verkman AS. Cloning of a novel rat kidney cDNA homologous to CHIP28 and WCH-CD water channels. Biochemical and Biophysical Research Communications. 197: 654-659, 1993. 44. Ma T, Song Y, Yang B, Gillespie A, Carlson EJ, Epstein CJ, Verkman AS. Nephrogenic diabetes insipidus in mice lacking Aquaporin-3 water channels. Proceeding of the National Academy of Sciences of the United States of America. 97: 4386-4391, 2000. 45. Matsuzaki T, Suzuki T, Tanaka K. Water channel protein AQP3 is present in epithelial exposed to the environment of possible water loss. Journal of Histochemistry and Cytochemistry. 47: 1275-1286, 1999. 46. Maurel C, Boursiac Y, Luu DT, Santoni V, Shahzad Z, Verdoucq L. Aquaporins in plants. Physiological Reviews. 95: 1321-1358, 2015. 47. Mutyam V, Puccetti MV, Frisbie J, Goldstein DL, Krane CM. Dynamic regulation of aquaglyceroporin expression in erythrocyte cultures from cold- and warm acclimated Cope’s Gray Treefrog, Hyla chrysoscelis. The Journal of Experimental Zoology. 315: 424-437, 2011. 48. Nejsum LN, Kwon TH, Jensen UB, Fumagalli O, Frokiaer J, Krane CM, Menon AG, King LS, Agre PC, Nielsen S. Functional requirement of aquaporin-5 in plasma membranes of sweat glands. Proceedings of the National Academy of Sciences of the United States of America. 99: 511–516, 2002. 49. Nelson DL, Cox MM. Lehninger: Principles of Biochemistry. 5th Edition. New York, NY: W.H. Freeman and Company, 2008, p. 1101-1105. 50. Nielsen S, DiGiovanni SR, Christensen EI, Knepper MA, Harris HW. Cellular and subcellular immunolocalization of vasopressin-regulated water channel in rat kidney. Proceedings of the National Academy of Sciences of the United States of America. 90: 11663-11667, 1993. 51. Nielsen S, Nagelhus EA, Amiry-Moghaddam M, Bourque C, Agre P, Ottersen OP. Specialized membrane domains for water transport in glial cells: high-resolution immunogold cytochemistry of aquaporin-4 in rat brain. The Journal of Neuroscience. 17: 140-141; 171-180, 1997. 52. Öberg F, Sjöhamn J, Fischer G, Moberg A, Pedersen A, Neutze R, Hedfalk K. Glycosylation increases the thermostability of human aquaporin 10 protein. Journal of Biological Chemistry. 286: 31915-31923, 2011. 53. Ogushi Y, Kitagawa D, Gasegawa T, Suzuki M, Tanaka S. Correlation between aquaporin and water permeability in response to vasotocin, hydrin and beta-adrenergic effectors in the ventral pelvic skin of the treefrog Hyla japonica. Journal of Experimental Biology. 213: 288-294, 2010.

NATURAL SCIENCES 54. Ogushi Y, Mochida H, Nakakura T, Suzuki M, Tanaka S. Immunocytochemical and phylogenetic analyses of an arginine vasotocin-dependent aquaporin, AQP-h2K, specifically expressed in the kidney of the treefrog, Hyla japonica. Endocrinology. 148: 5891-5901, 2007.

69. Willumsen NJ, Davis CW, Boucher RC. Selective response of human airway epithelia to luminal but not serosal solution hypertonicity. Possible role for proximal airway epithelia as an osmolality transducer. The Journal of Clinical Investigation. 94: 779-787, 1994.

55. Pandey RN, Yanganti S, Coffey S, Frisbie J, Alnajjar K, Goldstein DL. Expression and immunolocalization of aquaporins HC-1, -2, and -3 in Cope’s Gray Treefrog, Hyla chrysoscelis. Comparative Biochemistry and Physiology Part A. 157: 86-94, 2010.

70. Xiong Y, Tan YJ, Ziong YM, Huang YT, Hu XL, Lu YC, Ye YH, Wang TT, Zhang D, Jin F, Huang HF, Sheng JZ. Expression of aquaporins in human embryos and potential role of AQP3 and AQP7 in preimplantation mouse embryo development. Cellular Physiology and Biochemistry. 31: 649-658, 2013.

56. Preston GM, Agre P. Isolation of the cDNA for erythrocyte integral membrane protein of 28 kilodaltons: Member of an ancient channel family. Proceedings of the National Academy of Sciences USA. 88: 11110– 11114, 1991. 57. Rojek A, Praetorius J, Frøkiaer J, Nielsen S, Fenton RA. A current view of the mammalian aquaglyceroporins. Annual Review of Physiology. 70: 301-327, 2008. 58. Roudier N, Bailly P, Gane P, Lucien N, Gobin R, Catron JP, Ripoche P. Erythroid expression and oligomeric state of the AQP3 protein. Journal of Biological Chemistry. 277: 7664-7669, 2002. 59. Roudier N, Vervavatz JM, Maurel C, Ripoche P, Tacnet F. Evidence for the presence of aquaporin-3 in human red blood cells. Journal of Biological Chemistry. 273:84078412, 1998. 60. Silverthorn DU, Johnson BR, Ober WC, Garrison CW, Silverthorn AC. Human Physiology: An Integrated Approach. 6th Edition. Glenview, IL: Pearson, 2013, p. 132-138, 153, 512-523, 663-664. 61. Singer SJ, Nicholson GL. The fluid mosaic model of the structure of cell membranes. Science. 175: 627-650; 720-731, 1972.

71. Yang BX, Ma TH, Verkman KS. Erythrocyte water permeability and renal function in double knockout mice lacking aquaporin -1 and aquaporin-3. Journal of Biological Chemistry. 276: 624-628, 2001. 72. Yasui H, Kubota M, Iguchi K, Usui S, Kiho T, Hirano K. Membrane trafficking of aquaporin 3 Induced by epinephrine. Biochemical and Biophysical Research Communications. 373: 613-617, 2008. 73. Yasui M, Hazama A, Kwon TH, Nielsen S, Guggino WB, Agre P. Rapid gating and anion permeability of an intracellular aquaporin. Nature. 402: 184-187, 1999. 74. Zeuthen T, Klaerke DA. Transport of water and glycerol in Aquaporin 3 is gated by H+. Journal of Biological Chemistry. 274: 21631-21636, 1999. 75. Zimmerman SL, Frisbie J, Goldstein DL, West J, Rivera K, Krane CM. Excretion and conservation of glycerol, and expression of aquaporins and glyceroporins, during cold acclimation in Cope’s Gray Treefrog Hyla chrysoscelis. American Journal of Physiology- Regulatory, Integrative and Comparative Physiology. 292: R544-R555, 2007.

62. Skowronski MT, Lebeck J, Rojek A, Praetorius J, Füchtbauer EM, Frøkiaer J, Nielsen S. AQP7 is localized in capillaries of adipose tissue, cardiac and striated muscle: implications in glycerol metabolism. The American Journal of Physiology-Renal Physiology. 292: F956-F965, 2007. 63. Smith BL, Agre P. Erythrocyte Mr 28,000 transmembrane protein exists as a multisubunit oligomer similar to channel proteins. Journal of Biological Chemistry. 266: 6407-6415, 1991. 64. Sougrat R, Morand M, Gondran C, Barrè P, Gobin R, Bonté F, Dumas M, Verbavatz JM. Functional expression of AQP3 in human skin epidermis and reconstructed epidermis. The Journal of Investigative Dermatology. 118: 678-685, 2002. 65. Spinello A, de Almeida A, Casini A, Barone G. The inhibition of glycerol permeation through aquaglyceroporin-3 Induced by mercury (II): A molecular dynamics study. Journal of Inorganic Biochemistry. 160: 78-84, 2016. 66. Storey KB, Storey JM. Natural Freezing Survival in Animals. Annual Review of Ecology and Systematics. 27: 365-386, 1996. 67. Sugiyama Y, Ota Y, Hara M, Inoue S. Osmotic stress up-regulates aquaporin-3 expression in cultured human keratinocytes. Biochimica et Biophysica ActaBiomembranes. 1522: 82-88, 2001. 68. Tanii H, Hasegawa T, Hirakawa N, Suzuki M, Tanaka S. Molecular and cellular characterization of a water-channel protein, AQP-h3, specifically expressed in the frog ventral skin. Journal of Membrane Biology. 188: 43-53, 2002.

Proceedings 2016

NATURAL SCIENCES

Resolving the Molecular Mechanisms by Which DNA Mutations Alter the Function of a Cis-Regulatory Element Emily E. Wey1,2 and Thomas M. Williams3,4 University of Dayton, 300 College Park, Dayton, OH, 45469 1. Department of Chemistry 2. University Honors Program 3. Department of Biology 4. Center for Tissue Regeneration and Engineering

Thesis Mentor: Thomas M. Williams, Ph.D. Department of Biology Corresponding Author: Emily E. Wey Email: weye02@udayton.edu

Abstract Each human genome possesses around a million mutations that are genetic baggage from DNA replication mistakes or “mutations” that occurred in the past. Each mutation can have one of three outcomes on an individual, these are to improve, reduce or have no effect on health. Moreover, the effects of such mutations can depend on the presence or absence of other mutations, so called epistatic interactions. A major goal of genomic medicine is to glean diagnostic or predictive health information from the genome sequences of individuals. However, this goal remains out of reach as the effects of mutations and epistatic interactions are difficult to predict without knowing the function of the DNA sequence they reside in. This difficulty is especially heightened for mutations occurring in cis-regulatory element sequences that act as switches to control gene transcription. The research I propose to perform for my thesis is to use a fruit fly model to test hypotheses about the molecular mechanisms by which mutations alter a cis-regulatory element’s activity and test whether certain mutations are subjected to the tyranny of epistatic interactions. I will study the Drosophila melanogaster dimorphic element, which is a transcription-regulating switch for the bric-à-brac genes. Three mutations in the dimorphic element were identified that individually alter the level of bric-à-brac transcription. The presence or absence of epistatic interactions will be determined by measuring the activity of Proceedings 2016

dimorphic elements from related species that have been engineered to possess the Drosophila melanogaster mutations. I will also test the hypothesis that these mutations impart their effects by creating or destroying binding sites for proteins known as transcription factors. The results will provide a sorely needed example where an understanding of molecular mechanisms bridges the gap between a DNA sequence and its in vivo function.

Introduction Mutation is an inevitable by-product of individual cells replicating their DNA, and when mutations occur in the DNA of the cells involved in reproduction, these new mutations have the opportunity to become part of the species gene pool. For any mutation three outcomes are possible. The mutation can be deleterious, making an individual less fit compared to its peers. A mutation can be advantageous, increasing the possessor’s fitness, or it can be functionally neutral. To complicate matters, the outcomes of mutations can depend upon the presence or absence of other mutations elsewhere in the genome, a phenomenon that is known as epistasis (Taylor & Ehrenreich 2015; Liu et al. 2012) (Figure 1). For humans, mutations are responsible for a considerable deal of variation in traits that impact health (Wellcome et al. 2007). Thus, a major challenge for genetics research is to identify which mutations in an individual’s genetic makeup will 67

X NATURAL SCIENCES to reside in CREs (Sethupathy & Collins 2008; Visel et al. 2009; Wellcome et al. 2007), including those contributing to obesity (Claussnitzer et al. 2015), asthma (Moffatt et al. 2007), lactose tolerance (Tishkoff et al. 2007) and heart attack risk (Musunuru et al. 2010).

Figure 1. Hypothetical outcomes for mutations. Courtesy of E.E. Wey and T.M. Williams, 2016.

impact their well-being (or “fitness”). Identifying these “functionally-relevant” mutations and their epistatic interactions requires an understanding of how these subtle differences in DNA can affect the molecular mechanisms of cellular functions. Problematically, individual genomes and the gene pools of species are vast and identifying the meaningful mutations remains a significant obstacle to using an individual’s DNA sequence effectively as a part of personalized medicine.

The recognition that CREs are a deep reservoir of genetic variation emerged in the past 10 years (Wray 2007; Carroll 2005; Carroll 2008; Stern 2010), the future challenge is to learn how to tap into this reservoir to extract the useful information (Sadee & Hartmann 2014). It is estimated that the human genome has over half a million CREs (Lindblad-Toh et al. 2011; Andersson et al. 2014; Kvon et al. 2014), and within a gene pool CREs possess an inordinate wealth of mutations. Thus, any individual can be expected to possess a figurative “haystack” of a hundred thousand or more mutations in its CREs, and the chore for geneticists and clinicians is to determine which mutations are the “needles” that affect fitness (Zhang et al. 2015; Wang et al. 2015; Smith et al. 2013). This requires understanding how both the DNA sequences of CREs function and how a change to these sequences can alter their normal switch activity.

Cis-Regulatory Elements as Reservoirs for Mutations That Impact Human Fitness The human genome has been found to possess over 22,000 genes that each encode a particular protein (Lander et al. 2001), though the DNA sequences encoding the information to make these proteins is estimated to comprise less than 3% of the genome (Pennisi 2012). A much greater proportion of the human genome is comprised of DNA sequences known as cis-regulatory elements (CREs) (Neph et al. 2012) that function as switches to turn ON and OFF the making of a gene or genes protein product(s) (Levine 2010; Maston et al. 2006), referred to as gene expression. CREs effectively control in which of the hundreds of different cell types in the body and when during an individual’s embryonic development or adult life that genes are expressed (Figure 2). As a consequence, most mutations that cause variation in health related traits seem 68

Figure 2. The control of gene expression by CREs. (A) The expressed sequences of genes (white boxes) are often controlled by multiple (colored ovals) CREs. (B-D) CREs can direct expression (colored regions) in stages and cell types of the developing embryo and (E) in cells, tissues and organs of an adult. Courtesy of E.E. Wey and T.M. Williams, 2016.

Proceedings 2016

NATURAL SCIENCES X General Features of CREs and Their Functions While the functional parts of most CREs remain unknown, several CREs have been well studied and together illustrate some general features of their functional components (Levine 2010). These double stranded DNA (base pairs) sequences tend to be around a hundred to a thousand base pairs in length, and the particular “A,” “C,” “T” or “G” nucleotide bases are more important in certain locations than others. The places of importance are often 5-15 base pairs in length and these short motifs function as binding sites for proteins known as transcription factors (Figure 3A). Animals such as fruit flies and humans have ~750 and ~1,400 unique transcription factors respectively, and these different factors bind to distinct binding site DNA sequences on which they can contribute either activator or repressor activity to the element’s overall transcriptionregulating activity (Vaquerizas, Juan M., Kummerfeld, Sarah K., Teichmann, Sarah A., Luscombe 2009; Pfreundt et al. 2010; Jolma et al. 2013). The activity of CREs in certain cell types, life times or environmental conditions requires a combination of transcription factors to bind to a number of binding sites within a CRE, what is referred to as the CRE’s regulatory logic (Figure 3B) (Arnone & Davidson 1997; Swanson et al. 2010; Small et al. 1992). The switch activity of a CRE in a given cell type or lifetime requires the correct combination of transcription factors to be present in a cell’s nucleus in order to have the CRE turn the regulated gene either ON or OFF. With this coarse understanding of CREs,

Figure 3. General features of CRE regulatory logic. (A) Representation of transcription factor binding sites within a CRE and (B) an overall regulatory logic of CRE-bound transcription factors. Colored shapes represent transcription factors. Courtesy of E.E. Wey and T.M. Williams, 2016.

Proceedings 2016

it is reasonable to suspect that many functionaltering CRE mutations create or destroy binding sites for a particular transcription factor protein and thereby alter the regulatory logic. At present it is difficult to infer any CRE’s regulatory logic from its DNA sequence alone, and therefore predicting which mutations alter regulatory logic remains beyond our grasp (Rebeiz & Williams 2011).

A Fruit Fly Model to Study CREs, Their Mechanism of Action and Function Altering Mutations Fruit flies of the genus Drosophila present an excellent model organism system to ask fundamental question about regulatory logic and its evolution through mutations. First these species share many of the same CREs, though the DNA sequences diverged since the species last shared a common ancestor (Stark et al. 2007). Second, fruit flies are multicellular animals that possess many of the same transcription factor genes as humans, and these shared proteins have been largely found to function in similar ways in both species. Third, the fruit fly species Drosophila (D.) melanogaster is a convenient organism in which to perform tests on CRE functions, as it is feasible to compare the switch activity of various CREs, including those for which a mutation or mutations have been introduced (Figure 2) (Rebeiz & Williams 2011). One well studied CRE is known as the dimorphic element because it activates the expression of the bric-à-brac (bab) genes in the female dorsal abdominal epidermis during metamorphic (pupal) development (Williams et al. 2008). This pattern of gene expression activation requires a regulatory logic that includes 14 binding sites for the transcription factor Abd-B and two for the transcription factor DSX. A previous study identified that dimorphic element sequences obtained from different D. melanogaster populations had measurably different abilities to activate the expression of a green fluorescent protein (GFP) reporter gene in transgenic fruit fly pupae (Rogers et al. 2013) (Figure 4). The increased and decreased activities were largely caused by three “derived mutations” that each altered a single base pair out of nearly 1,500, what became known as the “D,” “F” and “L” mutations. More than 20 additional mutations appeared to be functionally neutral 69

NATURAL SCIENCES X

Figure 4. Mutations that alter the function of the dimorphic element. (A) Phylogenetic relationship of D. melanogaster dimorphic element alleles with respect to an outgroup species (D. simulans). The drived (B) "D," (C) "F" and (D) "L" mutations. Expression of GFP in transgenic D. melanogaster pupae driven by the (E) wild type dimorphic element and the wild type element modified to posses the (F) "D," (G) "F" and (H) "L" mutations. The CRE activity was measured as the level of fluorescence in intensity among replicate specimens compared to that for the wild type. Standard error of the mean (%) included for each activity. Figure was based upon that published in Rogers et al., PLoS Genetics (2013). Courtesy of E.E. Wey and T.M. Williams, 2016.

with respect to this CRE’s switch activity and thus it remains a mystery why these three mutations are functionally meaningful but not the other 18. Moreover, it remained unclear whether the D, F and L mutations only had their expression-altering effects in the presence or absence of certain mutations, so called epistatic interactions.

A Thesis Investigating the Occurrence of Epistasis and Molecular Mechanisms for CRE Mutations My thesis research aims to answer four questions pertaining to the derived D, F and L mutations. Does the dimorphic element’s sequence environment have epistatic effects on the regulatory activity outcomes of these three mutations? Do these derived mutations create or destroy binding sites for transcription factors that function as activators or repressors? What is the identity and activity of the transcription factor that has gained or lost a binding site due to each derived mutation? How do these mutations differ from the others that were found to be neutral? Collectively, this research delves into one of the most difficult challenges facing modern genetics research. 70

It remains largely unknown whether CRE mutations are rigidly constrained by their local DNA sequence environment. The first hypothesis that my research aimed to test was that the effects of the derived D, F and L mutations are subjected to widespread epistasis with other natural dimorphic element mutations. I am testing this hypothesis using a DNA mutagenesis approach to separately graft these derived mutations into the dimorphic elements of D. mauritiana, D. yakuba and D. auraria, three species with increasingly more DNA divergence to that of the D. melanogaster dimorphic element (Figure 5). The modified dimorphic elements of these divergent species will be tested for their ability to activate the expression of the GFP reporter gene in transgenic D. melanogaster by the Williams Lab’s standard protocol (Rogers & Williams 2011) (Figure 6). If the mutations have no effect on the regulatory activity of these

Figure 5. Phylogenetic relatedness of the fruit fly species to be studied. Courtesy of E.E. Wey and T.M. Williams, 2016.

Proceedings 2016

NATURAL SCIENCES X

Figure 6. Representation of a reporter transgene. Each reporter transgene will include a dimorphic element CRE sequence adjacent to a minimal promoter (arrow) and the protein coding sequence for a nuclear-localized Green Fluorescent Protein (called GFP). These will be introduced into D. melanogaster in order to observe CRE activities from the patterns and levels of the GFP produced. Courtesy of E.E. Wey and T.M. Williams, 2016.

heterologous dimorphic elements or an effect opposite to what was found when tested in the wild type D. melanogaster dimorphic element (Figure 4), then I will conclude that the derived mutations encounter widespread epistasis. If the mutations behave similarly to that observed for the D. melanogaster sequence, then I will conclude that these mutations encounter littleto-no epistasis from other CRE mutations. This latter outcome would show a resilience of the three mutations to cohabiting CRE sequence differences as the species share a nucleotide identity to the D. melanogaster sequence of only 88%, 82% and <80% for D. mauritiana, D. yakuba, and D. auraria respectively. This divergence makes the melanogaster CRE sequence different from these three species at over ~180, ~270 and >300 nucleotide sites. A second hypothesis that my thesis will test is that the D, F and L mutations each altered the D. melanogaster dimorphic element by creating or destroying a binding site for a transcription factor protein. For example, the L mutation was shown to increase the activity of the dimorphic element (Figure 4). If this mutation affected a binding site sequence, then I can anticipate that a mutation to the flanking base pairs of this mutation would result in a loss of the gained activity upon expression of the GFP reporter gene (Figure 7). This outcome would also indicate that the transcription factor interacting with the created binding site acts as an “activator” of gene expression. To test this hypothesis I will create

Figure 7. Example of a flank mutation scheme and the inferences that can be made regarding the transcription factor binding site effects that the derived L mutation had. Courtesy of E.E. Wey and T.M. Williams, 2016.

Proceedings 2016

a mutant version of the CRE for which four of five base pairs on each side of the L mutation is altered (referred to as the “L mutant + flanks mutation”) and analyzing the resulting level of GFP expression in transgenic D. melanogaster. It is also possible that the L mutation did not create a binding site, but rather destroyed an existing one. This would be seen if the flanks mutation to the wild type dimorphic element resulted in the GFP expression increasing to a magnitude comparable to that caused by the L mutation on its own (Figure 7). Moreover, this outcome would suggest that the binding site that was destroyed was bound by a transcription factor that acts as a “repressor” of gene expression. I will take the same experimental approach to study the significance of the D and F mutations. If the flanks mutations produce data indicative of the D, F and/or L mutations creating or destroying a transcription factor binding sites, my goal will be to identify which of the ~750 D. melanogaster transcription factors interacts with the binding sites gained or lost by the derived mutations. This will reveal how the dimorphic element’s regulatory logic of Abd-B and DSX binding sites was supplemented or streamlined by these new mutations. By traditional methods, studying an interaction between one DNA sequence and one protein is time consuming and resource draining. In order to test the thousands of potential interactions that may occur in vivo, I will collaborate with the lab of Dr. Bart Deplancke to use their high throughput yeast one-hybrid assay to identify transcription factors that can bind to these sequences in nuclei of yeast cells (Deplancke et al. 2004; Hens et al. 2011). This method allows nearly 700 D. melanogaster transcription factors to be tested one-by one for interactions with a specific DNA sequence in yeast nuclei in a single experiment. Any transcription factor found to interact in yeast cells with a dimorphic element sequence will be considered a “candidate” for a possible in vivo function. I will then test these candidate transcription factors for functional binding within the abdominal epidermis of D. melanogaster by an RNA-inhibition (RNA-i) approach (Rogers et al. 2014). Here RNA-i will be used to reduce the expression of the transcription factor in the D. melanogaster abdomen and the activity (ability to drive GFP reporter gene expression) of the dimorphic element will be measured in order to see whether activity has increased or decreased compared to a control abdomen. 71

NATURAL SCIENCES X One final goal of my thesis will be to investigate why these three derived mutations were special, whereas twenty or more other identified mutations are seemingly neutral. This investigation will include looking at the conservation of the base pairs surrounding each mutation between related species, use of bioinformatic tools to predict whether the neutral mutations failed to make or destroy a transcription factor binding site or to see whether binding sites were made but for a transcription factor that is not expressed in the necessary cell types. Ultimately, this thesis will provide a thorough characterization of mutations that effect a D. melanogaster CRE, highlighting a role for or absence of epistasis, and elucidating molecular mechanisms by which mutations have their functional effects. Success here may reveal instructive lessons that scientists can apply to studies of CREs and their mutations in any animal species, notably for those of our species which may facilitate using genomic DNA sequence data in routine medical care.

Acknowledgments The authors would like to thank the Berry Family for their support during the 2016 summer. Thanks are owed to Dr. Will Rogers for making this project possible by his research into the dimorphic element CRE and sharing some of his previous data for including into figures. Additional thanks are owed to Dr. Eric M. Camino and Dr. Bart Deplancke for their efforts in advancing the yeast one-hybrid experiments for this project. E.E. Wey is supported by the University of Dayton Honor’s Program and The Berry Summer Thesis Institute.

References Andersson, R. et al., 2014. An atlas of active enhancers across human cell types and tissues. Nature. 507(7493), pp.455–461. Available at: http://www.nature.com/doifinder/10.1038/ nature12787 [Accessed March 26, 2014]. Arnone, M.I. & Davidson, E.H., 1997. The hardwiring of development: organization and function of genomic regulatory systems. Development (Cambridge, England). 124(10), pp.1851–64. Available at: http://www.ncbi.nlm.nih. gov/pubmed/9169833. Carroll, S.B., 2008. Evo-devo and an expanding evolutionary synthesis: a genetic theory of morphological evolution. Cell. 134(1), pp.25–36. Available at: http://www.ncbi. nlm.nih.gov/pubmed/18614008 [Accessed July 24, 2011].

Carroll, S.B., 2005. Evolution at two levels: on genes and form. PLoS Biology. 3(7), p.e245. Available at: http://www. pubmedcentral.nih.gov/articlerender.fcgi?artid=1174822&to ol=pmcentrez&rendertype=abstract [Accessed July 6, 2011]. Claussnitzer, M. et al., 2015. FTO Obesity Variant Circuitry and Adipocyte Browning in Humans. New England Journal of Medicine. p.150819140043007. Available at: http://www.nejm. org/doi/abs/10.1056/NEJMoa1502214. Deplancke, B. et al., 2004. A Gateway-Compatible Yeast One-Hybrid System. Genome Research. 14(508), pp.2093–2101. Hens, K. et al., 2011. Automated protein DNA interactions screening of Drosophila regulatory elements. Nature Methods. 8, pp.1065–1070. Jolma, A. et al., 2013. DNA-Binding Specificities of Human Transcription Factors. Cell. 152(1-2), pp.327–339. Available at: http://linkinghub.elsevier.com/retrieve/pii/ S0092867412014961 [Accessed January 17, 2013]. Kvon, E.Z. et al., 2014. Genome-scale functional characterization of Drosophila developmental enhancers in vivo. Nature. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/24896182 [Accessed July 9, 2014]. Lander, E.S. et al., 2001. Initial sequencing and analysis of the human genome. Nature. 409(6822), pp.860–921. Available at: http://www.ncbi.nlm.nih.gov/pubmed/11237011. Levine, M., 2010. Transcriptional enhancers in animal development and evolution. Current Biology: CB. 20(17), pp.R754–63. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/20833320 [Accessed July 22, 2014]. Lindblad-Toh, K. et al., 2011. A high-resolution map of human evolutionary constraint using 29 mammals. Nature. 478(7370), pp.476–481. Available at: http://dx.doi.org/10.1038/ nature10530. Liu, Y. et al., 2012. Gene , pathway and network frameworks to identify epistatic interactions of single nucleotide polymorphisms derived from GWAS data. BMC Systems Biology. 6(Suppl 3), pp.1–12. Maston, G.A., Evans, S.K. & Green, M.R., 2006. Transcriptional Regulatory Elements in the Human Genome. Annu. Rev. Genomics Hum. Genet. 29, pp.29–59. Moffatt, M.F. et al., 2007. Genetic variants regulating ORMDL3 expression contribute to the risk of childhood asthma. Nature. 448(July), pp.470–474. Musunuru, K. et al., 2010. From noncoding variant to phenotype via SORT1 at the 1p13 cholesterol locus. Nature. 466(7307), pp.714–9. Available at: http://www.pubmedcentral. nih.gov/articlerender.fcgi?artid=3062476&tool=pmcentrez&r endertype=abstract [Accessed June 11, 2011]. Neph, S. et al., 2012. An expansive human regulatory lexicon encoded in transcription factor footprints. Nature. 489(7414), pp.83–90. Available at: http://www.pubmedcentral.nih.gov/ articlerender.fcgi?artid=3736582&tool=pmcentrez&renderty pe=abstract [Accessed July 11, 2014]. Pennisi, E., 2012. ENCODE Project Writes Eulogy For Junk DNA. Science. 337(7 September), pp.1159–1160. Pfreundt, U. et al., 2010. FlyTF: improved annotation and enhanced functionality of the Drosophila transcription factor database. Nucleic Acids Research. 38(Database issue), pp.D443–7. Available at: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2808907&tool=p mcentrez&rendertype=abstract [Accessed July 13, 2012].

Proceedings 2016

NATURAL SCIENCES X Rebeiz, M. & Williams, T.M., 2011. Experimental Approaches to Evaluate the Contributions of Candidate Cis- regulatory Mutations to Phenotypic Evolution. In V. Orgogozo & M. V. Rockman, eds. Methods in Molecular Biology. Totowa, NJ: Humana Press, pp. 351–375. Available at: http://www. springerlink.com/index/10.1007/978-1-61779-228-1 [Accessed November 9, 2011]. Rogers, W.A. et al., 2014. A survey of the trans-regulatory landscape for Drosophila melanogaster abdominal pigmentation. Developmental Biology. 385(2), pp.417–432. Available at: http://www.ncbi.nlm.nih.gov/pubmed/24269556 [Accessed December 18, 2013]. Rogers, W.A. et al., 2013. Recurrent Modification of a Conserved Cis-Regulatory Element Underlies Fruit Fly Pigmentation Diversity M. Levine, ed. PLoS Genetics. 9(8), p.e1003740. Available at: http://dx.plos.org/10.1371/journal. pgen.1003740 [Accessed September 1, 2013]. Rogers, W.A. & Williams, T.M., 2011. Quantitative Comparison of cis-Regulatory Element (CRE) Activities in Transgenic Drosophila melanogaster. Journal of Visualized Experiments. (58), pp.2–7. Available at: http://www.ncbi.nlm. nih.gov/pubmed/22215325 [Accessed January 22, 2012]. Sadee, W. & Hartmann, K., 2014. Missing heritability of common diseases and treatments outside the protein ‑ coding exome. Human Genetics. 133, pp.1199–1215. Sethupathy, P. & Collins, F.S., 2008. MicroRNA target site polymorphisms and human disease. Trends in Genetics: TIG. 24(10), pp.489–97. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/18778868 [Accessed September 6, 2011]. Small, S., Blair, a & Levine, M., 1992. Regulation of evenskipped stripe 2 in the Drosophila embryo. The EMBO Journal. 11(11), pp.4047–57. Available at: http://www. pubmedcentral.nih.gov/articlerender.fcgi?artid=556915&tool =pmcentrez&rendertype=abstract.

Vaquerizas, Juan M., Kummerfeld, Sarah K., Teichmann, Sarah A., Luscombe, N.M., 2009. A census of human transcription factors: function, expression and evolution. Nature Reviews Genetics. 10, pp.252–263. Visel, A., Rubin, E.M. & Pennacchio, L.A., 2009. Genomic views of distant-acting enhancers. Nature. 461(7261), pp.199–205. Available at: http://www.pubmedcentral.nih.gov/ articlerender.fcgi?artid=2923221&tool=pmcentrez&renderty pe=abstract [Accessed June 17, 2011]. Wang, E. et al., 2015. Seminars in Cancer Biology Predictive genomics: A cancer hallmark network framework for predicting tumor clinical phenotypes using genome sequencing data. Seminars in Cancer Biology. 30, pp.4–12. Available at: http://dx.doi.org/10.1016/j. semcancer.2014.04.002. Wellcome, T., Case, T. & Consortium, C., 2007. Genome-wide association study of 14,000 cases of seven common diseases and 3,000 shared controls. Nature. 447(7145), pp.661–78. Available at: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2719288&tool=pmcentrez&rendertype=abs tract [Accessed July 9, 2014]. Williams, T.M. et al., 2008. The regulation and evolution of a genetic switch controlling sexually dimorphic traits in Drosophila. Cell. 134(4), pp.610–23. Available at: http://www. pubmedcentral.nih.gov/articlerender.fcgi?artid=2597198&t ool=pmcentrez&rendertype=abstract [Accessed August 4, 2011]. Wray, G.A., 2007. The evolutionary significance of cis-regulatory mutations. Nature Reviews. Genetics. 8(3), pp.206–16. Available at: http://www.ncbi.nlm.nih.gov/ pubmed/17304246 [Accessed July 17, 2011]. Zhang, X., Kuivenhoven, J.A. & Groen, A.K., 2015. Forward Individualized Medicine from Personal Genomes to Interactomes. Frontiers in Physiology. 6(December), pp.1–11.

Smith, J.J. et al., 2013. Sequencing of the sea lamprey (Petromyzon marinus) genome provides insights into vertebrate evolution. Nature Genetics. 45(4), pp.415–421. Available at: http://www.ncbi.nlm.nih.gov/pubmed/23435085 [Accessed February 27, 2013]. Stark, A. et al., 2007. Discovery of functional elements in 12 Drosophila genomes using evolutionary signatures. Nature. 450(7167), pp.219–32. Available at: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2474711&tool=pmce ntrez&rendertype=abstract [Accessed March 4, 2012]. Stern, D.L., 2010. Evolution, Development, & the Predictable Genome 1st ed. Greenwood Village: Roberts & Company Publishers. Swanson, C.I., Evans, N.C. & Barolo, S., 2010. Structural rules and complex regulatory circuitry constrain expression of a Notch- and EGFR-regulated eye enhancer. Developmental Cell. 18(3), pp.359–70. Available at: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2847355&tool=pmce ntrez&rendertype=abstract [Accessed August 11, 2011]. Taylor, M.B. & Ehrenreich, I.M., 2015. Higher-order genetic interactions and their contribution to complex traits. Trends in Genetics. 31(1), pp.34–40. Available at: http://dx.doi. org/10.1016/j.tig.2014.09.001. Tishkoff, S.A. et al., 2007. Convergent adaptation of human lactase persistence in Africa and Europe. Nature Genetics. 39(1), pp.31–40. Available at: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2672153&tool=p mcentrez&rendertype=abstract [Accessed July 7, 2011].

Proceedings 2016

HEALTH SCIENCE

Development of an Evidence-Based Strength Training Program for Individuals with Dementia Participating in Adult Day Services Jaclyn H. Franz1,3,4 and Kurt Jackson2 University of Dayton, 300 College Park, Dayton, OH, 45469 1. Department of Health and Sport Science 2. Department of Physical Therapy 3. Berry Summer Thesis Institute 4. University Honors Program

Thesis Mentor: Kurt Jackson, Ph.D., PT, GCS Department of Physical Therapy Corresponding Author: Kurt Jackson, Ph.D., PT, GCS Email: kjackson1@udayton.edu

Abstract Falling and loss of mobility present serious risks for elderly adults, especially those with cognitive impairments such as dementia. These risks have been shown to be significantly reduced when elderly adults participate in exercises focusing on strength and balance of sufficient intensity. Despite these potential benefits, many adult day programs do not incorporate exercise in a systematic and progressive fashion to achieve desirable improvements in function. The purpose of this project was to develop an evidence-based exercise program, later titled Simply Strong, for reducing fall risk and improving mobility in elderly adults with dementia participating in Goodwill Easter Seals adult day services. An extensive literature review of current research into the implementation and resulting outcomes of exercise for older adults with dementia was conducted. A supplementary survey of Goodwill Easter Seals program managers regarding barriers and needs was conducted. Barriers to providing such a program were identified through the survey and addressed in the creation of the program so that this program, Simply Strong, and other programs of a similar nature, have an increased likelihood of being utilized long-term. Based on the current literature, an evidencebased training program, titled Simply Strong, was developed to meet the needs of older adults 74

with dementia and through the results of the staff survey was specifically tailored for individuals with dementia at Goodwill Easter Seals Adult Day Service. Staff members of Goodwill Easter Seals were instructed in providing the program so that the program remained self-sustaining after the conclusion of this project. Additionally, a training manual, an accompanying video and an equipment cart to assist in the implementation of the program was fabricated and then donated to one Goodwill Easter Seals location.

Background and Purpose An Aging America America is currently facing a dramatic rise in its population of adults aged 65 and over. By the year 2030 this single population is expected to make up 20% of America’s total population; this is a steep growth from the comparative 13% in 2010, or even the 9.8% in 1970. In 2050, this population is expected to encompass nearly 83.7 million people, nearly double of its 43.1 million estimate from 2012 (Ortman 2014). This upsurge of America’s elderly population raises several concerns, one of which being the expected toll of health care systems as a result of this population’s susceptibility to injury. The high prevalence of injury can be traced to this population’s increased fall risk due to muscle reduction. Proceedings 2016

HEALTH SCIENCE Fall Risk in Elderly Adults With age, an individual’s overall muscle mass decreases, thus increasing the likelihood that this person will fall as a result of muscle weakness. It is expected that one in three seniors will fall each year and of those falls, one in five will result in serious injury. (Why You Fall 2015). In the year 2000, America reported 10,300 fatal and 2.6 million non-fatal, but still medically treated, injuries as result of falls in the population of adults aged 65 and over (Karlsson 2013). This is the exact population that is expected to increase considerably in the years to come. While the danger of falls presents little risk to the otherwise healthy adult, fall risk can be especially threatening to cognitively impaired adults.

Dementia and Alzheimer’s Influence on Fall Risk Up to 5.3 million Americans have Alzheimer’s disease, the most common form of dementia (Herbert 2003). Dementia is now the sixth leading cause of death in the United States, killing more than breast and prostate cancer combined (Alzheimer’s Association 2016). The prevalence of the disease is only set to rise dramatically within the upcoming years due to the aging population. Elderly adults with dementia are at an increased risk for falls and have much lower mobility than their peers without dementia (Tinetti 1988, van Dijk PT 1993). The risk of injury due to falls, including fractures, is greater in people with impaired cognitive function (Buchner DM, Myers AH). Adults with dementia have a prevalence of falling that is two to three times higher than their peers (Harlein et al. 2009). This loss of mobility, with the related risk of falls and injury, creates a steep burden on immediate family members and society as a whole (CDC 2014). It is estimated that in 2016 dementia will cost the United States 236 million dollars in health care. This is approximately $5,000 per family caring for a loved one with the disease (Alzheimer’s Association 2016).

The Role of Physical Activity and Adult Day Centers for Improving Function Current clinical guidelines indicate that exercises emphasizing lower body strength and balance of sufficient intensity can serve as Proceedings 2016

an effective primary intervention for reducing falls and improving independence in elderly adults, both with and without dementia (Burton 2015). Strength training programs are shown to be the most effective and least expensive fall prevention strategy in the elderly community as well as the only intervention that reduces both the number of falls and fall rate (Karlsson, 2013). It is, therefore, imperative that elderly adults commit to strength training to reduce their chance of falling and the overarching pressure on societal medical care caused by fall risk. In response to an increasing elderly population and resulting pressure on caregivers, adult day service programs are increasing in number. These programs form a crucial aspect of support for families as well as needed social exposure and physical activity for elderly persons. Since many adults, the majority of which are experiencing dementia in some form, participate in day service programs, these programs have a unique opportunity to provide exercise programs that allow elderly adults to engage in strength training in a safe environment without additional strain on the adult’s caretakers and families. However, most adult day service programs do not include these types of evidencebased exercises as part of their physical activity sessions. Physical activity provided in these programs typically focus on general movement in a seated position, well-being and socialization, rather than much needed progressive strength and balance training. Barriers such as staffing, safety, cost, time and training may prevent day programs from implementing these types of exercise programs.

Program Development Using current recommendations on strength training for elderly adults the program Simply Strong was created to take advantage of the unique opportunity adult day services present in reaching adults vulnerable to falls. Drawing inspiration from the Otago Exercise Program, Simply Strong focuses on large, anti-gravity muscle groups of the upper and lower body. Progress made by participants can be tracked to allow for sufficient progression. All the exercises can be performed sitting or standing based on the comfort and strength of the individual.

HEALTH SCIENCE A functional cart was developed to store all program materials including the Simply Strong manual with individual tracking sheets, progression board, exercise equipment, video guide and television monitor. To make the program appropriate for adults with cognitive impairment, the exercises are simple and program materials were designed to ensure the program is easy to follow. Program materials were streamlined for simplicity and to allow for the program’s administrator to comfortably lead sessions with minimal training. It is hoped that anyone given the program materials would be able to effectively implement Simply Strong among whatever population of adult he/she is working with. However, during the duration of this project the focus was placed solely on the population of elderly adults participating in Goodwill Easter Seals in Beavercreek, Ohio.

Methods Literature Review Initially, a comprehensive review of the current research was performed. This review focused on falls and mobility impairments in individuals with dementia and the potential benefits of strength and balance exercises for this population. Needs Assessment A survey and interview of Goodwill Easter Seals program managers was conducted to identify possible barriers and concerns regarding implementation of an exercise program. These concerns were taken into consideration during the creation of the program so that potential barriers could be tackled before a problem was presented. The survey requested the program managers list experience in leading group exercises and activities in order to gauge the incoming comfort level with leading Simply Strong. It was revealed that there was little to no prior experience among program managers so program materials were developed to assist in leading. Development of the Exercise Program Based on the research conducted during the extensive literature review and the needs recognized during the assessment stage, a comprehensive and evidence based strength training program was developed so that it is appropriate for individuals with dementia. This program was taught to the Goodwill Easter Seals staff members who will work to implement the 76

program in the Beavercreek location on a trial basis. Implementation of Mobility Assessments Prior to participating in the exercise program, participants’ gait speed, mobility and lower body strength were tested using the “Timed Up and Go” and “Five Repetition Sit to Stand” assessments. These assessments were used to establish a baseline level for participants and to assess their individual fall risk. The assessments should be re-performed with participants every six to twelve weeks to monitor progress. Development of Training and Resource Materials Goodwill Easter Seals staff were educated in the methods of the strength training program so that the staff members may implement the program with minimal guidance. Upon the conclusion of the training the staff member(s) were comfortable with leading the program so that it could be continued independently at Goodwill Easter Seals. Along with the development of the program itself, the materials to be used in the implementation of the program were developed. These materials include a fully equipped cart containing various weights, strength bands, exercise wands and a television monitor (Figure 1). Supplemental training materials and forms for assessing and tracking participant’s balance, mobility and exercise progression were also developed and added to a comprehensive manual detailing the program.

Figure 1. Completed Simply Strong exercise cart and materials. Courtesy of Jaclyn Franz, 2016.

Proceedings 2016

HEALTH SCIENCE

Figure 2. Title slide from the Simply Strong instructional exercise video. Courtesy of Jaclyn Franz and Kurt Jackson, 2016.

An exercise video showing step-by-step guiding instructions for the program, specifically tailored to meet the needs of both staff members and participants, was created to be viewed on the cartâ&#x20AC;&#x2122;s monitor during the program's implementation. Upon the completion of the project, the cart created for the implementation of the program was donated to the Goodwill Easter Seals Beavercreek location.

Warm-Up Forward Rowing (1 set of 10) Backward Rowing (1 set of 10) Twists (1 set of 10) Marching, Seated or Standing (1 set of 10) Kicks, Seated or Standing (1 set of 10)

Strength Training

The Exercise Program

Overhead Press (2 sets of 10)

The Simply Strong exercise program focuses on the large, antigravity muscle groups of the upper and lower body through simple to perform exercises preceded by a short warm-up routine. Upper body muscle groups targeted were the deltoids, triceps, biceps, upper and middle trapezius and rhomboids. Strengthening these groups should allow for improved posture, reduced thoracic kyphosis, enhanced ability to reach overhead items and increased arm extension. Lower body muscle groups targeted include the gluteus, quadriceps and gastrocnemius. Improving muscle quality in these groups should improve balance, mobility, mediallateral stability and sit-to-stand performance.

Curls (2 sets of 10)

Proceedings 2016

Band Stretches (2 sets of 10) Squats and/or Knee Extensions (2 sets of 10) Hip Abduction, Seated or Standing (2 sets of 10) Heel Raises, Seated or Standing (2 sets of 10)

HEALTH SCIENCE

Figure 3. Demonstrating an overhead press (standing) as seen in exercise video. Courtesy of Jaclyn Franz and Kurt Jackson, 2016.

Figure 5. Demonstrating a band stretch (seated) as seen in exercise video. Courtesy of Jaclyn Franz and Kurt Jackson, 2016.

Figure 7. Demonstrating a knee extension as seen in exercise video. Courtesy of Jaclyn Franz and Kurt Jackson, 2016.

Figure 4. Demonstrating a curl (standing) as seen in exercise video. Courtesy of Jaclyn Franz and Kurt Jackson, 2016.

Figure 6. Demonstrating a squat as seen in exercise video. Courtesy of Jaclyn Franz and Kurt Jackson, 2016.

Figure 8. Demonstrating a hip abduction (standing) as seen in exercise video. Courtesy of Jaclyn Franz and Kurt Jackson, 2016.

Proceedings 2016

HEALTH SCIENCE References Alzheimer’s Association. 2016 Alzheimer’s Disease Facts and Figures. Alzheimer’s Association website. http://www.alz.org/ facts/. 2016. Buchner DM, Larson EB. Falls and fractures in patients with Alzheimer-type dementia. JAMA. 1987; 257:1492-95. Burton E, Cavalheri V, Adams R, et al. Effectiveness of exercise programs to reduce falls in older people with dementia living in the community: A systematic review and meta-analysis. Clin Interv Aging. 2015; 10:421-434. CDC. Dementia/Alzheimer’s Disease. http://www.cdc.gov/ mentalhealth/basics/mental-illness/dementia.htm. 2014. Hebert L, Scherr PA, Bienias JL, Bennett DA, Evans DA. Alzheimer disease in the US Population. Arch Neurol. 2003; 60:1119-1122.

Figure 9. Demonstrating a heel raise (standing) as seen in exercise video. Courtesy of Jaclyn Franz and Kurt Jackson, 2016.

Progress and Outcome At the conclusion of the project, an evidencebased strength training program specifically targeting adults with dementia was developed for Goodwill Easter Seals Adult Day Services. During this project the program was implemented with supervision for three weeks at which point the staff were trained in leading the program and were allowed to assume responsibility for leading. The program was implemented in such a way that it is able to be independently led by the staff members of Goodwill Easter Seals into future years. This project provided deeper insight into the barriers that prevent adult day centers from taking an active role in decreasing fall risk in adults with dementia and provided a possible model for implementation in adult day centers across the country.

Harlein J, Dassen T, Halfens RJ & Heinze C (2009) Fall Risk Factors in Older People with Dementia or Cognitive Impairment: A Systematic Review. Journal of Advanced Nursing. 65, 922-933. Karlsson M, Vonschewelov T, Karlsson C, Cöster M, Rosengen B. Prevention of falls in the elderly: A review. Scandinavian Journal Of Public Health [serial online]. July 2013; 41(5):442-454. Available from: Health Source: Nursing/ Academic Edition, Ipswich, MA. Accessed May 24, 2016. Myers AH, Baker S, Van Natta M, Abbey H, Robinson EG. Risk factors associated with falls and injuries among elderly institutionalized persons. Am J Epidemiol. 1991; 133:1179-90. Ortman JM, Velkoff VA. An Aging Nation: The Older Population in the United States. Census Bureau, US Department of Commerce; 2014. Tinetti ME, Speechley M, Ginter SF. Risk factors for falls among elderly persons living in the community. N Engl J Med. 1988; 319(26): 1701-1707. van Dijk, P.T., Meulenburg OG, van de Sande, H.J., Habbema JD. Falls in dementia patients. Gerontologist. 1993; 33(2):200-204. Why you fall—and what you can do about it. Harvard Women's Health Watch [serial online]. December 2015; 23(4):4-5. Available from: Health Source: Nursing/Academic Edition, Ipswich, MA. Accessed May 24, 2016.

Acknowledgement I would like to thank the Berry Foundation as well as the University of Dayton Honors Program for supporting and organizing this incredible experience. Special thanks to my advisor, Dr. Kurt Jackson, for all his patience and gentle guidance in the navigation of this project. I am also very grateful for my extraordinary BSTI cohort members and all the shared laughter. Lastly, I would like to thank the faculty of Goodwill Easter Seals, and most especially, the gracious participants of the GWES adult day center — you are all unforgettable to me.

Proceedings 2016

ENGINEERING

Design and Protocol for the Utilization and Setup of a Low-Cost Slip Trainer for Fall Prevention Stephen T. McFadden1,2,3 and Kimberly Bigelow3 University of Dayton, 300 College Park, Dayton, OH, 45469 1. Berry Summer Thesis Program 2. University Honors Program 3. University of Dayton Mechanical and Aerospace Engineering Department

Thesis Mentor: Kimberly Bigelow, Ph.D. Department of Mechanical and Aerospace Engineering Corresponding Author: Stephen McFadden Email: mcfaddens2@udayton.edu

Abstract Recently, the idea of a “fall vaccine” has been studied as a way to proactively rehabilitate and possibly prevent falls for older adults. Previous work has shown that the effects created by a slip trainer, which is a device designed to recreate a falling situation safely in order to train a patient’s reflexes, carried over for more than a year, leading to a reduction in falls. However, the facilities to perform this kind of training are only accessible in certain labs and the equipment therein is also extremely expensive, so not many doctor’s offices or local therapy centers could afford to purchase such devices. There is therefore a need to design a reduced cost and more accessible slip trainer which functions analogously to the current lab setups. Mass Rehab Inc. has already developed a prototype of a manual slip trainer. Considering the manual nature of this prototype, the consistency and assessment capabilities of that slip trainer are limited. In addition, an effective slip trainer needs to have the capability to provide a consistent and repeatable slip depending on the heights, weights and abilities of the patient. The objective of our research is to modify this existing slip trainer into a low tech, mechanically controlled device which allows for repeatable slips and reliable assessment of a patient’s abilities. Other aspects of our research are to gain an understanding of the relation between the magnitude of the slip and the weight on the trainer to maximize the effectiveness of the slip trainer, to provide guidance for future studies in 80

this area and to begin to relate the weight on the board to the type of reaction we might observe patients utilizing in reaction to a sudden slip.

Introduction Every year approximately half of individuals age 85 or over fall [2]. In addition, over 30% of adults over the age of 65 fall each year [3]. Around twenty percent of those individuals who fall sustain physical injuries which decrease mobility and thereby independence [4]. Falls present not only physical injuries for the elderly, but also psychological effects. These effects include reduction of habitual social and physical activities, along with anxiety and the fear of falling again [4]. A high number of factors influence the chance of falling. Risk factors for falling include feelings of anxiety, drugs used in medical care, previous medical conditions, balance issues and advancing age [2]. These factors can increase when an independent individual engages in everyday activities. Most research in this area concentrates on rehabilitating those who have already fallen at least once. Current strategies for preventing falls mostly involves changing or adapting to the environment around an elderly individual, including adding rails, adding carpet and other adaptations. During everyday activities, such as simply walking, a major factor in falling is the risk of tripping or slipping. While rehabilitation techniques aid individuals in recovery, few Proceedings 2016

ENGINEERING techniques can prepare individuals, especially those with reduced reflex times such as older adults, from sustaining serious injuries from a sudden trip or slip. While individuals fall for a number of reasons, a primary factor between the many different types of falls is balance. Falls often depend on the body’s systems for balance, including visual, proprioception and vestibular systems. The visual system takes into account how an individual observes their surroundings and how they adjust depending on their sensory intake, especially in terms of environmental conditions [17]. Proprioception is the body’s sense of where limbs are in relation to the rest of the body. This system is regulated by a system of mechanoreceptors in the capsules and ligaments of joints [17]. The vestibular system includes the semi-circular canals found in the inner ear which regulate the sense of being upright and the relation of the body in a 3D plane system [17]. In particular importance to those systems and to reflexes in general is the role of the central nervous system, or CNS. The CNS, in particular to its ability to adapt to changing balance situations, is critical in maintaining an upright stance [14]. Therefore, it is critical to maintain the CNS as to prevent losses in balance, whose effects can be disastrous.

Slip Trainers The very first slip trainers were a variety of devices utilized to induce a slip/fall in a study participant, usually to gather data on how to study how individuals fell in a variety of circumstances. These devices include lean and release mechanisms [20], ones involving a virtual reality device to create the visual sensation of a slip [18], and a lab setup which involves randomly moving plates on a walkway as the participant

would walk around on that walkway [1,19]. Many of these devices are very effective in their application, but they lack the ease of use, mobility,and reduced cost that a widely available and accessible slip trainer would need to possess. A slip trainer addresses the need for a clinically effective counterpart to proactive balance training as a reactive measure. In particular, this kind of study studies the effect of the CNS and coordinated muscle system responses to regain balance in the event of an unexpected perturbation [14]. The adaptations shown in previous studies to an unexpected perturbation include decreased braking impulse, fast onset latency muscle responses, foot angle and elevating COM [14]. The learning of locomotion and reflex skill acquisition is a continuous process (Newell 1991). The key to this training is to establish a stationary control over an individual’s balance. This state of control can be defined as the position and the velocity of the COM over one’s BOS [15]. The most widely regarded slip training work has been done by Clive Pai [1]. In their slip training paradigm, they have people walk on a walkway consisting of static plates and a set of moveable side by side plates embedded near the midpoint of the walkway. As the participant passed over the moveable plates, an assistant would trigger the plates and would thereby slip the individual. Sit to Stand (STS) trials were also conducted. Data concerning position and velocity of the participant’s COM was collected and analyzed. They have found that participants adjusted to the slip and the incidence of falling or balance loss was greatly decreased, regardless of age or task after the slip training [1]. A current low cost alternative would be the slip trainer designed and produced by Mass Rehab, Inc. This manual slip trainer, as shown in Figure 1, would be hand operated by a clinician. This

Figure 1. Photograph of the commercially available and manually controlled Slip Trainer. Photograph courtesy of Stephen McFadden, 2016.

Proceedings 2016

ENGINEERING pushing and pulling on the handle in a back and forth, random fashion, would simulate a slipping scenario in an effective pattern. While this random pattern stimulates an individualâ&#x20AC;&#x2122;s reflexes very efficiently, it is impossible to use as an assessment tool.

Study Objective The long term goal of this work was to modify a low cost slip trainer to create a reliable and effective assessment tool for clinical professionals in regard to determine the fall risk of older adults. In particular, this slip trainer would be able to separate faller and non-faller populations based on their performance during a set of slips set at a particular acceleration. Work on this project included designing, building, and testing a low-cost slip trainer which can mimic the effect the current lab slip trainers provide in addition to developing a protocol for physical therapists to utilize this new device effectively. Before the start of this project, our belief was that, depending on the weight of the individual or stand-in on the board, the acceleration would vary greatly.

Prototyping and Experimental Procedure First, background research and a literature review provided context to the development of the slip trainer and biomechanics in general. I also began to gather the parts I needed to make this project a success. These included a snap shackle (Figure 2), a safety harness (Figure 3), and a weight stack (Figure 4).

Figure 2. Snap Shackle mechanism utilized for releasing the weight stack to initiate slip. Photograph courtesy of Stephen McFadden, 2016.

Figure 3. A safety harness utilized to ensure safety of slip trainer participants. Photograph courtesy of Stephen McFadden, 2016.

Figure 4. A standard weight stack being used for rehabilitation was used as the drop mechanism. The weight stack had weights of 2 â&#x20AC;&#x201C; 12 kg, with individual plates each weighing 2 kg. Photograph courtesy of Stephen McFadden, 2016.

Proceedings 2016

ENGINEERING Prototype Development Initial construction of a prototype version was tested. This prototype is shown below in Figure 5. This design fulfilled the basic function as a slip trainer but lacked the necessary safety equipment, a high tension cord to allow for higher accelerations and forces, and a system for releasing the weight stack in a repeatable manner.

However, this prototype required the clinician or operator to hoist the weight stack manually, and the cord being utilized was stretching significantly, lowering the maximum forces needed. A third prototype, as shown in Figure 7, was developed by switching out the rope cord for a metal threaded cable and the addition of a block and tackle pulley system to assist in the raising of the weight stack. The metal cable, especially in comparison to the ropes utilized before, have a very low stretch nature. This property will become important over time, as a rope system under almost constant tension would likely stretch and perhaps break under the strain. The pulley system improves the ability of the doctor/physical therapist operating the device, as repeatedly hoisting a weight stack up to the snap shackle would put them under considerable physical stress.

Figure 5. A photograph of the first prototype of the slip trainer. Photograph courtesy of Stephen McFadden, 2016.

From this prototype, Dr. Bigelow, Dr. Jackson and I worked on a final design involving a safety harness, rails for patients to use to steady themselves initially and a box structure to house the weight drop/release mechanism. SolidWorks was utilized to provide initial prototyping design work. A second prototype, as shown in Figure 6, was created in order to increase safety and improve the overall design. The addition of a safety harness and the upgrade of the simple rope on the original weight stack to 1200 pound cord were essential to improving the safety of the device. A snap shackle was added in order to provide a way to hold up the weight stack mechanically. This device is a fast action loop which can be released under a stress/release.

Figure 7. A photograph of the third prototype of the slip trainer. Photograph courtesy of Stephen McFadden, 2016.

Testing was conducted in comparison with the second prototype test results, at a 22 inches drop height. This testing was done in order to test tensile properties of wire cable in comparison to rope. In general, the wire rope provided a smoother and more effective slip in addition to a higher force threshold, so it was chosen over the rope. The final drop height of the wire cord was set at 60 centimeters, and the rear slack cord length was set at 15 centimeters.

Experimental Procedure to Determine Acceleration

Figure 6. A photograph of the second prototype of the slip trainer. Photograph courtesy of Stephen McFadden, 2016.

Proceedings 2016

APDM Opal Sensors were utilized to test the slip trainer. Opal sensors are wearable inertial measurement units, or IMUs, which can record acceleration and three-dimensional rotation. In all trials, one Opal sensor was strapped to the board to record acceleration data. Data was 83

ENGINEERING measured over 100 to 280 pounds in 20 pounds increments on the board, 2 to 12 kilograms weight dropped at 2 kilograms intervals, at a set drop height of 60 centimeters. Another three trials were conducted from 120 to 280 pounds at 40 pounds increments, drop weights between 2 to 10 kilograms, and the same drop height of 60 centimeters as a comparison. Data was imported into Microsoft Excel from the Motion Studio software. The acceleration data in the forward and backward direction was utilized and analyzed as both a function of the drop height and the weight on the slip trainer. From this acceleration data, the impact of the slip trainer was measured in gâ&#x20AC;&#x2122;s.

Results Because the Opal IMU measures linear acceleration in three different directions and because the slip trainer was only moving in the forwardbackward direction, we could easily isolate the linear acceleration component that we cared about. When the slip trainer went from stationary to the initiated slip movement, it experiences a sharp acceleration in one direction. This was seen on the acceleration plot versus time as a sharp peak. This first spike peaked in the negative direction at first because of how the Opals were placed on the board. Consider the negative direction the same as the forward direction. This was the result of the board being pulled forward. The positive spike right after was the slack at the back of the board catching and pulling the board slightly back. Therefore, the first step was to examine the data from each trial to determine the acceleration experienced at that first peak. This was done for every trial and all three were averaged. The first trial revealed several correlations, but mostly displayed the insignificance of the 20 pounds increments and the 12 pounds drop weight. In general, the three following trials revealed that the weight on the board was relatively insignificant to the weight dropped from the weight stack concerning the acceleration the board experienced. While this model is lacking the high COM and other human factors that a real person would display on the board, these trials helped to establish a protocol for utilizing the board and determining a range between 6 to 8 kilograms as the drop weight as the most effective acceleration generator. Figure 8 graphically shows the average values of acceleration between the three trials as compared to the weight on the board. 84

Figure 8. A graph of the weight on board as compared to the acceleration of the board. Courtesy of Stephen McFadden, 2016.

Figure 9 showcases the average values of acceleration as compared to the weight dropped from the weight stack. Below is the total acceleration data from the three trials, plus the average acceleration over the three trials and the standard deviation between the three trial values. Acceleration is in meters per second squared (m/s2), weight dropped in kilograms (kg), and the weight on the board in pounds (lbs.). Table 1 provides the raw numbers from each trial and the average, in addition to the +/- standard deviation for each combination of weight on the slip trainer and the drop weight. Figure 9 defines the beginning of the protocol portion of this project. Clinical professionals can utilize this chart to personalize slip training depending on their individual patientâ&#x20AC;&#x2122;s physical characteristics, especially weight. All of the accelerometer data can easily be converted into gâ&#x20AC;&#x2122;s, or g forces, which are a measure of a vector acceleration that a person experiences when under a mechanical stress.

Figure 9. A graph of the weight dropped from the weight stack as compared to the acceleration of the board. Courtesy of Stephen McFadden, 2016.

Proceedings 2016

ENGINEERING Table 1. Raw numbers and the average from each trial. Courtesy of Stephen McFadden, 2016.

Discussion The effectiveness of the slip trainer can be analyzed as how it functions as a medical device and its ease of use for clinical professionals. Over this summer, the slip trainer was designed with certain engineering principles in mind. Of the highest concern were safety, cost, and ease of use. Safety was chosen as this is a medical device which could injure the patient if designed incorrectly. As this design was made to be affordable in comparison to more high cost lab setups, cost was a principle concern. As this device is made to be operated by clinical professions such as doctors or physical therapists and not engineers, any design made must be simple to operate and not be over engineered. Safety was accomplished with the addition of a safety harness and an operating procedure which included safety briefings for before any slipping was to occur. The cost of the device was kept Proceedings 2016

low by utilizing everyday available materials for most of the construction and a simplistic design. Ease of use was accomplished by the utilization of a simple snap shackle release mechanism. Testing on the device consisted of an acceleration test and a user rating test. The success of this device was considered accomplished if it fulfilled those principles well, passed all tests and was comparable to its competitors. The limitations of this project include the prototype nature of this project, the lack of human participants on the slip trainer, and the limited time and budget. Since this was just the beginning of our thesis project, the slip trainer I have designed is still not ready for clinical work. Due to IRB and time constraints, I was unable to test with human participants, instead using a set of weights to mimic a humanâ&#x20AC;&#x2122;s weight. The results gathered make sense. As the weight dropped increases, the board moves forward as the cord exerts a pulling force. 85

ENGINEERING Further Investigation Now that this work has laid the foundational relationships between weight on the trainer, weight dropped, and trainer acceleration the next step is confirming if this relationship holds in people. This will be achieved by an IRB-approved human subject study that will first be done on healthy, young college aged students of varying weights. Testing those participants on the device would make sure it worked and performed as expected. Then both the device and the Opal Sensors would be utilized to measure fall acceleration and position. Accelerometers would be placed on the actual sled part of the slip trainer to measure the g’s produced by the slip trainer and how that affected the subjects. This will establish the weight-acceleration relationship proposed during this summer’s work. Another human subject study will then be started to examine the acceleration necessary to cause an appropriate amount of instability. Once this range has been established, testing on recovery methods and potential long-term training studies will be done. If these results support our hypotheses, this device would be ready to be tested independently or used by local physical therapists, with whom I could work with to make future improvements. Of particular importance to this researcher is what kind of recovery strategy do participants utilize under the effect of a novel slip. If possible, the Bertec balance plate in the Engineering Wellness Through Biomechanics Lab will be placed on the slip trainer to provide an analysis of how a patient’s COM and balance change over the duration of the slip. Additional components to be added include a raised step platform for participants to start walking as they stepped on the slip trainer, allowing both static and dynamic testing situations. This is important as static training may not be quite as effective as dynamic testing in terms of presenting a realistic test for the reflexes.

weight stack does significantly affect the acceleration. Further research should focus on the efficiency and effectiveness of this kind of slip trainer in comparison to more expensive models. Continued work on clinical applications will depend on the effectiveness of the slip trainer. The main outcome of this project was the design of the slip trainer, which was accomplished. To assist in the utilization of this slip trainer, a protocol was developed from the weight/acceleration ratio gathered from data collection.

Acknowledgements I would like to thank Dr. Kimberly Bigelow for mentoring me during this experience and all her help with this project. Also, a special thanks for Dr. Kurt Jackson for his guidance and aid during the design process of the slip trainer, in conjunction with Steve Heinrichs and Mass Rehab, Inc. Also, I would like to thank my fellow Engineering Wellness through Biomechanics lab assistant, Lianna Nordwig and Courtney Golembiewski. In addition, without the University Honors Program, the Berry Family, and the Honors staff which put on the Berry Summer Thesis Program, I would not have been able to participate in such a special undergraduate experience. This experience also would not have been the same without the rest of the BSTI cohort, who inspired me in ways I did not expect.

Appendix Here are some basic formulas/diagrams for friction and forces acting on a slipping individual. List of Abbreviations BOS: Base of Support COF: Coefficient of Friction RCOF: Required Coefficient of Friction

Conclusion

COM: Center of Mass

During this project, a low cost prototype of a slip trainer was designed, built and tested. Testing was done to determine the relationship between the weight on the board and the maximum acceleration obtained using a set drop height. A main takeaway was while the weight on the board does not significantly impact the acceleration the board undergoes, the weight dropped from the 86

Proceedings 2016

ENGINEERING References 1.

Pai, Yi-Chung; Bhatt, Tanvi; Wang, Edward; Espy, Deborah; Pavol, Michael. (2010). Inoculation Against Falls: Rapid Adaptation by Young and Older Adults to Slips During Daily Activities. Arch Phys Med Rehabil, 452-459. Retrieved January 20, 2016.

Iinattiniemi S, et al. Scandinavian Journal of Primary Health Care, 27, 25-30. 2009.

White Paper â&#x20AC;&#x201D; Falls Among Older Adults: A National Problem. Presented by the National Safety Council to the 2005 White House Conference on Aging. December 11-14, 2005.

Iinattiniemi S, et al. International Journal of Circumpolar Health, 67, 2-3. 2008.

Dembling, Robert and Warner, Kenneth. The Pathophysiology of Free-Fall Injury. Annals of Emergency Medicine, 15, 1088-1093. 1986. Retrieved January 27, 2016.

Tinetti M, Speechley M, Ginter SF. Risk factors for falls among elderly persons living in the community. N Engl J Med, 319: 1701-7. 1988.

Pai, Y and Iqba,l K. Simulated movement termination for balance and recovery: Can movement strategies be sought to maintain stability even in the presence of slipping or forced sliding? J Biomech, 32: 779-86. 1999.

Bhatt, T. and Y.-C. Pai. Long-term retention of gait stability improvements. J Neurophysiol, 94: 1971â&#x20AC;&#x201C;1979. 2005.

Parijat, P. and Lockhart, T. Effects of Moveable Platform Training in Preventing Slip-Induced Falls in Older Adults, 2011. Annals of Biomedical Engineering, 2012 Vol: 40 (5) pp: 1111-1121.

16. Yi-Chung Pai. Simulated movement termination for balance recovery: can movement strategies be sought to maintain stability in the presence of slipping or forced sliding? Journal of Biomechanics, 1999 (32) pp: 779-786. 17. Lockhart, T. Slips and Falls. APA Handbook of Human Systems Integration, 2015 pp: 211-225. 18. Parijat P, Lockhart T, and Liu J. Effects of PerturbationBased Slip Training Using a Virtual Reality Environment on Slip-induced Falls 2014. Annals of Biomedical Engineering, Vol. 43, No. 4, April 2015 (2014) pp. 958-967. 19. Lockhart T, et al. Evaluation of Gait Characteristics and Ground Reaction Forces in Cognitively Declined Older Adults With an Emphasis on Slip-Induced Falls. Assistive Technology, 2009 Vol: 21 (4) pp: 188-195. 20. King G Luchies C Stylianou A Schiffman J Thelen D. Effects of step length on stepping responses used to arrest a forward fall. Gait & Posture, 22 (2005) 219-224.

10. Pavol M, Runtz E, and Pai Y. Young and Older Adults Exhibit Proactive and Reactive Adaptations to Repeated Slip Exposure. The Journals of Gerontology Series A: Biological Sciences and Medical Sciences, 2004 Vol: 59 (5) pp: M494-M502. 11. Pai Y and Bhatt T. Repeated-slip training: an emerging paradigm for prevention of slip-related falls among older adults. Physical Therapy, 2007 Vol: 87 (11) pp: 1478-91. 12. Pai Y, Wang E, Espy D, and Bhatt T. Adaptability to perturbation as a predictor of future falls: a preliminary prospective study. Journal of Geriatric Physical Therapy, (2001) 2010 Vol: 33 (2) pp: 50-5. 13. Lin L, Chiou F, and Cohen H. Slip and Fall Accident Prevention: A Review of Research, Practice, and Regulations. Pergamon Journal of Safety Research. 1995 vol: 26 (4) pp: 203-212. Hsiao E, Robinovitch S. Biomechanical infuences on balance recovery by stepping. Journal of Biomechanics, 1999 Vol: 32 pp: 1099-1106. 14. Marigold, D and Patla, A. Strategies for dynamic stability during locomotion on a slippery surface: effects of prior experience and knowledge. Journal of Neurophysiology, 2002 Vol: 88 (1) pp: 339-53. 15. Bhatt T, Wang E, and Pai Y. Retention of adaptive control over varying intervals: prevention of slipinduced backward balance loss during gait. Journal of Neurophysiology, 2006 Vol: 95 (5) pp: 2913-22.

Proceedings 2016

ENGINEERING

Influence of Reverse Shoulder Implant Positioning on Patient-Specific Muscle Forces: A Simulation Study Kayla M. Pariser1,2,3 and Allison L. Kinney1 University of Dayton, 300 College Park, Dayton, OH, 45469 1. Department of Mechanical and Aerospace Engineering, University of Dayton, Dayton OH 45469 2. Berry Summer Thesis Institute 3. University Honors Program

Thesis Mentor: Allison L. Kinney, Ph.D. Department of Mechanical and Aerospace Engineering Corresponding Author: Allison L. Kinney, Ph.D. Email: akinney2@udayton.edu

Abstract

Introduction

A reverse shoulder arthroplasty (RTSA) is a common treatment for patients with severe shoulder injuries such as rotator cuff muscle tears and/or severe arthritis. The goal of a RTSA is to stabilize the shoulder and improve a patient’s range of motion. The stability of the shoulder relies on the muscles of the shoulder and with rotator cuff tears, the deltoid muscle becomes the shoulder’s primary stabilizer. The positioning of the implant is important as it directly affects the length of the deltoid muscle moment arm, which determines the amount of deltoid muscle force required to produce the necessary torque about the shoulder for it to function after a RTSA. However, it is impossible to experimentally determine the force that the deltoid muscle will be able to produce after surgery. The overall goal of this work is to use simulation and optimization methods to optimize RTSA implant placement and deltoid muscle forces for 15 RTSA patients. For each of the 15 patients, a model of the shoulder joint and muscles and an optimization framework will be used to calibrate muscle model parameters and predict the shoulder muscle forces. As a first step toward achieving the overall goal, the specific purpose of this study was to perform a parameter sensitivity study to improve the optimization methods.

Tsa vs. Rtsa

Total shoulder arthroplasty (TSA) is a shoulder joint replacement surgery procedure used for patients with severe shoulder injuries such as rotator cuff muscle tears and/or severe arthritis [10]. In a TSA, the surgeon replaces the patient’s shoulder joint with an artificial one that resembles a normal shoulder joint. In a normal shoulder, the ball component of the ball-and-socket shoulder joint is the rounded head of the humerus and the socket is a concave section of the scapula called the glenoid cavity. The purposes of this TSA procedure are to relieve pain, increase the patient’s range of motion (ROM) and improve the patient’s ability to complete upper extremity functional tasks. Recently, surgeons have begun to perform a modification of the TSA called a reverse total shoulder arthroplasty (RTSA). This procedure got its name because the locations of the ball and socket of the shoulder implant are reversed from that of a normal shoulder. In a RTSA, the ball-and-socket glenohumeral (i.e., shoulder) joint is formed by implanting a glenosphere (ball) on the glenoid cavity of the scapula and drilling a stem into the humerus with a concave articulation (socket) on the head of the humerus [5]. The ball and socket in the normal shoulder joint allows for large ROM of the arm, but the socket is shallow rather than deep; as a consequence, the normal shoulder joint has limited bony stability. The shoulder joint’s stability is derived from strong muscles. When a person with a normal shoulder joint Proceedings 2016

ENGINEERING reaches upwards, the head of their humerus translates upwards on the socket. In people with weak shoulder muscles (which is common in this patient population), this upward translation may be excessive and may cause rotator cuff muscle tears and arthritis. The RTSA implant is designed to reduce stress on the shoulder joint and its musculature by increasing stability of the joint by enabling the deltoid muscle to become the primary stabilizer and eliminating upward translation of the head of the humerus [11].

Deltoid Muscle Function, Force and Moment Arm Muscles in the body move limbs by contracting and producing a torque about a joint. This torque (τۡ ) produced by the muscle is given by Equation: τۡ = rۡ × Fۡ

(1)

subregions. This study determined that in RTSA patients the deltoid moment arms have the same signs as those with healthy shoulders. A RTSA is designed to stabilize the shoulder by increasing the moment arm of the deltoid muscle thereby decreasing the force the deltoid is required to produce in order to get the same torque about the shoulder joint. This increase in its leverage enables the deltoid muscle to compensate for torn rotator cuff muscles without having to exert an extremely high amount of force to lift the arm [2]. This is essential because compensatory high muscle forces could deleteriously contribute to notching of the scapula bone [4]. A muscle’s ability to produce force is also related to its length via the force-length curve (Figure 1). A muscle produces its optimal isometric force (FMo) at its maximum fiber length (LMo). The ideal range of the LMo values is between 0.5LMo and 1LMo where the muscle will produce only active force; from 1LMo to 1.5LMo the muscle produces passive force which can overstretch the muscle and lead to injuries such as muscle tears [17].

In Equation (1), rۡ represents the moment arm and Fۡ is muscle force produced. Torque is equal to the cross product of the moment arm and muscle force vectors. A longer moment arm results in improved ability of a muscle to exert a Proper positioning of the RTSA implant is torque about a joint [2]. The deltoid is one of the complex since it involves three position and primary muscles responsible for arm elevation three rotation variables (i.e., degrees of Anatomically, the deltoid is divided into three freedom) for both the glenoid and the humerus. subregions: anterior, middle, and posterior There are functional consequences to the deltoid deltoid. In normal healthy shoulders it has been muscle if the RTSA implant is placed such that found that the anterior and middle deltoid regions the moment arm of the deltoid is too short or too have positive abduction moment arms, meaning that they contribute muscle force to aid in abduction, while the posterior deltoid has a negative abduction moment arm, indicating that it is an adductor [1, 8]. In previous cadaveric studies with reverse total shoulder patients, researchers concluded that all three subregions of the deltoid had positive abduction moment arms [2]. However, while these studies are beneficial there are many limitations when using cadaver specimens. Recent studies have used musculoskeletal models to calculate shoulder muscle moment arms. One study conducted by Walker et al., [14] used in vivo motion capture data to create scaled OpenSim models for 14 patients and calculated the moment arms for each of the deltoid Figure 1. Force-length curve relating muscle force to muscle length. Courtesy of A. Kinney, 2016. Proceedings 2016

ENGINEERING long [7]. Previous research suggests that a more medial and inferior placement of the center of rotation (COR) for the RTSA implant will make the moment arm of the deltoid muscle longer and as a result the muscle will not have to produce as much force to generate the required torque for shoulder activities [5]. Surgeons try to avoid placing the COR of the implant laterally, because in this position external shear forces and torques could more easily loosen the new joint; but even with medial and inferior placement of the implant many RTSA patients do not regain full ROM and some experience scapular notching leading to bone loss [4]. It is not clear why these negative outcomes occur. One possible reason for negative outcomes may be implant positioning. Determination of the best placement of RTSA implants is complex given the multiple degrees of freedom of the shoulder joint. In addition, implants come in various sizes and shapes to fit the patient’s body and there is no standardized or objective assessment for surgeons to use to fit an implant to a specific patient [4-5, 7].

Methodology Simulation and Optimization

Experimental Data

There is a need to provide surgeons an objective way to optimally place an implant in a consistent manner. During surgery, it is possible to test for scapular notching and how the location of the implant will affect the patient’s ROM. However, a patient’s muscle function and forces cannot be easily measured while in surgery. Simulation and optimization methods have been used in understanding moment arms and how they affect a muscle’s functionality, predicting muscle forces and analyzing how surgeries affect the muscle moment arms [9]. Specifically for this study, simulations let modification of the placement and design of the implant occur in a virtual environment prior to surgery. The optimization framework could allow surgeons to determine the location of the RTSA implant that would optimize a specific patient’s shoulder muscle forces.

The participants in this study were 15 people who have a RTSA. Rehoboth Innovations LLC collected muscle electromyography (EMG) data, limb motion capture data [16] and fluoroscopic data [15] during scaption for each of the participants (Figure 2). Scaption is defined as arm elevation in the scapular plane [1]. A combination of the fluoroscopic images and the motion capture data were used to define the motion in a shoulder model for each patient. These models were modified from Holzbaur et al., 2005 [6] to include the RTSA and each patient’s model was scaled to their dimensions. Each model includes two degrees of freedom at the shoulder (abduction/adduction and flexion/extension) and 15 major shoulder muscles: anterior, lateral and posterior deltoid; four rotator cuff muscles; thoracic, lumbar and iliac latissimus dorsi; clavicular, sternal and costal pectoralis major; upper trapezius and teres major (Figure 3). The software OpenSim [3] was used to perform biomechanical modeling and to calculate quantities such as muscle moment arms, joint moments, musculotendon lengths and musculotendon velocities of each patient’s shoulder during the scaption.

The overall goal of this study is to investigate how the deltoid muscle forces relate to RTSA placement for 15 patients using simulations and an optimization framework. As a preliminary step, a parameter sensitivity study was performed to improve the optimization methods.

Proceedings 2016

ENGINEERING Optimization The overall goal of this study is to optimize the RTSA implant placement and deltoid muscle forces for all 15 patients. To accomplish this goal, the data from the models and a MATLAB optimization framework will be used to calibrate muscle model parameters (maximum isometric force, optimal fiber length and tendon slack length) for the 15 patients and then predict muscle forces during scaption. The optimization framework will take place in two phases (calibration and prediction). The calibration phase will involve calibrating muscle model parameters to match the data collected by Rehoboth Innovations LLC while the patients performed isometric contractions of their shoulder muscles. The calibration phase will use a two-level optimization: the outer-level optimization optimizes the muscle model parameters and the inner-level optimization uses the muscle model parameters from the outer-level optimization to optimize the muscle activations needed to match the experimental data; this occurs in a cyclic pattern until the optimization converges [13]. Figure 3. A representative OpenSim model for one of the RTSA patients. All 15 muscles are included in the model as depicted by the red lines and the pink sphere represents one of The second phase, prediction, will the motion capture markers. The rest of the model represents the bones and RTSA implant. use the calibrated parameters from Courtesy of Rehoboth Innovations LLCÂŠ . the first phase to predict muscle initial guess that would decrease convergence forces during scaption. Once both phases have been completed, implant location time. The original muscle model parameters and muscle forces will be compared across all (optimal fiber length, maximum isometric force, 15 patients to determine trends that may aid tendon slack length, pennation angle) were surgeons in identifying the exact location that obtained from Saul et al. [12]. First, the optimal a RTSA implant should be placed to optimize fiber length (LMo) and the tendon slack length the muscle forces in the shoulder and improve (LTs) were scaled by the same multiplier to function in the shoulder. As a first step towards adjust the muscle tendon length values (LMtilda) completing the calibration and prediction to be within the ideal range of 0.5LMo-1LMo for phases, a parameter sensitivity study was needed all seven muscles that were studied (DELT1, to improve the optimization methods. DELT2, DELT3, four rotator cuff muscles) and for eight of the patients. Then, the maximum isometric forces (FMo) for two patients were Parameter Sensitivity Study altered to determine the effect that increasing Due to the fast workplace environment, surgeons or decreasing the FMo value would have on do not have time to wait for optimizations that matching the predicted muscle activation for take a long time to converge. Prior to the muscle the three deltoid muscles with the experimental model parameter calibration phase, a parameter EMG data. sensitivity study was performed to provide the two-level calibration optimization with a good Proceedings 2016

ENGINEERING Results and Discussion With the original LMo and LTs parameters from Saul et al. [12], the LMtilda values were not within the ideal range of 0.5LMo -1LMo (Figure 4). At the conclusion of the parameter sensitivity study all eight patients had LMtilda values for each muscle in the ideal range (Figure 5). Across the eight patients a common multiplier was found for each of the seven muscles studied except for the anterior deltoid (DELT1). The differences in anterior deltoid multipliers between the patients can be attributed to the diversity of dimensions amongst the patients. By adjusting the multipliers, we achieved the goal of having all LMtilda values in the range of 0.5LMo to 1LMo as this is the range on the force-length curve where the muscle is producing active force. Modifying the FMo values did not prove to make a clear difference in matching the predicted muscle activation for the three deltoid muscles with the EMG data for either of the two patients. Because altering the FMo parameter was not conclusive, it was decided that one of the next

steps should be to change the muscles included in the calibration phase by adding in the latissimus dorsi, pectoralis major, and upper trapezius to contribute to the muscle force needed for each patients to complete the scaption motion.

Further Investigation The next goal is to use the two-level optimization to calibrate the parameters for all 15 patients. The results of the parameter sensitivity study will be used to modify the original parameters in order for the calibration optimizations to converge quicker. The next steps for this research are to alter the muscle groupings to determine the best combination of muscles for which the predicted deltoid muscle activations align with the EMG data and calibrate the parameters for all 15 patients. Then the calibrated parameters will be used to predict muscle forces for the scaption motion. Overall the goal of this investigation is to use simulation and optimization methods to optimize the deltoid muscle force for all 15 RTSA patients and generate a tool that orthopedic surgeons could use to determine the optimal patient-specific placement of the implant. With this tool, a surgeon would have the ability to determine the implant placement prior to surgery. This would make the procedure more efficient and improve outcomes for the patient.

Acknowledgements Figure 4. LMtilda plots for a representative patient generated with the LTs and LMo values as described by Saul et al. [12]. Note that the LMtilda values are outside of the desired range of 0.5LMo-1LMo. Courtesy of K. Pariser, 2016

I would like to thank my advisor Dr. Kinney for all of her support during this research process. I would also like to extend a special thank you to the Berry Family and the Berry Family Foundation as well as the University Honors Program for making this summer research experience possible. Finally, I would like to thank Dr. David Walker and Rehoboth Innovations LLC for their generosity and support in allowing me to work on this study.

Figure 5. Modified LMtilda plots for a representative patient after the LMo and LTs values were altered to achieve the desired LMtilda range (0.5LMo-1LMo) on the force-length curve. Courtesy of K. Pariser, 2016

Proceedings 2016

ENGINEERING References 1.

Ackland, D. C., Pak, P., Richardson, M., Pandy, M. G. (2008). Moment arms of the muscles crossing the anatomical shoulder. Journal of Anatomy, 213(4), 383-390. doi: 10.1111/j.1469-7580.2008.00965.x

Ackland DC, Roshan-Zamir S, Richardson M, Pandy MG. (2010). Moment arms of the shoulder musculature after reverse total shoulder arthroplasty. Journal of Bone Joint Surgery Am., 92(5), 1221-1230. doi:10.2106/ JBJS.I.00001

Delp SL, Anderson FC, Arnold AS, Loan P, Habib A, John CT, et al. (2007). OpenSim: open-source software to create and analyze dynamic simulations of movement. IEEE Trans Biomed Eng, 54(11), 1940-1950. doi: 10.1109/ TBME.2007.901024

Henninger HB, King FK, Tashjian RZ, Burks RT. (2014). Biomechanical comparison of reverse total shoulder arthroplasty systems in soft tissue-constrained shoulders. J Shoulder Elbow Surg., 23(5), e108-e117. doi: 10.1016/j.jse.2013.08.008

Hoenecke HR, Flores-Hernandez C, D’Lima DD. (2014). Reverse total shoulder arthroplasty component center of rotation affects muscle function. J Shoulder Elbow Surg, 23(8), 1128-1135. doi:10.1016/j.jse.2013.11.025

Holzbaur KRS, Murray WM, Delp SL. (2005). A Model of the Upper Extremity for Simulating Musculoskeletal Surgery and Analyzing Neuromuscular Control. Ann Biomed Eng., 33(6), 829-840. doi:10.1007/ s10439-005-3320-7.

Kontaxis a., Johnson GR. The biomechanics of reverse anatomy shoulder replacement—A modeling study. (2009). Clin Biomech, 24(3), 254-260. doi: 10.1016/j. clinbiomech.2008.12.004

Otis J., Jiang C., Wickiewicz T., Peterson M., Warren R., Santner T. (1994). Changes in the moment arms of the rotator cuff and deltoid muscles with abduction and rotation. Journal of Bone and Joint Surgery, Am., 76(5), 667-676.

Pandy, M. G., & Andriacchi, T. P. (2010). Muscle and joint function in human locomotion. Annual Review of Biomedical Engineering, 12, 401-433. doi: 10.1146/ annurev-bioeng-070909-105259

14. Walker, D. R., Struk, A. M., Matsuki, K., Wright, T. W., & Banks, S. A. (2016). How do deltoid muscle moment arms change after reverse total shoulder arthroplasty? Journal of Shoulder and Elbow Surgery, 25(4), 581-588. doi: 10.1016/j.jse.2015.09.015 15. Walker, D., Matsuki, K., Struk, A. M., Wright, T. W., & Banks, S. A. (2015). Scapulohumeral rhythm in shoulders with reverse shoulder arthroplasty. Journal of Shoulder and Elbow Surgery, 24(7), 1129-1134. doi: 10.1016/j. jse.2014.11.043 16. Walker, D., Wright, T. W., Banks, S. A., & Struk, A. M. (2014). Electromyographic analysis of reverse total shoulder arthroplasties. Journal of Shoulder and Elbow Surgery, 23(2), 166-172. doi: 10.1016/j.jse.2013.05.005 17. Zajac F. (1989). Muscle and tendon: properties, models, scaling, and application to biomechanics and motor control. Critical Reviews in Biomedical Engineering 17(4), 359-411.

10. Quental C, Folgado J, Ambrósio J, Monteiro J. Multibody system of the upper limb including a reverse shoulder prosthesis. (2013). J Biomech Eng, 135(11), 111005. doi:10.1115/1.4025325 11. Roche C., Flurin, P.-H., Wright, T., Crosby, L. a, Mauldin, M., & Zuckerman, J. D. (2009). An evaluation of the relationships between reverse shoulder design parameters and range of motion impingement, and stability. Journal of Shoulder and Elbow Surgery, 18(5), 734–41. doi:10.1016/j.jse.2008.12.008 12. Saul, K. R., Hu, X., Goehler, C. M., Vidt, M. E., Daly, M., Velisar, A., & Murray, W. M. (2014). Benchmarking of dynamic simulation predictions in two software platforms using an upper limb musculoskeletal model. Computer Methods in Biomechanics and Biomedical Engineering, 5842(May 2016), 1-14. doi: 10.1080/10255842.2014.916698 13. Serrancoli, G., Kinney, A., Fregly, B., Llagunes, J. (2016). Neuromusculoskeletal Model Calibration Significantly Affects Predicted Knee Contact Forces for Walking. Journal of Biomechanical Engineering, 138(August), 1-11. doi: 10.1115/1.4033673

Proceedings 2016

CREDITS PHOTOGRAPH AND ILLUSTRATION CREDITS Cover Row 1, left to right OpenSim Model. Courtesy of Rehoboth Innovations LLC© . Preliminary Confirmatory Factor Analysis Model for NEO-PI-3 and PID-5. Chart courtesy of Lisa Stone, 2016. Snap Shackle Mechanism. Photograph courtesy of Stephen McFadden, 2016. Row 2, left to right A Solution of the Blue-900 βC-TFA Complex in Benzene Solvent. Photograph courtesy of Lauren Hoody, 2016. Experiential Data. Photograph courtesy of Lucy Bratton, 2016. Gracis a la Vida. Image courtesy of Elizabeth Turnwald, 2016. Simply Strong. Graphic courtesy of Jaclyn Franz and Kurt Jackson, 2016. Drosophila Life Cycle. Illustration courtesy of Lydia Payton and Amit Singh, 2016. Row 3, left to right ICC Images of Erythrocytes. Photograph courtesy of Christopher Turley, 2016. The Control of Gene Expression by CREs. Illustration courtesy of E.E. Wey and T.M. Williams, 2016. Running Wheel. Photograph courtesy of Caroline Lynch, 2016.

Articles Page 2: Coca-Cola. 2009. Image courtesy of brandsoftheworld.com. Page 2: Apple. 2014. Image courtesy of brandsoftheworld.com. Page 2: Starbucks. 2011. Image courtesy of brandsoftheworld.com. Page 2: I Love New York. 1977. Image courtesy of iloveny.com. Page 3: Blackout. 1977. Photograph courtesy of NYC Council for Public Safety, council.nyc.gov. Page 3: Fear City: A Survival Guide for Visitors to the City of New York. 1975. Photograph courtesy of NYC Council for Public Safety, council.nyc.gov. Page 3: Milton Glaser. 2006. Photograph courtesy of Sam Haskins, Sam Haskins Estate. Page 3: I (Heart) NY Concept Sketch. Milton Glaser, 1976. Photograph courtesy of moma.org. Page 3: I (Heart) NY Concept Layout. Milton Glaser, 1976. Photograph courtesy of moma.org. Page 3: LOVE. Robert Indiana, 1966. Photograph courtesy of pixabay.com. Page 4: I Love UD. 2016. Image courtesy of the University of Dayton. Page 4: Berry Summer Thesis Institute Proceedings 2015 Cover. 2015. Image courtesy of the University Honors Program, University of Dayton. Page 4: Berry Summer Thesis Institute Logo. 2013. Image courtesy of the University Honors Program, University of Dayton. Page 5: Collage. Photograph courtesy of Lucy Bratton, 2016. Page 5: Logos: University of Dayton, Flyer Enterprises, University Honors Program Seal, The Blend, Berry Summer Thesis Institute. Images courtesy of the University of Dayton, University Honors Program and Flyer Enterprises, 2016. Page 6: Sketches. Photographs courtesy of Lucy Bratton, 2016. Page 11: Joan Baez Performs “Gracias a la Vida” in Bretagne, France. 2000. Photograph courtesy of https://www.youtube.com/watch?v=DFZxBvUMIG0. Page 13: Mercedes Sosa’s Live Performance of “Gracias a la Vida.” 1971. Photograph courtesy of https://www.youtube.com/watch?v=clrGQD84F1g. 94

Proceedings 2016

CREDITS Page 14: Figure of Nuestra Señora de Luján, Buenos Aires, Argentina. Photograph courtesy of paraclito.org, 2016. Page 14: Statue of the Virgin Mary on Top of Cerro San Cristóbal, Santiago, Chile. Photograph courtesy of wikipedia.org, 2016. Page 14: Painting of Our Lady of Guadalupe in Mexico City, Mexico. Photograph courtesy of thecatholiccatalogue.com, 2016. Page 15: Music Video Stills of “Gracias a la Vida” Performed by “Voces Unidas por Chile.” 2010. Photograph courtesy of https://youtube.com/watch?v=_7-vTDV_aSA. Page 22: Distance Run. Photographs courtesy of Caroline Lynch, 2016. Page 23: Elevated Plus Maze. Photograph courtesy of Caroline Lynch, 2016. Page 41: ß-Carotene. Photograph courtesy of Lauren Hoody, 2016. Page 49: Alzheimer’s Disease Neuropathology. Illustration courtesy of the National Institute on Aging through http://neurosciencenews.com/buphenyl-possible-treatment-alzheimers-diseaseneurotrophic-factors/, 2016. Page 50: Expressing Genes in the Drosophila Model. Illustration courtesy of L. Payton and A. Singh. Page 50: Drosophila Life Cycle. Illustration courtesy of L. Payton and A. Singh. Page 51: Wild Type Drosophila Eyes. Photographs courtesy of Sarkar & Singh, unpublished data. Page 52: The Electron Transport Chain. Courtesy of L. Payton and A. Singh. Page 53: Mitochondrial Turnover. Illustration courtesy of L. Payton and A. Singh. Page 60: Hour Glass Figure of AQP HC-3. Image courtesy of C. Turley, 2016. Page 62: Western Blot Results. 2016. Images courtesy of C. Turley, 2016. Page 62: ICC Images of Erythrocytes. Images courtesy of C. Turley, 2016. Page 68: The Control of Gene Expression by CREs. Illustration courtesy of E.E. Wey and T.M. Williams, 2016. Page 69: General Features of CRE Regulatory Logic. Illustration courtesy of E.E. Wey and T.M. Williams, 2016. Page 70: D. Melanogaster Mutations. Images courtesy of E.E. Wey and T.M. Williams, 2016. Page 71: Representation of a Reporter Transgene. Illustration courtesy of E.E. Wey and T.M. Williams, 2016. Page 76: Simply Strong Exercise Cart and Materials. Photograph courtesy of Jaclyn Franz. 2016. Page 77: Simply Strong. Graphic courtesy of Jaclyn Franz and Kurt Jackson, 2016. Page 78: Standing Overhead Press. Photograph courtesy of Jaclyn Franz and Kurt Jackson, 2016. Page 78: Standing Curl. Photograph courtesy of Jaclyn Franz and Kurt Jackson, 2016. Page 78: Seated Band Stretch. Photograph courtesy of Jaclyn Franz and Kurt Jackson, 2016. Page 78: Squat. Photograph courtesy of Jaclyn Franz and Kurt Jackson, 2016. Page 78: Knee Extension. Photograph courtesy of Jaclyn Franz and Kurt Jackson, 2016. Page 78: Standing Hip Abduction. Photograph courtesy of Jaclyn Franz and Kurt Jackson, 2016. Page 79: Standing Heel Raise. Photograph courtesy of Jaclyn Franz and Kurt Jackson, 2016. Page 81: Slip Trainer. Photograph courtesy of Stephen McFadden, 2016. Page 82: Snap Shackle. Photograph courtesy of Stephen McFadden, 2016. Page 82: Safety Harness. Photograph courtesy of Stephen McFadden, 2016. Page 82: Standard Weight Stack. Photograph courtesy of Stephen McFadden, 2016. Page 83: First Prototype of Slip Trainer. Photograph courtesy of Stephen McFadden, 2016. Page 83: Second Prototype of Slip Trainer. Photograph courtesy of Stephen McFadden, 2016. Page 83: Third Prototype of Slip Trainer. Photograph courtesy of Stephen McFadden, 2016. Page 90: RTSA Patient. Photograph courtesy of Rehoboth Innovations LLC ©. Page 91: OpenSim Model. Illustration courtesy of Rehoboth Innovations LLC ©.

Proceedings 2016