Indianapolis Project

SEED

2012 Poster Session

Thursday, July 26, 2012 4:00 PM – 7:30 PM

VanNuys Medical Science Building Atrium

Indianapolis Project SEED Website: www.indyprojectseed.org

E-mail: indyprojectseed@yahoo.com

The Indianapolis Project SEED An educational outreach program of the

American Chemical Society Indiana Section

with institutional, administrative, financial, staff or research mentor support from the

Indiana Clinical and Translational Sciences Institute (Indiana CTSI)

IUPUI School of Science IU School of Medicine

IUPUI School of Education

and scientific research mentors from the

IU School of Dentistry IUPUI School of Informatics

IUPUI School of Physical Education and Tourism Management The Polis Center at IUPUI

The Veterans Administration Medical Center Eli Lilly and Co.

Heritage Research Group Methodist Research Institute at IU Health

Ball State University Indiana University

cordially invites you to attend the

2012 Summer Research Poster Session Thursday the 26th of July 4:00-8:00 PM*

*4:00 pm Poster Session, 6:00 pm Awards Ceremony

Details and Registration at

www.indyprojectseed.org/Poster

Indianapolis Project SEED

40 years

Project SEED was established in 1968 by the American Chemical Society (ACS) to encourage high

school students to explore careers in Science. SEED is an acronym for: Summer Experience for the

Economically Disadvantaged. The Indianapolis program was started by Professor Edwin Harper

(retired) of the Indiana University School of Medicine in 1972 as an educational outreach program

of the Indiana Section of the ACS.

With administrative support and funding from the IUPUI School of Science, this all volunteer

program continues to thrive on the campus of IUPUI and the IU School of Medicine with additional

support from the IU School of Education, corporate, public and private donors.

The 2012 program was substantially supported by the Indiana Clinical and Translational Sciences

Institute (CTSI) in partnership with Dr. Kim Nguyen, LSAMP director for the School of Education.

The Indiana CTSI is funded in part by an award from the National Institutes of Health (NIH) and

operates as a partnership of the IU School of Medicine, IU-Bloomington, IUPUI, Purdue University

and the University of Notre Dame. The overarching goal of the Indiana CTSI is to improve the health

of people throughout the State of Indiana by supporting research, education and community

outreach.

The Project SEED Committee would like to thank the dedicated volunteer team of research

scientists who invest their time in the mentorship of these students, the science leaders of our

future.

2012 Advisory Committee

James McAteer, I U School of Medicine

Rosie Bonjouklian, Eli Lilly (retired)

Lisa Dinsmore, CTSI, I U School of Medicine

Kim Nguyen, IUPUI School of Education

Kathy Marrs, IUPUI School of Science

Jeff Watt, IUPUI School of Science

Linda Monroe, Warren Central H.S.

Elmer Sanders, Southport HS

2012 Parent Committee

Nancy Kassab (Nathan & Hannah)

Eddy and Kathy Cabello (Gigi)

Karen Lucas (Jacob & Ryan)

Anushree Bag (Roshni)

2012 Staff

Jennifer Yu, Program Assistant

Laura Slocum, Student Coordinator

Erin McCrea Gantz, SEED Fellow

Philip Sanders, Webmaster

Brent Rogers, Technology Intern

Project SEED 2012 Program Acknowledgements

When I joined SEED as a volunteer 12 years ago, I had no idea of the journey I began. I was welcomed by some of the most respectful, cultured, educated and dedicated public servants I had ever met.

Once I saw how transformative the SEED experience was for the students, I needed no more convincing. Over the years I continue to meet even more of these dedicated public servants that have allowed the program to grow from an average of 12 to 60 this year.

Project SEED is a triumph of public service and humanitarian generosity. The Preceptors mentoring the students and the volunteer committee members have logged over 20,000 hours of volunteerism!

As an educational program outreach of the American Chemical Society Indiana Section, Project SEED is thankful for the support of its full membership and the executive committee. We are especially thankful to the 2012 ACS Indiana Section Chair, Sibel Selcuk, PhD. Sibel is the Chief Science Officer for the Heritage Research Group and an enthusiastic ambassador to the scientific community. We are also thankful to Eli Lilly and Company, Dow Agro Sciences and Heritage Environmental Services for supporting the Indiana Section’s outreach programs.

Since 2009, The Indianapolis Project SEED has now quadrupled in growth, serving more young and aspiring scientists than ever before, with substantial support from the Indiana Clinical Sciences and Translational Sciences Institute (CTSI). We especially want to thank Anantha Shekhar, Lisa Dinsmore, Joe Hunt, and Samantha Scahill for their personal support for the student interns and special program invitations. The overarching goal of the Indiana CTSI is to improve the health of people throughout the State of Indiana by supporting research, education and community outreach. The interns research was made possible in part by Grant Number TL1 TR000006 (A. Shekhar, PI) from the National Institutes of Health.

The IUPUI School of Science has been a longtime supporter of Project SEED since the early 1970’s. Professors W.K. Fife, T. Cutshall and E. Boschman from the Department of Chemistry and R. Samuels from the Department of Biology, were among the inaugural mentors for the Indianapolis program.

The IUPUI School of Science continues to provide administrative and financial support to the program in addition to the many research faculties who mentor students in their laboratories. We are especially thankful to Natalie Mazanowski for facilitating computer network support for the students, Jon Berg and Amelia Miller for facilitating the students reception on the campus, Lindsay Heinzman and Anne Marie Chastain for facilitating support from the IU Foundation, and Mark Federwisch for his assistance in printing the scientific posters for the student interns. Associate Deans Kathy Marrs and Jeff Watts serve on the Committee and provide valuable support and guidance.

The IUPUI School of Education has played a very special role over the years, especially since 2009. Dr. Kim Nguyen from the UCASE office and LSAMP program has been member of the Committee for over 20 years. She takes a special interest in each student intern and especially those who enroll at IUPUI for their university studies. Dr. Nguyen has facilitated the support from the Indiana CTSI and the School of Education fiscal officer, Pat Stites provided fiscal oversight.

The Indianapolis Project SEED is exceptional in the long term commitment of its volunteers and committee members. We are most especially thankful to Dr. Rosanne ‘Rosie’ Bonjouklian, medicinal chemist (retired) from Eli Lilly and Company, and Dr. James ‘Jim’ McAteer, Professor of Anatomy at the School of Medicine who have served the program over 30 years.

On behalf of the committee, to these and everyone who have helped the SEED program over the years, we say thank You! Elmer Sanders Program Coordinator, Committee Chair

The Future…

We all know that the future lies within the youth! We all were

there once. Now it is our job to create a safe and creative

environment that gives them a chance to be a part of today’s

scientific problems so that they become the innovators of

tomorrow. Project SEED is here to give us that opportunity. It is an

exceptional platform for today’s scientists to create that

environment for tomorrow’s scientists. And ACS Indiana Local

Section’s SEED Program is a huge success because of all the

volunteering mentors and incredible young chemistry fans. It is a

privilege to be a part of this scientific community.

Sibel Selcuk, PhD. ACS Indiana Local Section Chair

The Indianapolis Project SEED Welcomes

The Purdue Project SEED 2012 Inaugural Program

Interns

Aneli Carrillo Jennifer Pham Kimberly Rios Tania Sotelo

Mentors

Suzanne C. Bart, PhD Stephen P. Beaudoin, PhD

Yue Wu, PhD Yu Xia, PhD

Program Coordinator

Bryan W. Boudouris, PhD Assistant Professor of Chemical Engineering

Purdue University

Other Guest Presenters:

Michael Blatchley, Indiana University-Purdue University Indianapolis

Steven X. Chen, Indiana University-Purdue University Indianapolis

Taylor Hurst, Indiana University

The Inaugural Year of Project SEED at

Purdue University

June 4, 2012 marked the beginning of the Project SEED program at Purdue University. During its first summer in existence, Purdue Project SEED was fortunate to recruit four talented students from Lafayette, Monticello, and Frankfort, Indiana to participate in our program. Critical to the successful recruitment of these individuals was Mr. William Bayley in the Department of Chemistry at Purdue University. His ability to connect with these talented students was instrumental in our success in the first year. Through their hard work inside and outside their research laboratories in the School of Chemical Engineering and Department of Chemistry at Purdue, this first year class of Purdue Project SEED students was an amazing success. Beyond their chemistry talent, the ability of the students to communicate their scientific work clearly and in a straightforward manner has been remarkable to observe. We anticipate that the wonderful foundation that these students have set will allow for the continued success of Project SEED at Purdue.

Of course, these initial positive results could not have been possible without the generous financial support of the local Purdue American Chemical Society and the School of Chemical Engineering and the College of Engineering at Purdue University. Specifically, we gratefully thank Dr. Beatriz Cisneros, Professor Michael Harris, and Professor Arvind Varma for their visions and desires to make Purdue Project SEED a success for years to come.

A great deal of thanks is due to our inaugural Project SEED mentors at Purdue University also. Professors Suzanne C. Bart (Chemistry), Stephen P. Beaudoin (Chemical Engineering), Yue Wu (Chemical Engineering), and Yu Xia (Chemistry) have led our students down an excellent path in order to train them to be the next generation of chemistry leaders in the United States. Their enthusiasm, talent, and motivational skills have inspired our students to make wonderful discoveries in a very short 8-week period of time. Importantly, Purdue Project SEED would not exist without the wonderful support, inspiration, and guidance of our well-established sister program in Indianapolis. The outpouring of advice from Indianapolis Project SEED was remarkable. Specifically, I owe an amazingly large debt of gratitude to Mr. Elmer Sanders for providing valuable insight, resources, and 24-hour advice and support during the start-up and implementation of Project SEED at Purdue. There is no doubt that without his guidance, Purdue Project SEED would not be so well-poised to continue the advancement of secondary education through university-level research experiences.

Because of the dedication and support of all of these wonderful individuals and the talent and hard work of our inaugural class of SEED students, the future of Purdue Project SEED is indeed very bright. We look forward to continuing to make a deep impact across our community through chemistry education. Sincerely,

Bryan W. Boudouris Assistant Professor of Chemical Engineering, Purdue University Program Coordinator, Purdue Project SEED

Congratulations 2012 Interns

Thank you 2012 Preceptors

Tomohiko Ai, MD, PhD Margaret Bauer, PhD Angela Bruzzaniti, PhD Christopher Burlak, PhD Jinhui Chen, MD, PhD Zhenhui Chen, PhD Neil Devadasan, PhD Scott Engum, MD Gabe Filippelli, PhD Anthony Firulli, PhD Shreevrat Goenka, PhD Jesus Gutierrez, PhD Stacey L. Halum, MD Debra Hickman, DVM, MS H. N. Jayaram, PhD Philip L. Johnson, PhD Melissa A. Kacena, PhD NiCole Keith, PhD

Murray Korc, MD Tim Lahm, MD Mark LaPack, PhD Jirong Lu, PhD Malika Mahoui, PhD Anna Malkova, PhD James Marrs, PhD Jason S. Meyer, PhD Steven J. Miller, PhD Amber Mosley, PhD Sharlene Newman, PhD Linda Osborn, MS C. Subah Packer, PhD Mathew J. Palakal, PhD Horia Petrache, PhD Irina Petrache, MD Meeta Pradhan, PhD Jingzhi Pu, PhD

Bruce Ray, PhD Maria T. Rizzo, MD Alexander G. Robling, PhD Randall J. Roper, PhD Elliot D. Rosen, PhD Robert E. Sammelson, PhD Rajesh Sardar, PhD Anantha Shekhar, MD, PhD Rafat Siddiqui, PhD Daniel Sliva, PhD Michael Statnick, PhD Gavriil Tsechpenakis, PhD Johnathan Tune, PhD Steven R. Wassall, PhD Cong Yang, PhD Feng-Chun Yang, MD, PhD Feng C. Zhou, PhD

Rebecca Abaddi Adenike Adeyemo Susanna Adjei Caleb Akers Abimbola Akintomide Samuel Arregui Roshni Bag Jesus Bazan Ephraim Belayneh Gabriela Cabello Ai-Xin Chen Rushvi Desai Lindsay Egan Sally England Karen Garcia

Priyanka Ghosh Devyn Hayes Leilah Hendricks-Forrest Emily Higgs Daniel Hnin Carmen Hu Charles Irving Dhwani Juthani Hannah Kassab Nathan Kassab Mariah Keane Jatana Ketterman Victoria Kreyden Akshay Kumar Jacob Lucas

Ryan Lucas Shizen Moh Umaymah Mohammad Ruben Naoye Collin Nguyen Phuong ‘Wendy’Nguyen

Biak Chin Par Rushil Patibandla Liliana Patino Sharmila Paul Pranoti Pradhan Imani Rameses Abinand Rejimon Munajj Siddeeq Petr Sliva

Olivia Smith Melinda Song Austin Stults Amitha Sundaram Naeem Tai Ninad Thanawala Tianyu ‘Tony’ Tan Stephanie Tinsley Morrent Thang Shreya Veera Carmen Vernon Jonas Wang Odette Zero Baizhen Zhu Van Dawt Zi

Comparing the incidence of cardiovascular disease risk factors among fitness center

members who are from high and low socioeconomic groups

Rebecca Abaddi1, Kisha Virgil3, Sarah Shore-Beck2, NiCole Keith, PhD3

1Hamilton Southeastern High School, Fishers, IN, and 2Department of Kinesiology, IUPUI School of Physical

Education and Tourism Management, Indianapolis, IN, 3IU School of Health and Rehabilitation Sciences, IUPUI, Indianapolis, IN

Introduction: Income and education are combined to determine socioeconomic status (SES). People with a lower SES are at a higher risk for CVD, when compared to higher SES individuals (Prasad, 2009). Purpose: This project evaluated whether differences in the number of CVD risk factors existed when SES groups were compared. Methods: All participants were members of a Midwest, urban fitness center and had equal access to the facility (e.g., location and financial barriers were eliminated). Participants completed a Demographics and Health History Questionnaire and participated in health and fitness assessments (SFT). Analysis: A one-way analysis of variance (ANOVA) for categorical variables and T-test for continuous variables were used in this cross-sectional study. Results: Of the 323 members evaluated, 46.25% were Non-Hispanic Black, 54.52% attained a high school diploma or had some college (e.g., Associate’s Degree or Trade School), and 27.86% were below the poverty threshold. People below the poverty threshold when compared to people above the poverty threshold had lower RHR (81.06+13.10 vs. 75.21+12.39, p<.001), SBP (127.10+11.03 vs. 123.89+16.43,p<.05), DBP (78.09+11.21 vs. 75.48+11.76,p=.07), BMI (36.92+10.73 vs. 32.38+9.76,p<.001), and more CVD risk factors (2.61+1.70 vs. 2.13+1.53,p<.05).This group also had lower SFT scores. The medium-level of education group (High School Diploma/GED, Some College, Associate’s Degree, or Trade School) had more CVD risk factors and a higher mean blood pressure and body mass index when compared to high and low education groups. All definitions and results are presented in the poster and paper. Conclusion: While income findings from this research are consistent with the literature, education findings are not. Future research should further evaluate whether access to a fitness center influences CVD risk factors more than education.

Development of a Novel Mouse Femoral Isograft Carrier Model for Assessing Bone Healing

Adenike O. Adeyemo1,2, Thomas Bemenderfer2, Alexander R. Wesssel2 , Jonathan Harris2, MD, Monique Bethel2, MD, Yinghua Cheng2, MD, PhD, Melissa A. Kacena2,PhD

1Ben Davis High School, Indianapolis, IN, 2 Orthopaedic Surgery, Indiana University School of Medicine, Indianapolis, IN

Critical-size defects (CSDs) in bones do not naturally heal within the lifespan of an animal without intervention. Despite advances in surgical techniques and improved implant technology, achieving bony union across such large gaps remains a challenge. Currently, clinicians use bone morphogenic protein (BMP) alone or coupled with native or cadaveric bone grafts to augment bone healing. Although these methods have shown efficacy in promoting healing, they also come with a unique set of limitations.

We previously found that thrombopoietin (TPO), the main megakaryocyte growth factor, was able to heal a femoral CSD in mice when delivered to the defect site by a specially designed composite scaffold. However, use of composite spacers is not an ideal model for moving this work into the clinical arena. Therefore, in this study we sought to develop a more translatable mouse model for studying bone healing. Briefly, we created a 4 mm defect in the femur, into which was placed an isograft (a segment of femur obtained from a genetically identical mouse) specially designed to deliver either BMP-2, TPO, or saline (control) to the defect site. The defect, with the graft in place, was then stabilized over a 21 gauge pin and the animals were allowed to heal for 15 weeks. Our studies demonstrate that this model allows for successful healing of CSDs. Development of animal models which more closely represent what is observed in the clinic is of critical importance for developing novel therapies to enhance bone healing.

Measurements of protein-lipid interactions

Susanna Adjei1, Bruce D. Ray, PhD2, Horia I. Petrache, PhD2

1Plainfield High School, Plainfield, IN, and 2Department of Physics, Indiana University Purdue University Indianapolis, Indianapolis, IN

Glycosylation is the most common post-translational protein modification. Its exact role in protein functions and interactions is still debated. Previous ion current measurements using the antibiotic channel gramicidin A in the presence of the glycosylated Serpin family proteins, alpha-1 antitrypsin (A1AT) and the three forms of ovalbumin have shown interference with ion channel transport, suggesting that there is some kind of interaction between the ion channel and the glycosylated Serpin family proteins. In contrast, this kind of interaction is not detected with glycosylated proteins from the Kazal family. The structural shapes of A1AT and ovalbumin in the Serpin family are ovoid, whereas ovomucoid in the Kazal family is more T-shaped because of its three domains. In this study, ion channel measurements of Gramicidin A were used to determine if a glycoprotein in another structural family would interfere with ion transport. Conalbumin, also known as Ovotransferrin, is a member of the transferrin group and is structurally related to metalloproteinases. Along with ovalbumin, it is one of the major proteins in egg white. Like ovalbumin, and unlike A1AT, ovotransferrin only has a single glycosylation that does not contain galactose and does not have a terminal sialic acid. Ion channel measurements of ovotransferrin showed an interference pattern similar to the ones observed for the three forms of ovalbumin and alpha-1 antitrypsin. This study can help determine the effect of glycosylation on protein-membrane interactions.

Bringing new information-generating technologies to Eli Lilly and Company

Caleb Akers1, Gregory Brown2, and Mark A. LaPack, PhD2

1 Roncalli High School, Indianapolis, IN, and 2 Eli Lilly and Company, Indianapolis, IN

Process analytical technology (PAT) is an important tool used in the development and manufacture of active pharmaceutical ingredients (API) and products, specifically to obtain real-time reaction profiles used to investigate process mechanisms and to generate models that lead to better understanding of the chemical process. PAT can be an integral part of the process control strategy to ensure safe operation of the process and high quality outputs. Recently, several sensors have been identified that could provide immediate value, including detecting off-gases that may present safety concerns (H2) or that adversely impact the process yield or product purity (CO2), or measuring the initiation of a process, its ability to maintain a steady state, and its completion. The goal of this study was to test the applicability of new technology for generating data to understand processes. These new technologies tested where the H2 Scan, Senova Systems’ pHit, Mettler Toledo Turbidity Transmitter, and the Vital Sensor CO2 monitor. The H2-Scan hydrogen gas sensor would be very useful in a gas stream process to indicate when a flammable or explosive level of hydrogen has been created. The other three systems would be used to improve reaction control or indicate when a process has reached completion. This study concluded that the H2 Scan and the Vital Sensor were acceptable for laboratory use. The H2-Scan sensor was quantitative down to about 3000 ppm of hydrogen, which has a lower flammability limit of 4% in air. The turbidity transmitter and pHit work with limited functionality and further development is required. Existing technologies coupled with new technologies tested in this study can effectively streamline the API processes for Eli Lilly and Company.

Force sensor in aide of chest compressions

Bim Akintomide1, Paul Curtis2, Paul Collins2, and Scott Engum, MD3

1Northwest High School, Indianapolis, IN, and 2,3Simulation Center, Indiana University Health,

Indianapolis, IN

Chest compression is a method used during cardiopulmonary resuscitation (CPR). CPR is used when a person has stopped breathing and has gone into sudden cardiac arrest, meaning their heart has stopped and they have no pulse. It is a very crucial technique and in order for it to be successful, it requires the individual performing it to know the required depth and the rate at which the compressions should be performed. On an average adult, the depth of compressions should be 2 inches. Usually after about 30 compressions or after a minute, oxygen is supplied to the patient. The main purpose of this project is to program a sensor and a display to illustrate how much force is applied to the chest when performing CPR. A microcontroller, Arduino UNO R3, was programmed to output the data provided by an FSR (Force Sensitive Resistors) into a graph. FSR sensors are the cheapest and easiest way to make this project a reality. It is made of two layers separated by a spacer, and it changes its resistive value depending on how much force is applied. In addition to the graph, an electroluminescent panel was used to determine if the resuscitator was compressing sufficiently. However, there is some editing to be done in the code written for both the microcontroller and the display to develop and improve their responses. In conclusion, the FSR and electroluminescent will provide informative feedback to resuscitators thereby increasing the effectiveness of CPR to save lives.

Evaluation of parenteral lipid emulsions for prevention of hypoxic and ischemic/reperfusion injury in cultured cardiomycytes

Sam Arregui1, Kevin Harvey2, Rafat Siddiqui, PhD 2, 3

1Brebeuf Jesuit High School, Indianapolis, IN, and 2Methodist Research Institute, Indianapolis, IN and 3Indiana University School of Medicine, Indianapolis, IN

Ischemic heart disease is a leading killer, yet clinical therapies to prevent ischemic damage of the heart remain elusive. Cardiac cells utilize fatty acids as predominant source of energy. A lowering of the energy occurs during hypoxia (reduced oxygen availability). Low O2 acts as a signal in triggering the activation of Hypoxia inducing factor 1alpha (HIF-1a) and AMP-kinase (AMPK). AMPK is the key sensor of cellular energy status and involved in the regulation of fatty acid and glucose metabolism. However, increased metabolism generates excessive ROS and that leads to cellular injury. The objective of this study is to evaluate how fatty acid fuel in the form of lipid emulsions containing diverse fatty acid composition modulates hypoxic and/or ischemic-reperfusion injury. We employed a soybean oil-based lipid emulsion, Intralipid ® (Fresenius Kabi, DE), and an olive oil based emulsion, ClinOleic® (Baxter Healthcare Corporation, FR), to investigate effect of PUFAs (Intralipid) and MUFAs (ClinOleic) on hypoxia-induced AMPK activation. H9C2 cardiomycytes were treated with 1:4000 dilution of lipid emulsions under normoxia (22hrs), hypoxia (22 hrs), and hypoxia (19

hrs)/reperfusion (3 hrs) conditions. Induction of Hypoxia was followed by expression of HIF-1 and Hemeoxygense (HO-1), whereas activation of AMPK was determined by following its phosphorylation. Our

data shows that expression of HIF-1 and HO-1 was reduced during hypoxia or hypoxia/reperfusion conditions by ClinOleic®, whereas Intralipid® has only a small effect. ClinOleic® also reduced phosphorylation of AMPK in a similar fashion. This data demonstrates that MUFAs could prevent hypoxia-induced cellular injury in cardiomycytes whereas PUFAs were less effective. Experiments are underway to study effects of these lipid emulsions on cellular uptake system of fatty acid (CD36) and glucose (Glut4). In conclusion our data suggest that energy supply in the form of MUFAs during hypoxia has beneficial effects on hypoxia-induced cellular injury.

Are niche cells critical for bone marrow failure?

Roshni Bag1,2, Yongzheng He2,3 MD, Mingjiang Xu2,3 MD PhD, Feng-chun Yang2,3 MD PhD

1University High School of Indiana, Carmel, Indiana, 2Herman B. Wells Center for Pediatric Research, Indianapolis, Indiana, and 3Department of Anatomy and Cell Biology, Indiana University School of Medicine,

Indianapolis, Indiana

Fanconi anemia (FA) is a heterogeneous recessive disorder that results from a genetic defect in a cluster of 13 mutated genes that interfere with the common pathway associated with homologous recombination of damaged DNA repair. Patients with FA develop a range of hematopoietic malignancies, including acute myeloid leukemia (AML), myelodysplastic syndrome (MDS) as well as bone marrow failure (BMF). It has been long-known that defective hematopoietic stem cells (HSPCs) are associated with the pathogenesis of these hematopoietic deficits in FA. Given the fact that ~75% FA patients suffer from growth retardation, osteopenia/osteoporosis, kidney malformation, and pigmentation in skin, we hypothesis that the bone marrow “niche” is also defected in FA patients, and the defective “niche” may contributes, at least in part, to the pathogenesis of hematopoiesis in FA. We have previously established that deficiency of Fancg genes (Fancg-/-) impairs hematopoietic reconstitution in murine system. In the present study, we analyzed “niche” functions in a Fancc deficient and Fancg deficient murine model (Fancc:g-/-) who has most severe hematopoietic phenotypes. We found that Fancc:g-/- mice had a low frequency of MSPCs in the bone marrow. Fancc:g-/- MSPCs had diminished proliferation and increased apoptosis. In addition, Fancc:g-/- MSPCs also had less potential to differentiate into osteoblasts, while the osteoclasts derived from the HSPCs were hyperactive, thus resulting in decreased bone mineral density. Our results demonstrate that the knockout of the 2 Fanc genes impairs MSPC and osteoblast functions, leading to prominent defects in the bone. The defective bone niche may contribute, in part, to the hematopoietic deficits.

Animal welfare: The foundation for scientific research

Jesus Bazan1, Melissa Swan2, R’nld Wheeler2, Hannah Becker3, Chris Boehm2, Debra Hickman, MS, DVM2

1Lawrence Central High School, Indianapolis IN,2 Laboratory Animal Resource Center, IU School of Medicine,

Indianapolis, IN 3University of Notre Dame, Notre Dame, IN

One main aspect to consider when dealing with animals in the laboratory research setting is to take into account animal welfare. Each species of animal has a recommended set of guidelines provided by the Institutional Animal Care and Use Committee (IACUC), which is based off The Guide for Care and Use of Laboratory Animals. These species specific guidelines must be followed in order to ensure that the needs of each species are optimized, both physically and emotionally. Physical needs may range from food, water, and shelter where as these regulations aim to avoid changes to the animal that cause stress, pain, and anxiety. At times, improving the environment or physical needs of an animal will also help enhance the emotional needs of an animal by increasing behaviors exhibited their wild counterparts.

At the Laboratory Animal Resource Center (LARC), the Hickman Lab aims to improve the overall welfare for animals used in the research setting. Various studies this summer have been conducted that address the improvement animal welfare. Two projects that will be presented today include the Sentinel Study and the Cognitive Bias Study, which both involve manipulating the environment to improve the welfare of each species. Observations made at LARC help justify that animal welfare needs to be considered in science as well as the science of animal welfare is gaining importance in the field of laboratory animal medicine.

Literature mining to correlate miRNAs in pancreatic cancer

Ephraim Belayneh1, Mathew J. Palakal, PhD2, Meeta P. Pradhan, PhD2

1Ben Davis High School, Indianapolis, IN, and Indiana University Purdue University Indianapolis School of Informatics, Indianapolis, IN

Pancreatic Cancer (PC) is the fourth common cancer across the world. Recent studies have correlated miRNAs with progression of PC. Our objective for this work is to obtain the global profile of PC miRNA by augmenting experimentally data with literature data. For 65 experimentally validated miRNAs in PC we performed literature mining using in-house built tool BioMAP and extracted additional 100 miRNAs. The targets of these miRNAs were identified using miRBASE. Around 10,000 genes were identified as targets and were analyzed based on their target scores (100, 95, 90, 80). Twenty one miRNAs were associated with all scores, 25 miRNAs were associated with only one score and 24 miRNAs were associated with only 100 target score. Our study identified miR-100-3p, miR-198, and miR-183-3p as novel PA miRNAs. Unique genes identified for each score were 41, 124, 221 and 491. Using PubMed we analyzed the 41 genes associated with target score 100 for their significance in PA. Only nine of these genes were reported to play a role in PA and the rest 32 are unique, for example, zinc finger proteins etc. We further ranked the miRNAs based on their target connectivity profile and validated their association with PA using PubMed. These miRNAs correlate with PA, breast cancer, lung cancer, gastric cancer, ovarian cancer, and colorectal cancer. The target genes were analyzed for their pathways using KEGG. The number of unique pathways identified was fifteen i.e. metabolic, MAPKs signaling etc. Our study identified novel miRNAs, and genes that can be validated in the experimental laboratory; at the same time it demonstrated the power of using literature data for obtaining the global profile of a PC.

The CTD phosphatase rtr1 is regulated by CTDK-I

Gabriela Cabello1, Whitney Rae Smith-Kinnaman2, Melanie J. Fox2, Jerry M. Hunter2, Amber L. Mosley, PhD2

1Ben Davis High School, Indianapolis, IN, 2Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN

Rtr1 is a recently described CTD phosphatase that functions during transcription elongation to remove

serine 5 phosphorylation from the RNAPII CTD. As such, Rtr1 plays an important although poorly understood

role in regulating the transition from RNAPII initiation to elongation during transcription. This study

investigated the role of CTDK-I in the regulation of Rtr1 function during transcription elongation, using

Saccharomyces cerevisiae as a template. Considering that CTDK-I is a kinase complex that directly

phosphorylates serine 2 in the CTD, it is possible that S2 CTD phosphorylation has a role in either the

recruitment or regulation of Rtr1 during transcription elongation. We have found that the deletion of the

catalytic subunit of CTDK-I, CTK1, results in reduced interaction between Rtr1 and RNAPII. Experiments in

this study focused on resolving the possibility that Rtr1 recruitment is regulated by direct modification by

the CTDK-I complex.

Our preliminary purifications of Rtr1-TAP from CTK1 deletion cells revealed that CTDK-I activity is required

for Rtr1 to interact with RNAPII and for Rtr1 phosphorylation. To confirm this result, we performed

additional biological replicates of these studies such that we could determine the statistical significance in

the change in Rtr1-RNAPII binding. In addition, we performed western blot analyses of the Rtr1-TAP

purifications from wild-type and ctk1Δ cells to confirm our finding from the mass spectrometry analysis. The

experiments confirmed that CTDK-I activity is required for recruitment of Rtr1 to RNAPII. The identification

of the proteins and/or modifications that control Rtr1 function is of broad significance since currently the

mechanisms that dictate Rtr1 function are not fully understood. Understanding the fundamental biology of

transcription elongation can provide targets for future drug treatments.

Craniofacial malformations as a result of perturbed Hand1 Dimer regulation

Ai-Xin Chen1, Beth A. Firulli1, Anthony B. Firulli Ph.D2

1Zionsville High School, Zionsville, IN, and 2Riley Heart Research Center, Indiana University School of Medicine, Indianapolis, IN

Neural Crest Cells (NCCs) are migratory cells that give rise to melanocytes, craniofacial cartilage and bone, the cardiac outflow tract, smooth muscle, peripheral and enteric neurons, and glia cells. Thus, NCCs play a crucial role in craniofacial structure and abnormalities in their development and migration lead to cleft palates and lips, one of the most common birth defects in the United States. Our study focuses on the twist-family bHLH transcription factors, specifically Hand1 and its role within the NCC during embryogenesis. For Hand1 to function, it must form a dimer with another bHLH factor, creating transcriptional complexes such as Hand1 homodimers or Hand1-E12 heterodimers. The Firulli lab has uncovered a post-translational phosphorylation circuit that influences the dimer choice of Hand1 in vitro. The task now is to investigate the molecular pathways of hypo-phosphorylated and phosphorylated Hand1 mutations and their morphological implications. We collected tissue from wildtype and Hand1 mutant mice models to isolate RNA and performed reverse transcriptase polymerase chain reaction (QRTPCR) analysis. To look at positional changes of gene expression we used whole mount in situ hybridization to examine changes in tissues within the early to mid-gestation wildtype and mutant embryos. Preliminary results indeed showed that the expression levels of genes related to the Hand1 pathway were altered. The expression of PDGFbR decreased, Etv4 and Gli1 increased, Etv5 and Gli3 were not statistically significant, while Shh and PDGFaR stayed constant, all with respect to the wildtype levels. These data suggest that disruption of dimer regulation of Hand1 alters several signaling pathways critical for craniofacial formation.

An aggregate outlook on the effects of risk factors on preterm births using statistical analysis

Rushvi Desai1, Malika Mahoui, PhD2

1Carmel High School, Carmel, IN, and 2The Polis Center, Indiana University-Purdue University, Indianapolis, IN

Preterm birth leads to an increased risk of health problems because the child is underdeveloped at less than 37 weeks old. The purpose of this study was to assess potential variables aggregated at the census tract geographical level to find factors responsible for preterm births in Marion County, Indiana. By investigating the variables affecting preterm births, intervention and adequate surveillance can be implemented into public health and obstetrician practices. The risk factors tested using statistical analyses include income, poverty level, substance abuse, ethnicity, level of education, and prenatal care. Data was pulled from the Central Indiana Community Information System (also known as SAVI). Using linear regression, the relationship between the health risk factors and preterm births was determined. Experimentation and testing for correlations, linear regressions, and regression diagnostics were done through the Statistical Package for the Social Sciences (SPSS). Keeping all other variables constant, an increase in the overall value of a particular risk factor was done through a change in its coefficient to prove an association. Results from the study showed that in addition to the mother’s race, the father’s race proved to be of even greater threat to the risk of preterm birth. Health risk factors such as the consumption of drugs and alcohol, STDs or AIDs, and smoking showed positive correlation to preterm birth rates. Limitations such as missing years, performing the tests at the census tract level, and functions of display may have an impact on the effectiveness of the results in this study. Data from this study need to be further investigated, thus contributing to lowering the rate of preterm births in Marion County.

Analysis of self-interaction of therapeutic antibodies by Dynamic Light Scattering

Lindsay Egan1, Chi-kin Chow2, and Jirong Lu, PhD2

1Westfield High School, Westfield, IN, 2Protein Optimization Team, Biotechnology Discovery Research

Division, Eli Lilly & Co., Indianapolis, IN

Dynamic Light Scattering (DLS) is a technique typically used to measure the diffusion coefficient and hence to determine the size of polymers in solution. The purpose of this study was two-fold: (A) To develop a method using DLS that can reproducibly determine the interaction parameter (kD) by measuring the diffusion coefficient at different concentrations. (B) To determine whether the kD values correlate with the behavior of a protein. Tests were done in three sets of antibodies in this study. Each individual set of antibodies binds to the same targets and only differs in a few amino acids. These sets are six bispecific antibody variants (BsAb A1-A6), three monoclonal antibody variants (mAb B1-B3), and three other monoclonal antibody variants (mAb C1-C3). In the first set, it was previously observed that BsAb A1 and BsAb A6 are more hydrophobic than the other bispecific antibodies in the set. BsAb A1 and BsAb A6 were found to have a relatively higher negative kD value which correlates to their higher hydrophobic interactions. In the second set, it was previously observed that mAb B1 and mAb B3 have a higher tendency to show phase separation. The kD values of these antibodies were highly negative, which correlates with their behavior. In the third set, mAb C2 was previously observed to have a higher tendency to aggregate. mAb C2 had a higher negative kD value which correlates with its behavior. In conclusion, kD values were successfully measured and they correlated well with the behavior of the proteins. A less negative kD value suggests that the protein is better behaved compared to a protein with a more negative kD value.

Gender differences in development of metabolic syndrome in ZDSD, a new rodent model

Sally A England1, David M Eaton, III2, Ashley N Rogers3, Ami E Rice, MD3, Kathy Coy 4, Richard G Peterson, PhD, 4 and C Subah Packer, PhD3

1Southport High School, 2Purdue University, 3Indiana University School of Medicine and 4PreClinOmics (PCO) Metabolic syndrome is a disease characterized by obesity, dyslipidemia, diabetes and hypertension. Interestingly, the development of metabolic syndrome and the impact of metabolic syndrome on cardiovascular health appears to be disparate for men compared with women. The gender dichotomy has received relatively little investigative attention. The ZDSD is a new leptin intact rat model of metabolic syndrome. An initial serial time course study showed that male ZDSD and control male CD have similar growth curves but that ZDSD develop hyperglycemia, hypercholesterolemia, dyslipidemia and high blood pressure. Accordingly, the current study addressed the Hypothesis: Male ZDSD develop hypercholesterolemia, hypertriglyceridemia, hypertension and diabetes earlier in development and manifest more severe symptoms with aging than do Female ZDSD. The Specific Objective was to compare Male and Female ZDSD blood pressures, circulating cholesterol, TG and glucose levels in a serial time course study from weaning until late adulthood. Results of this on-going study show Male ZDSD manifest characteristics of metabolic syndrome early in life. Males had significant elevations in TG and BP before 60 days of age while Female values remained in the normal range at least until after 70 days of age. Results suggest that in predisposed populations, males are at higher risk than females of developing full blown metabolic syndrome. (Funded by PCO; David Eaton is a NSF Louis Stokes Alliance for Minority Participation, LSAMP, Scholar.)

Method optimization to improve recoveries of high molecular-weight polycyclic aromatic hydrocarbons from an asphalt matrix

Karen G. Garcia1, Michael A. Brinton2, Lisa A. Smith2 and Linda V. Osborn2

1Ben Davis High School, Indianapolis, IN, and 2Heritage Research Group, Indianapolis, IN

Europe has a regulatory program that deals with the Registration, Evaluation, Authorization and Restriction of Chemical substances (REACh), which seeks to improve the protection in human and environmental health. Asphalt is one substance that is registered within the REACh program such that the concentrations of eight individual Polycyclic Aromatic Hydrocarbons (PAHs) must be determined. In order to comply, the sum of these eight PAH concentrations should not exceed 10 ppm and the level of benzo[a]pyrene should be below 1 ppm. Studies show that these PAHs may be harmful to the body possibly resulting in damage to lungs, liver, skin, and kidneys with significant exposure. Understanding the composition of asphalt helps scientist predict the potential risk of different types of asphalts. Detection of these eight PACs was achieved using gas chromatography/time-of-flight mass spectrometry. Heritage Research Group has previously used a modification of the BS 2000-346 to extract these PAHs from an asphalt matrix. However, recovery of some of the higher molecular weight PAHs was poor. The objective of this study was to modify the existing extraction method in order to improve recoveries of higher molecular-weight PAHs from an asphalt matrix. In order to improve the existing method, four variables were investigated. First, additional extractions were added. Secondly, conical vials were used to improve separation. Thirdly, the dimethylsulfoxide was heated to 80oC prior to use. Finally, the last modification involved replacing iso-octane with dichloromethane during the latter part of the extraction procedure. Results show that replacing iso-octane with dichloromethane at the latter stage of the extraction procedure shows the most promise to improving recoveries of the higher molecular weight PAHs. Future research will involve validating this method modification by performance of repeatability and method detection limit studies.

Exploring the relationship between phonological ability and arithmetic ability in children

Priyanka Ghosh1, Roy Seo2, Sharlene Newman, PhD2

1Bloomington High School South, Bloomington, IN, and 2Department of Psychological and Brain Sciences,

Indiana University, Bloomington, IN

Reading disability (RD) and mathematic disability (MD) have long been found to co-occur. Neuroimaging studies have supported this co-occurrence, as both RD and MD in children with dyslexia have been linked to a common origin, the inferior parietal cortex. Since difficulty with phonological processing often underlies RD, in this study we examined the relationship between phonological processing and simple arithmetic in children. The subjects (n=45) performed a pencil and paper task assessing phonological ability and a timed simple addition test. In the results, a positive correlation was found between phonological and arithmetic ability. In order to investigate the neural mechanism behind the relationship, functional MRI (fMRI) scans were taken while subjects performed a rhyme-recognition task and a simple arithmetic task.

Mechanisms of double-strand break repair in yeast

Devyn Hayes1 , Munajj Siddeeq2 , Cynthia Sakofsky3 Soumini Vasan3 , Yu-Hsiang Chen3, Anna Malkova, PhD3

1Charles A. Tindley Accelerated School, Indianapolis, IN, 2Lawrence Central High School, Indianapolis, IN, and

3Department of Biology, IUPUI, Indianapolis, IN Double-Strand DNA breaks (DSBs) are breaks affecting both strands of the double-helix of DNA. DSBs are one of the most severe forms of DNA damage. This type of damage is normally repaired by different mechanisms within the cell. However, if left unrepaired, DSBs can lead to cell death. Additionally, defective repair can cause genome instability. One important DSB repair pathway is Break-Induced Replication (BIR). BIR is a replication-based repair pathway, such that one of the broken DNA strands invades a homologous region of DNA and copies that region in order to repair the DNA break. Although BIR can be beneficial to the cell, this pathway can also be dangerous because it can lead to several types of genome instabilities, similar to those that promote cancer. Thus, determining the mechanisms of BIR and its regulations is critical for understanding how genomes become destabilized. Our research aims to identify the genes that drive BIR into pathways of genome instability. We used helicase (pif1Δ) and translesion synthesis DNA polymerase (rev3Δ) deletion mutants to determine how these mutations affected the frequency of BIR. We scored and calculated frequencies of various DSB repair outcomes, such as gene conversion, BIR, chromosome loss, and half-crossovers, in wild-type strains and mutant strains. Finally, we compared the frequencies between mutants and wild-type strains to determine how the specific DNA replication protein mutation affects BIR. Our results show that the mutants lowered the frequencies of BIR and raised the frequencies of chromosome losses and half-crossovers, which can be considered deleterious DSB repair outcomes. These data suggest that the absence of these DNA replication proteins (Pif1 and Rev3) is involved in driving BIR into pathways of genome instability.

Micro-spatial analysis of urban gardens

Leilah-Olivia Hendricks1, Jessica Adamic2, Gabriel Filipelli, PhD2

1Broad Ripple High School, Indianapolis, IN, and 2Department of Earth Sciences, Indiana University School of Science, Indianapolis, IN

Despite great advancements in the remediation of lead sources in Indianapolis, the number of children ages 0-5 with lead poisoning is still much higher than the national average. The most common ways people are exposed to lead are through exposure to lead based paints, gasoline, and industry. The research in this project was conducted with the Urban Gardening Initiative. This study focuses on urban gardens in the 46218 zip code area where a lead smelting facility (National Lead Industries) exploded in 1971. This area has since been remediated by the Marion County Health Department (MCHD)and Indiana Department of Environmental Management (IDEM) but residents still had concerns. Using an ICP (Inductively Coupled Plasma)-OES instrument. Lead, Manganese, Zinc, and Copper values were analyzed. These results will then allow analysis of individual gardens and entire neighborhoods. Five different locations were samples from each house; these locations included a “road side”, “drip line”, and 3 “backyard” samples. Micro-spatial analysis testing results will provide insight into how lead and other elemental values vary within individual yards and neighborhood. This will help narrow down the location of lead sources and which parts of a property are safest for planting.

miR-192 increases pancreatic cancer cell growth in 3-D culture

Emily Higgs1, A. Jesse Gore, PhD2, Samantha Deitz, MS2, Murray Korc, MD2

1Zionsville Community High School, Zionsville, IN, 2Department of Medicine, Indiana University School of

Medicine and the Pancreatic Cancer Signature Center, Indianapolis, IN

Pancreatic ductal adenocarcinoma (PDAC) is a deadly malignancy with a poor prognosis, which is due to the retroperitoneal location of the pancreas, making an early diagnosis difficult, and the inherent resistance of pancreatic cancer cells to conventional therapies. Moreover, the dense stroma surrounding PDAC interferes with effective delivery of therapeutics to the cancer cells. Unfortunately, PDAC is often diagnosed at advanced stages when the tumors are sufficiently large to cause symptoms, yet even small tumors have a propensity to metastasize. Mouse models provide an effective way to study PDAC, since they recapitulate human disease and can be engineered with similar mutations, such as Kras (~99% of patients), and Smad4 (50-55%). Because micro-RNAs (miRNAs) may serve as novel therapeutic targets and biomarkers in PDAC, and may be oncogenic or tumor suppressing, we conducted microarray studies to identify novel miRNAs in pancreatic cancer cell lines derived from PDAC mouse models with Kras mutations alone or Kras and Smad4 mutations. Pancreatic cancer cells from a mouse model with concomitant Kras and Smad4 mutations expressed low levels of miR-192 compared to cells harboring mutated Kras alone, but higher levels compared with normal cells, suggesting that miR-192 expression may be modulated by Smad4. In support of this conclusion, BxPC3 human pancreatic cancer cells, which are Smad4-deficient, expressed low levels of miR-192. Given that Smad4 is a tumor suppressor gene, we next sought to determine whether up-regulation of miR-192 would inhibit cancer cell proliferation. Accordingly, we overexpressed miR-192 in BxPC3 cells and observed the effects on colony formation and growth in a 3-D system. Surprisingly, in this system, miR-192 enhanced growth, suggesting that miR-192 may promote tumor progression in PDAC.

Fibrin network formation under flow conditions

Daniel V.C. Hnin1, Horia Petrache, PhD2, Bruce D. Ray, PhD2, Elliot D. Rosen, PhD2

1Southport High School, Indianapolis, IN, 2Medical and Molecular Genetics, IU School of Medicine

Indianapolis, IN

Blood clotting evolved to prevent excessive bleeding following break of a blood vessel. The process is rapid, but it also regulated to prevent the growth of a clot inside the vessel (thrombus) following minor damage. By using fibrin, clots can form in the blood. Fibrin comes from an inactive precursor called fibrinogen. The body of the fibrinogen molecule has caps on its ends that mask fibrin from fibrin binding sites. Fibrin monomers polymerize to form fibrin polymers when the caps are removed. Thrombin and calcium are required in this process. Thrombin converts fibrinogen to fibrin. Calcium is used as glue to hold the fibrin monomers to each other, forming the polymeric fiber. The purpose of this project is to study if polymerized fibrin network forming on the surface of a growing clot provides a barrier limiting growth. To explore hypothesis, I have worked to develop an experimental system to form fibrins networks under flow condition. I have designed and taste microfluidic chambers to develop a system that can be used experimentally.

Epicardial perivascular adipose tissue impairs coronary vasodilation of voltage-dependent K+ channels

Carmen Hu1, Rebecca Krenzke2, Meredith C. Kohr3, Johnathan D. Tune, PhD3

1Lawrence North High School, Indianapolis, IN, 2St. Malachy Parish School, Brownsburg, Indiana, and 3Department of Cellular & Integrative Physiology, IUSM, Indianapolis, IN

Recent evidence from our laboratory suggests coronary perivascular adipose tissue releases constricting factors that influence vascular smooth muscle tone. The present investigation was designed to examine the contribution of perivascular adipose tissue (PVAT) to voltage-dependent potassium channel (KV)-mediated relaxation in porcine coronary arteries. We performed isometric tension studies on isolated coronary arteries from Ossabaw swine in the presence and absence of PVAT (0.3g). Arteries were isolated, cleaned of surrounding connective tissue and incubated 30 min in the presence or absence of PVAT. Following the incubation period, arteries were precontracted with U46619 (1uM) and after reaching steady-state maximum tension, stimulated with increasing concentrations of H2O2, a KV channel agonist (10nM-1mM). Our data reveal that the presence of PVAT did not affect overall tension generated with U46619, however, PVAT significantly diminished H2O2-induced relaxation (at 100uM and 1mM) relative to arteries not incubated with PVAT. These data suggest that factor(s) released from PVAT influence the activation state of KV channels.

Catalytic activity of polymer functionalized anisotropic gold nanoparticles

Charles Irving1, Gayatri Koshi2, Kimberly Smith2, Rajesh Sardar, PhD2

1Irvington Community High School, Indianapolis, IN, and 2Department of chemistry, Indiana University

School of Science, Indianapolis, IN

Metal nanoparticles have been used as catalysts for a variety of different reactions such as, reduction of alkenes, water splitting, and the conversion of solar energy to electricity. In this project PAA (polyallyamine) and PSS (polysodium 4-styrenesulfonate) were used to functionalize LbL (layer by layer) self-assembly of gold nanoprisms on a glass substrate as a heterogeneous catalyst for the reduction of p-nitrophenol to p-aminophenol. It was found that as the number of layers increased the catalytic activity of gold nanoprisms decreased. We have also found that gold nanoprisms are a more efficient catalyst as compared to spherical gold nanoparticles.

Characterization of antimicrobial peptide resistance conferred by phosphoethanolamine transferases in Haemophilus ducreyi

Hannah D. Kassab¹, Sherri D. Rinker², Michael P. Trombley², and Margaret E. Bauer, PhD²

¹Pike High School, Indianapolis, IN, and ²Department of Microbiology and Immunology, Indiana University

School of Medicine, Indianapolis, IN Haemophilus ducreyi (H. ducreyi) is a gram-negative bacterium and the causative agent of the sexually transmitted ulcer disease chancroid, which facilitates the transmission of HIV. Previously, the Bauer lab has identified putative resistance mechanisms within H. ducreyi against antimicrobial peptides (APs), one of which is phosphoethanolamine (PEA) transferases. PEA is a cationic functional group and PEA transferases are enzymes that transfer PEA to the lipooligosaccharide (LOS) of H. ducreyi to modify its surface charge. Since the human immune system interacts with the highly negative surface charge of invading bacteria via small, cationic APs, H. ducreyi defends itself using PEA transferases to transfer PEA onto the LOS. This changes the surface charge of the bacteria from its natural anionic state to a less anionic state, thereby causing electrostatic repulsion away from the APs and conferring resistance to APs during infection. To understand the relationship between surface charge modifications and AP resistance, a nonpolar, unmarked deletion mutant of H. ducreyi was constructed, wherein three genes (HD0852, HD0371, and HD1598) that encode PEA transferases were deleted. The PEA transferase mutant, MEB311, was less resistant than the parent strain to the highly charged β-defensin HBD-3 (+11). In this study, we attempted to characterize the LOS of the deletion mutant by optimizing different protocols to detect differences in surface charge between mutant and parent. The isogenic mutant MEB311 is more negatively charged than pathogenic clinical isolate 35000HP, which supports our hypothesis that PEA transferases lessen the negativity of the surface of H. ducreyi. These findings could contribute to future creation of novel therapeutics that disrupt H. ducreyi’s mechanisms of AP resistance and assist the host immune response in killing the bacteria.

Profiling GLP1 Peptides by Mass Spectrometry in Tissues

Nathan Kassab1, Zhaoyan Jin2, Jesus Gutierrez, PhD2

1Pike High School, Indianapolis, IN, and 2Department of Translational Science and Technology, Eli Lilly and

Company, Indianapolis, IN

Glucagon-like Peptide-1 (GLP-1) is one of two major incretin hormones and is secreted from

enteroendocrine cells through gastrointestinal tract in response to nutrient ingestion. GLP-1 is also found to

be secreted in selected neurons in brain. GLP-1 participates in numerous biological activities including

stimulation of glucose-dependent insulin production and secretion, inhibition of glucagon secretion, and

inhibition of food intake. Upon secretion, the active GLP-1 forms (7-36NH2and 7-37OH) forms are rapidly

converted to inactive forms (9-36NH2 and 9-37OH) by DDPIV and NEP proteases. Frequently, immunoassays

are used to detect and quantify the distinct forms of GLP-1. However, there does not exist a universal assay

to profile simultaneously the multiple GLP-1 forms in tissues. Instead, the available assays detect subset of

GLP-1forms and multiple assays are often required to profile these proteins. Even though GLP-1 peptides in

circulation have been extensively studied, the precise GLP-1 profile in tissues has not been systematically

studied. In this study, we developed a sensitive immuno-precipitation assay coupled with mass

spectrometry (IPMS) to profile GLP-1 forms in rat tissues.

Effects of nociceptin receptor agonist Ro64,6198 treatment on opioid peptide expression in the CNS of alcohol preferring rats

Mariah Keane1, Susan Gackenheimer2, Todd Suter2, Roberto Ciccocioppo, PhD3, and Michael A. Statnick PhD2

1Warren Central High School, Indianapolis, IN, 2Eli Lilly and Company, Indianapolis, IN,

3University Camerino, Camerino, Italy

Nociceptin (NOC) is a 17 amino acid peptide agonist for the Gprotein-coupled receptor (GPCR) ORL1. Activation of ORL1 inhibits the formation of cyclic AMP, which leads to reduced neuronal excitability and neurotransmitter release. In rat models of ethanol dependence dosed with Nociceptin and/or Ro64,6198 (a non-peptide nociceptin receptor agonist) decreases alcohol consumption (Murphy, 2010). The msP rat used in our study is a widely accepted animal model for the study of ethanol drinking and dependence. The focus of the present study is to determine if chronic treatment with Ro64,6198 would alter gene expression levels of ORL1 or NOC in msP rats. We dissected the amygdala, hypothalamus, and frontal cortex from msP rats. RNA was isolated from the samples and cDNA was synthesized using standard protocols. Primers specific for ORL1 and NOC were used in polymerase chain reaction (PCR) to determine relative levels of gene expression. Three days following a week of Ro64,6198 treatment at 24 hours, ethanol consumption was reduced with no effect on food or water intake. The NOC expression was unchanged in all three brain regions at this time point. In contrast, ORL1 expression was selectively decreased by Ro64,6198 in the pre-frontal cortex of ethanol naïve rats. Surprisingly in ethanol drinking msP rats, expression of ORL1 was unchanged, therefore it appears that the transcriptional regulation of Ro64,6198 is affected by ethanol drinking. Which suggests ethanol drinking regulates the ability of Ro64,6198 to down regulate ORL1 expression, which commonly occurs following chronic agonist stimulation of GPCRs. Our data supports that agonist stimulated receptor internalization is affected by ethanol consumption, suggesting that treatment with an ORL1 agonist compound may provide a novel treatment for alcoholism.

Examining the role of PAX6 in neural development using human induced pluripotent stem cells

Jatana M. Ketterman¹, Akshayalakshmi Sridhar², Melissa M. Steward², Jason S. Meyer, PhD2-4

¹Warren Central High School, ²Department of Biology, IUPUI, 3Indiana University Department of Medical and Molecular Genetics, 4Stark Neurosciences Research Institute, Indianapolis, IN

The development of the vertebrate nervous system is an elaborate and complex process requiring the actions of numerous genes and signaling factors, with the expression of these genes in a coordinated and sequential manner. Among the first events in neural development is the specification of a neural fate from the primitive ectoderm. Previous studies have implicated the transcription factor PAX6 as playing an instrumental role in human neural development. To further investigate the role of this gene, we utilized human induced pluripotent stem cells (hiPSCs) as a model system to mirror those events that occur in human neurogenesis. To elucidate the role of PAX6, we utilized previously established protocols to direct the differentiation of hiPSCs to a neural fate. In control experiments, wild-type lines of hiPSCs were used, whereas PAX6 was knocked down in experimental lines of hiPSCs through the use of shRNA technology. Samples were collected at Day 0, Day 10, and Day 20 of differentiation, analogous to critical stages of human neural development, including the pluripotent phase, neural plate, and neural tube. Immunocytochemistry (ICC) and RT-PCR techniques were used to analyze the acquisition of a neural fate in the absence of PAX6. Such experiments illustrated that the adoption of a neural fate was prevented in the absence of PAX6, and that the lack of PAX6 expression affected other transcriptional networks as evidenced by the decreased expression of other important neural transcription factors such as LHX2, SIX3, and FOXG1. This study establishes the critical role of PAX6 in neural development and demonstrates the use of hiPSCs as a model system for studies of human development.

Effects of hypoxia and sex hormones on rat pulmonary artery cell numbers

Victoria Kreyden1, Jordan A. Brown2, Marjorie Albrecht2, Irina Petrarche, PhD,2 Tim Lahm, MD2

1University High School of Indiana, Carmel, IN, 2 Division of Pulmonary, Allergy, Critical Care, Occupational and Sleep Medicine, Indiana University School of Medicine, Indianapolis, IN

Exposure to chronic hypoxia leads to pulmonary hypertension (PH), a condition characterized by abnormally high blood pressure in the arteries of the lungs. If left untreated, PH leads to right heart failure and death. Interestingly, women are more susceptible to PH development; however, the reason for this gender difference is unknown. Since one of the hallmarks of PH is uncontrolled growth of lung artery wall cells, we investigated the effects of the sex hormones 17beta-estradiol (E2), testosterone, and dehydroepiandrosterone (0.1nM-1microM) on rat pulmonary artery endothelial cell (RPAEC) counts during normoxia (21% oxygen) and hypoxia (1% oxygen). Cell numbers were counted manually and/or with an automated cell counter at 24, 48, or 72 hours. Manual and automated cell counts correlated well. In general, hypoxia (time-dependently) decreased cell numbers. Since hypoxia did not kill RPAECs (measured by trypan blue exclusion), we suspect that the hypoxia-associated decrease in cell numbers is due to decreased cell growth. E2 effects on cell numbers during hypoxia were mixed, with E2 increasing cell counts at lower concentrations, but decreasing numbers at higher concentrations. However, the latter effect may have been confounded by vehicle effects. Testosterone did not decrease counts during hypoxia, but tended to increase counts during normoxia. Dehydroepiandrosterone effects are currently under investigation. Hypoxia decreases RPAEC growth, and RPAEC numbers are influenced by sex hormones. Differential effects of E2 and testosterone on RPAEC growth may provide a partial explanation as to why women are more frequently affected by PH.

Identification of a specific protein motif that is modified by PARP-14

Akshay Kumar1, Nahid Akhtar2, Jonathan Riley2, Purvi Mehrotra2, Shreevrat Goenka, PhD2

1Carmel High School, Carmel, IN, and 2Herman B. Wells Center For Pediatric Research, Indianapolis, IN

Interleukin-4 (IL-4) is a cytokine that plays a role in immune responses and in allergic disorders. IL-4 activates the JAK-STAT pathway, specifically STAT6 to transcribe IL-4 dependent genes. Previously it has been determined that PARP-14 acts as a transcriptional switch to regulate STAT6 dependent transcription. PARP-14 recruits HDAC2 and 3 to repress transcription initially, but in the presence of STAT6 the enzyme activity (poly ADP-ribosylation) contained in the C-terminal portion of PARP-14 modifies its N-terminal and HDAC2 and 3. These modifications result in the dissociation of PARP-14, HDAC2 and 3 from the promoter, which allows for the activation of transcription. The identity of the protein motif on PARP-14 N or the HDACs that are modified is not known. The goal of this project was to identify the specific region that is modified by the C-terminal of PARP-14. Our approach was to express large quantities of PARP-14 N and C terminals as separate polypeptides and purify them, carry out in vitro poly ADP-ribosylation reaction followed by mass spectrometry to identify regions within PARP-14 that are modified. We have been able to express the N and C termini of PARP-14 using a bacterial expression system and partially purify these proteins. We are further optimizing the expression and purification of these polypeptides. Once we have suitable protein preparations, we will carry out ADP-ribosylation reactions followed by mass spectroscopy. We anticipate that we will identify the region of PARP-14 N that is ADP-ribosylated. Using this data we will also identify the regions of HDAC 2 and 3 that are ribosylated, and in the future we propose to use bioinformatics tools to find other substrates of PARP-14.

Molecular dynamics simulations of small acid soluble proteins

Jacob Lucas1, Pedro Ojeda-May2, Jingzhi Pu2, PhD2

1Arsenal Technical High School, Indianapolis, IN, and 2School of Science, Department of Chemistry and

Chemical Biology, IUPUI, Indianapolis, IN

The small acid soluble proteins, or SASPs, are known to contribute to the long-time survival of bacterial spores by providing key protection to the spores’ DNA from a multitude of environmental insults, including desiccation, heat, toxic chemicals, enzymes, and UV radiation. Existing as an intrinsically disordered protein in its monomeric form, the SASP undergoes conformational transition to a well defined helical structure upon binding to DNA. The purpose of this project was to determine if the disorder-order transition of the SASP is regulated intimately by DNA-protein and protein-protein interactions in the nucleoprotein complex. This was tested by computer simulations of the SASP protomer by removing the DNA component from the crystallized SASP-DNA complex structure. Key interactions from amino acid residues in the loop and the interhelical regions of the SASP were also investigated with a series of simulations carried out for the wild-type as well as two double amino acid mutants (E23A/R47A and G29A/N37A). Comparisons between the simulated structures and the reference x-ray crystal structure were made by monitoring the root mean square deviation (RMSD) of the structures along the simulation. The results showed that the wild-type differed from the crystal structure in the absence of DNA, and the mutated systems displayed significantly larger RMSD than the wild-type system, suggesting that both DNA and the mutated residues play important roles in maintaining the structural integrity of SASPs. Contributing to a deeper understanding of how DNAs are protected in bacterial spores, this work may help us design new strategies that selectively target SASPs in battling spore-related diseases such as anthrax and tetanus.

Effects of ethanol on cell adhesion in zebrafish embryos

Ryan Lucas1, Liliana Patino2, Courtney Curtis3, Sonya Gilbert3, Swapnalee Sarmah3, Pooja Muralidharan3, and James Marrs, PhD3

1Arsenal Technical High School, 2Warren Central High School, 3Department of Biology, Indiana University-

Purdue University Indianapolis (IUPUI), Indianapolis, IN

Fetal Alcohol Syndrome (FAS) occurs when human embryos are exposed to ethanol during development, causing significant developmental defects, particularly cognitive and heart defects. Ethanol exposure in model systems, like zebrafish, can be used to study the consequences of embryonic ethanol exposure and FAS. Ethanol affects epiboly (the first coordinated cell movement) and gastrulation cell movements in the embryo. Previous studies show that cell adhesion often contributes to epiboly and gastrulation defects. The purpose of this study was to determine if cell adhesion defects contribute to epiboly and gastrulation delay in ethanol treated embryos. To do this, embryos were raised to 4.5 hours post fertilization (hpf). Then, cells inside the embryos were dissociated. Cells were split into two treatment groups. A control group received normal L-15 medium, which keeps cells healthy, while the other receives L-15 supplemented with a 100 mM ethanol. Cells were placed on fibronectin-covered plates, and images were collected every 3 minutes for 3 hours, using DIC (Differential Interference Contrast) microscopy. The total number of cells and objects (groups of cells) were counted. A cell-to-object ratio for each image was then analyzed. Overall, our research showed that the group of cells exposed to ethanol had a lower cell-to-object ratio than control cells, meaning that ethanol reduces cell adhesion in embryonic cells. Therefore, our experiment indicates that adhesion defects contribute to ethanol-induced delays in epiboly and gastrulation.

Role of the lal gene in cancer metastasis

Shizen Moh1, Akira Moh2, Ting Zhao2, Ding Xinchun2, Linyuan Wu2, Hong Du PhD2, and Cong Yan PhD2

1Carmel High School, Carmel, IN, and 2Department of Pathology, Indiana University School of Medicine,

Indianapolis, IN

Cancer metastasis, the spreading or relocation of cancer, is the leading cause of cancer related mortality and unlocking its secrets is the final frontier in cancer research. Macrophages of the immune system play a key role in fighting cancer. The lal (Lysosomal Acid Lipase) gene creates the LAL enzyme in macrophages, which breaks down lipids to produce fatty acids to be used as cell energy. When this gene is knocked out of the genome of macrophage cells, we observed a dramatic increase in cancer cell proliferation. Based on this observation, we hypothesized that the lal gene plays a role in cancer metastasis. We observed the relation between macrophage cells from mice and B16 melanoma cancer cells in co-cultures. Preliminary results confirmed our original hypothesis; cancer proliferation had increased by as much as 70% in the presence of knock-out macrophages. This suggests that the LAL gene plays a key role in cancer metastasis. This knowledge may be used in the future to potentially discover ways to slow or even prevent cancer metastasis.

Prostaglandin E2-dependent signaling pathways involved in protecting human glioblastoma cells from endoplasmic reticulum stress-induced death

Umaymah Mohammad1 and Maria Theresa Rizzo, MD2

1Hamilton Southeastern High School, Fishers, IN, and 2Signal Transduction Laboratory, Methodist Research

Institute, Indiana University Health and Department of Medicine, Indiana University School of Medicine, Indianapolis, IN

Glioblastoma multiforme (GBM) is an aggressive primary brain tumor characterized by a high propensity to survive in unfavorable environmental conditions and to rapidly recur after treatment. The underlying molecular mechanisms are not understood. Our previous work demonstrated that prostaglandin-E2 (PGE2) stimulates growth of the human GBM cell lines U87-MG and T98G. Preliminary results indicate that PGE2 increases U87-MG cell survival upon their exposure to stimuli that induce endoplasmic reticulum (ER) stress-dependent cell death. Therefore, experiments were performed to understand the mechanisms fundamental to the protective effects of PGE2 against ER stress-induced death. Luciferase reporter assays were performed to characterize the transcriptional response of U87-MG cells to ER stress. Cells were transiently transfected with a luciferase reporter containing a consensus sequence for Activating Transcription Factor 6 (ATF6), one of the signaling arms of the Unfolded Protein Response (UPR) activated by ER stress. Stimulation of U87-MG cells with ER stress inducers, such as thapsigargin, tunicamycin or brefeldin A, increased luciferase activity. Because PGE2 inhibits phosphorylation of the extracellular signal-regulated kinase (ERK) in U87-MG and T98G cells, we explored the consequences of ERK inhibition on ER stress. PD98059 alone enhanced ATF6-dependent luciferase activity relative to control cells. Thapsigargin-, tunicamycin- or brefeldin A-induced luciferase activities were increased after treatment with PD98059. Together these results suggest that U87-MG cells are sensitive to induction of ER stress. Furthermore, PGE2-dependent protective effects are not mediated by ERK inhibition. However, additional studies are necessary to examine the contributions of ERK inhibition on the other signaling arms of UPR. Understanding the protective mechanisms involved in ER stress-induced death may lead to novel strategies to improve response to therapy and decrease tumor recurrence.

Identifying the targets of human antibodies in xenotransplantation

Ruben Naoye, A. Joseph Tector, MD, PhD, and Christopher Burlak, PhD

Department of Surgery, Indiana University School of Medicine, Indianapolis, IN

Xenotransplantation (transplantation of organs from one species to another) may offset the current shortage of human organs available for allotransplantation. Human antibody recognition of pig carbohydrates and proteins cause xenograft rejection when pig organs are transplanted into a human host. In order to have a xenograft compatible with a human recipient, genetic modifications are needed to prevent human antibodies from binding to the transplanted organ. The purpose of our research is to identify the targets of human antibodies that would bind to and cause rejection of a pig organ. To accomplish this we captured human antibodies from serum on protein L columns. We identified human protocadherin alpha C1 precursor, PCDH-alpha-C1 (PCDC1) as a target of human antibody on genetically modified pig cells. This confirms an earlier study that identified protocadherin C1 as a potential xenoantigen.

Effects of a suffocation stimulus (brief hypercapnia exposure) on cellular responses in the brain

Collin Nguyen1, Lauren Federici2,3, Stephanie Fitz3, Anantha Shekhar, MD, PhD3, Philip Johnson, PhD2,3

1Avon High School Avon, IN, 2Department of Anatomy and Cell Biology, and 3Department of

Psychiatry, Indiana University School of Medicine, Indianapolis, IN

Since dramatic changes in the concentration of CO2 in the blood (hypercapnia) can be fatal, this system is highly regulated. Initially small changes in blood CO2 lead to increases in respiration rate to “blow off” excess CO2, but more dramatic changes increase arousal and can provoke panic and lead to death. Sudden Infant Death Syndrome (SIDS) is a condition where the infant fails to arouse and reposition as CO2 levels increase, but panic disorder is associated with hyperactive panic responses to lower concentrations of CO2. Yet, little is known about the neural circuits that respond to an acute hypercapnic stimulus. In order to elucidate this circuitry, we exposed one group of adult rats to a 5 min 7.5% CO2 challenge and the control group to 5 min of atmospheric air, then assessed cellular responses in different brain regions ex vivo by immunostaining coronal brain sections for the protein product of the immediate early gene c-fos, which is a nuclear marker for cellular activity. Our initial results show that the hypercapnic stimulus is increasing brain activity in regions associated with arousal and panic responses (e.g. posterior hypothalamus). We are currently investigating other regions. These findings will provide insight into which brain regions respond to acute hypercapnia to regulate adaptive responses.

The effect of EMMPRIN on lung vascular endothelial growth factor expression

Phuong Nguyen1, Kelly S. Schweitzer2, Zorina Galis 2, 3, Irina Petrache, PhD2, 4

1Franklin Central High School, Indianapolis, IN, 2Department of Medicine, Indiana University School of

Medicine, 3Vascular Biology and Hypertension, National Institutes of Health, Bethesda MD, and 4Richard Roudebush VA Medical Center, Indianapolis, IN

Chronic obstructive pulmonary diseases (COPD) are the third leading cause of death in the US and occur mostly as a result of prolonged cigarette smoking. Emphysema is defined by the irreversible destruction of alveoli and the vascular network of the lung, associated with a loss of parenchyma vascular endothelial growth factor (VEGF), an essential factor that promotes blood vessel growths in the alveoli. Loss of VEGF signaling has been associated with emphysema due to apoptosis of structural lung cells. In addition, the support matrix of the lung becomes destroyed by matrix metalloproteinases, (MMP). However, MMPs are also needed for tissue repair and remodeling. The extracellular matrix metalloproteinase inducer, EMMPRIN, is known to upregulate both MMPs and VEGF. It has been reported that EMMPRIN is increased in lungs of COPD patients; however its role in lung pathology is unknown. We hypothesize that EMMPRIN has a protective effect by providing vasculoprotective and remodeling factors required for lung maintenance and repair. To test our hypothesis, we investigated wild type and EMMPRIN knockout mice that were exposed to cigarette smoke for 6 months. We performed ELISA to quantify VEGF in mouse lung homogenates, and bronchoalveolar lavage (BAL). Cigarette smoke exposure for 6 months significantly decreased lung elastance, suggestive of airspace destruction and emphysema development. Cigarette smoke-exposed mice which lacked EMMPRIN exhibited a significant worsening in lung elastance compared to those with intact EMMPRIN. Cigarette smoke increased VEGF in BAL fluid and total lung homogenates. In conclusion, EMMPRIN may have protective effects against emphysema development, since its loss caused worsening cigarette smoke-induced lung dysfunction. This effect may be associated with a decreased availability of VEGF in certain lung compartments.

Ovomucoid NMR spectral differences with deglycosylation

Biak C. Par1, Horia I. Petrache, PhD2, Bruce D. Ray, PhD2

1Southport High School, Indianapolis, IN, and 2Department of Physics, Indiana University – Purdue University

Indianapolis, Indianapolis, IN

Although protein glycosylation is the most common post-translational modification, its roles are not well understood. Alpha-1-anti-trypsin (A1AT) and ovalbumin, both glycosylated members of the serpin family of serine proteinase inhibitors, display transient ion currents in lipid ion channel measurements, and interfere with gramicidin ion channels. In contrast, a common Kazal family serine proteinase inhibitor, the quatra-glycosylated form of hen egg white ovomucoid, does neither of these. This difference is thought to be related to differences in structure. Previous measurements on quatra-glycosylated ovomucoid combined with theoretical modeling of the ovomucoid peptide chain indicated the positions and alignments of the four asparagines that are always glycosylated and the position and alignment of the partially glycosylated asparagine. In this research, NMR measurements on penta-glycosylated ovomucoid, and on chemically deglycosylated tetra-glycosylated and penta-glycosylated ovomucoid are compared to prior results in an attempt to assess glycosyl group orientations and possibly relate those to observations in ion channel measurements.

Pharmacological depletion of serotonin in the basolateral amygdala reduces social anxiety and fear conditioning

Rushil Patibandla1, Stephanie Fitz2, Philip Johnson, PhD2,3, Anantha Shekhar, MD, PhD2

1Hamilton Southeastern High School Fishers, IN, 2Department of Psychiatry, 3Department of

Anatomy and Cell Biology, Indiana University School of Medicine, Indianapolis, IN

The basolateral amygdala is implicated in a number of emotional responses including conditioned fear and social anxiety. Within the BLA, serotonin plays an important role in regulating these emotional responses. In the present studies, we hypothesized that pharmacologically depleting serotonin in the BLA using 5,7 DHT injections would lead to diminished anxiety and a reduction in fear conditioning. Methods – Disruption of serotonin synthesis was induced bilaterally by 5,7-dihydroxytryptamine (5,7-DHT), a serotonin depleting agent, through stereotaxic injections into the BLA. After a 5 day recovery period the animals were tested in social interaction (SI) and open field (OF) to assess anxiety like behavior. The animals then went through the 5 day experimentation of fear conditioning, Day 1 Habituation; Day 2 Conditioning; Day 3 Fear recall testing; Day 4 Extinction training; Day 5 Extinction recall test. Results – Rats with prior injections of 5,7 DHT into the BLA had less baseline social anxiety, as indicated by an increase in the duration of social interaction. These rats also had reduced freezing durations during the acquisition of fear (5 unconditioned shock stimulus + 5 conditioned tone stimulus) on day 2, and reduced freezing on day 4 where the rats were only exposed to the tone. This data provides additional support that serotonin in the BLA promotes anxiety and is critical for normal fear conditioning.

The role of Wnt signaling in zebrafish fin regeneration

Liliana Patino1, Ryan Lucas2, Swapnalee Sarmah3, Pooja Muralidharan3, Courtney Curtis3, and James A. Marrs, PhD3

1Warren Central High School, 2Arsenal Technical High School, and 3Department of Biology, Indiana

University-Purdue University Indianapolis (IUPUI) Indianapolis, IN

Bone growth can be impaired due to different factors, such as alcohol and disease, like osteoporosis. Wnt signaling pathways regulate bone growth. The parathyroid hormone (PTH) is therapeutic for anabolic bone growth (bone building). PTH activates Wnt signaling, which leads to bone growth in human patients. GSK3b (glycogen synthetase kinase 3 beta) protein inhibitors activate Wnt signaling in bone growth models. Our study utilized a zebrafish model system to study Wnt activated fin regeneration and bone growth. Wnt signaling is the first genetically identified step in fin regeneration, and bony rays are the main differentiated cell type in fins. Thus, zebrafish fin regeneration may be a useful model to study Wnt signaling mediated bone growth. To conduct the experiment, zebrafish embryos were treated with GSK3b inhibitor at various times. Embryos with a Wnt responsive promoter driving GFP (green fluorescent protein; TOP:GFP) were used as a Wnt responsive gene reporter. TOP:GFP embryos were then divided into three groups to undergo GSK3b inhibitor treatment. Two groups were treated with different concentrations of the drug, and the third group was the control (DMSO vehicle control). After treatment, embryos were imaged, and their fluorescence intensity was measured. Short-term treatment (2-6 hours) showed no change in Wnt responsive gene expression, but changes were detected in overnight treatments. Next, the caudal fins of embryos were cut to allow regeneration. In this case, Wnt responsive GFP expression occurred in short times (2-4 hours), indicating a transcriptional response. Finally, adult zebrafish caudal fin regeneration was tested. GSK3b inhibitor treatment increased fin regeneration at 4 and 7 days post-amputation. Together, these data indicate that bone growth in zebrafish fin regeneration is improved by activating Wnt signaling. Zebrafish Wnt signaling experiments are a good model for human bone growth and bone repair, and may provide an efficient drug discovery platform.

Role of Tet2 in lymphomagenesis

Sharmila Paul1, Ling Li2, Mingjiang Xu M.D. Ph.D.2, Feng-Chun Yang MD. Ph.D.2

1Carmel High School, Carmel, Indiana, and 2IU Herman B. Wells Center for Pediatric Research, Indianapolis,

Indiana

Current conventional treatments for lymphoma are chemotherapy, radiotherapy, and bone marrow transplantation. However, since these treatments lack target specificity, they are not completely successful and have adverse side effects. Therefore, scientists are trying to identify molecules that play a role in lymphoma development which could be used as targets for developing more efficacious treatments for lymphoma. The Tet2 gene, a tumor suppressor and epigenetic regulator, appears to be one such potential candidate. Although mutations/deletions of Tet2 gene has been mainly implicated in myeloid malignancies, a recent study (Quivoron et.al., Cancer Cell, 2011) has shown that a loss of Tet2 function can also lead to lymphoid disorders. The goal of this study is to study the role of Tet2 in lymphoma development/progression. Our labs generated Tet2 knockout (KO) mice, and we analyzed these mice to get an insight into the role of Tet2 in lymphomagenesis. Examination of the Tet2KO mice revealed that ~5% of these mice had higher lymphocyte counts in the blood than age and sex matched WT mice. Moreover, the spleen and thymus of these mice were enlarged, and the liver, spleen, and bone were pale. Furthermore, histological examination of these tissues showed a massive expansion/infiltration of lymphocytes. Flow cytometric analysis revealed that the increased lymphocyte population comprised mostly of T-lymphocytes, specifically Double Positive and Negative T-lymphocytes. Thus, the loss of Tet2 indeed leads to excess T cell expansion in mice. Current studies are underway to determine whether the abnormal T cell infiltration due to loss of Tet2 is a consequence of transformation of lymphocytes that could generate lymphoma. The pre-clinical model also provides us a platform to test novel therapeutics for Tet2-associated lymphoma.

Parathyroid hormone efficacy in mice lacking Wnt co-receptors LRP5 and LRP6

Pranoti Pradhan1, Lauren Deal2, Shana Bolt2, Raj Kedlaya, PhD2, and Alexander Robling, PhD3

1Hamilton Southeastern High School, Fishers, IN, and 2Department of Anatomy and Cell Biology, Indiana

University School of Medicine, Indianapolis, IN Parathyroid hormone (PTH) is a bone-active endocrine factor produced by the parathyroid glands, and serves to regulate calcium levels in the bloodstream. When calcium levels are reduced, PTH is released into the circulation where it causes osteoclasts to increase bone resorption. Osteoclastic resorption serves to liberate calcium ions from the bone matrix and restore serum calcium levels to an appropriate level. PTH also plays a role in bone formation, but it must be presented to the system intermittently for the anabolic (osteoblastic) effect. This observation has prompted several pharmaceutical companies to pursue intermittent PTH as an anabolic therapy for low bone mass disorders (e.g., Forteo® by Eli Lilly & Co.). While Forteo and other PTH fragments are clearly efficacious in improving bone mass, it is unclear how the compound exerts their effects on a cell-biological level. Recently published experiments have implicated the participation of the Wnt signaling pathway in the anabolic action of intermittent PTH. Wnt signaling is mediated extracellularly by the Wnt co-receptors Lrp5/Lrp6 and Frizzled. The objective of our study was to determine whether intermittent PTH can increase bone formation in the absence of the Wnt co-receptors Lrp5 and Lrp6. To delete Lrp5 and Lrp6, we used mice that were homozygous for loss-of-function floxed alleles for both Lrp6 and Lrp6 (Lrp5f/f and Lrp6f/f). These mice also carried a transgene for an inducible version of cre-recombinase, driven by the ubiquitously expressed Rosa promoter (Rosa-CreERt2). This transgene exercises its endonuclease activity (Lrp5 and Lrp6 deletion) only after induction by tamoxifen. 10-wk old male and female mice were treated with tamoxifen or vehicle (corn oil) to delete or maintain Lrp5/6 expression, and intermittent PTH treatment (60 ug/kg/dy; 7dy/wk; 4 wks) or vehicle treatment began 3 days later. DEXA and pQCT-based bone scans were collected at the beginning and end of the study to determine the change in global and regional bone mineral content (BMC) and bone mineral density (BMD) induced by intermittent PTH. Mice in which the Lrp5/6 receptors were recombined via Cre activation (tamoxifen injection) exhibited a negligible increase in BMD in response to PTH (from 11.8% increase in vehicle-treated to 12.8% increase in PTH-treated). Non-recombined mice exhibited the expected PTH-induced increase in BMD (from 3.0% increase in vehicle-treated to 9.3% increase in PTH-treated). Although our sample sizes are small, it appears that PTH might be less efficacious in Lrp5/6-deficient mice, suggesting that PTH anabolic action might require Wnt signaling for its full effects.

The correlation between cardiac and neurological deficits in infants with Down syndrome

Imani Rameses1, Nicole Shepherd2, Randall J Roper, PhD2

1Warren Central High School Indianapolis, IN, 2Department of Biology, Indiana University- Purdue University

Indianapolis, IN Down syndrome (DS) is one of the most common chromosomal disorders, occurring in approximately 1 out of 750 live births. Infants with DS experience several abnormalities with varying levels of severity and occurrence such as cardiac anomalies, cognitive defects, and craniofacial abnormalities. General cardiac deficiencies include ventricular septal abnormalities, and atrioventricular canal malformations. Cognitive defects include impaired memory and mental retardation. This study tested the hypothesis of a relationship between cardiac and neurological defects, therefore demonstrating a correlation among these phenotypes. Phenotype assessments of these particular abnormalities were taken from 174 infants with DS between birth and 6 months of age participating in the Down Syndrome Program at Riley Hospital for children from August 2005 to July 2010. Cardiac data was analyzed and categorized as severe or non-severe and compared with gross developmental assessments of the infants. APGAR scores were also compared with developmental quotients and differences in the period between the first and second visitations to the clinic. We hypothesized a correlation between cardiac defects and developmental quotients, as well as APGAR scores and developmental quotients. Our analyses revealed differences between severe and non-severe cardiac defects when compared with developmental quotients. Our research also showed a correlation between APGAR scores and developmental quotients. This data suggest a relationship between cardiac and neurological defects and the need for further research to identify genetic and phenotypic connections between these deficits in DS.

Characterization of small and non-small cells in lung cancer using systems biology approaches

Abinand Rejimon1, Akshay Desai2, Meeta Pradhan PhD2, Mathew Palakal, PhD2

1Carmel High School, Carmel, IN, and 2School of Informatics, Indiana University Purdue University Indianapolis, IN

Lung cancer is the highest cause of cancer-related death in the United States. Non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) are two categories of lung cancer. In this work, we used systems biology approaches to characterize NSCLC and SCLC by identifying the significant genes and their associated pathways. By using our in-house built text-mining tool, BioMAP, we extracted cells that might be correlated to NSCLC and SCLC. Through literature reading and with the use of KEGG database we collected genes and pathways related to these cells. We found that there were 456 common genes between the NSCLC and SCLC. Looking at the pathways, we found three that were common: p53 signaling, cell cycle, and apoptosis pathways. Using the degree centrality feature in network analysis; we found 22 significant genes which played regulatory roles in lung cancer pathways. Utilizing PubMed we validated these genes and identified 16 to play a role in cancer stem cells while RAC1, ATM, and P38 were specifically related to lung cancer stem cells. Also we discovered that the p53 signaling pathway, a pathway that is common to both NSCLC and SCLC, is associated with lung cancer stem cells. Characterization of cells helped us to understand the broader view of cellular machinery yet allowed us to contemplate details at the sub-cellular level. This understanding at the sub-cellular level will help to identify more specific therapeutic targets.

Mechanisms of double-strand break repair in yeast

Munajj Siddeeq1, Devyn Hayes2, Cynthia Sakofsky3, Soumini Vasan3, Yu-Hsiang Chen3, Anna Malkova, PhD3

1Lawrence Central High School, Indianapolis, IN, 2Charles A. Tindley Accelerated School, Indianapolis, IN, and

3Department of Biology, IUPUI, Indianapolis, IN Double-Strand DNA breaks (DSBs) are breaks affecting both strands of the double-helix of DNA. DSBs are one of the most severe forms of DNA damage. This type of damage is normally repaired by different mechanisms within the cell. However, if left unrepaired, DSBs can lead to cell death. Additionally, defective repair can cause genome instability. One important DSB repair pathway is Break-Induced Replication (BIR). BIR is a replication-based repair pathway, such that one of the broken DNA strands invades a homologous region of DNA and copies that region in order to repair the DNA break. Although BIR can be beneficial to the cell, this pathway can also be dangerous because it can lead to several types of genome instabilities, similar to those that promote cancer. Thus, determining the mechanisms of BIR and its regulations is critical for understanding how genomes become destabilized. Our research aims to identify the genes that drive BIR into pathways of genome instability. We used helicase (pif1Δ) and translesion synthesis DNA polymerase (rev3Δ) deletion mutants to determine how these mutations affected the frequency of BIR. We scored and calculated frequencies of various DSB repair outcomes, such as gene conversion, BIR, chromosome loss, and half-crossovers, in wild-type strains and mutant strains. Finally, we compared the frequencies between mutants and wild-type strains to determine how the specific DNA replication protein mutation affects BIR. Our results show that the mutants lowered the frequencies of BIR and raised the frequencies of chromosome losses and half-crossovers, which can be considered deleterious DSB repair outcomes. These data suggest that the absence of these DNA replication proteins (Pif1 and Rev3) is involved in driving BIR into pathways of genome instability.

Impact of age-related oxidant stress on mechanisms mediating collateral growth

Petr Sliva1, Mary J. Wenning2, Bruce Henry2, Randall G. Bills2, Joseph L. Unthank2, and Steven J. Miller, PhD2

1Lawrence Central High School, Indianapolis, IN, and 2Department of Surgery, Indiana University School of Medicine, Indianapolis, IN

A natural compensatory response to a blockage of a large artery due to injury or disease is the enlargement of pre-existing arteries, which bypass the site of the blockage and restore blood flow to the tissues that lack oxygen. This process, known as collateral growth, is stimulated by an increase in blood flow within the bypass arteries. However, collateral growth is inhibited by risk factors such as aging. Aging is linked with an increase in reactive oxygen species (ROS), and a major source of ROS is the enzyme NADPH oxidase (Nox). Nox-mediated overproduction of ROS may interfere with a great amount of processes involved in normal collateral growth including metabolic signaling pathways that ultimately regulate gene expression essential for collateral growth. The goal of this project is to analyze to what extent Nox-derived ROS impact the PI3-kinase/mTOR/NFκB signaling pathway and the ultimate regulation of transcription factors relevant to collateral growth. A combination of quantitative PCR, immunohistochemistry, biochemical assays, and animal models were used in conjunction with specific inhibitors of PI3-kinase, mTOR, and NFκB to determine the impact of these pathways on collateral growth. Current results show that inhibitors of the PI3-kinase/mTOR/NFκB signaling pathway can regulate the promotion or impairment of collateral growth in young and aged rats respectively. Enhanced knowledge of the role of the PI3-kinase/mTOR/NFκB signaling pathway on collateral growth will aid in developing targeted therapeutic molecular approaches to promote collateral growth in older patients.

Correlates of selecting BodPod assessment among urban fitness center members

Olivia Smith1, Kisha Virgil2, Sarah Shore-Beck3, NiCole Keith, PhD3

1Pike High School, Indianapolis, IN, 2IU School of Health and Rehabilitation Sciences, IUPUI, Indianapolis, IN, 3Department of Kinesiology, IUPUI School of Physical Education and Tourism Management, Indianapolis, IN

Introduction: Currently 34.0% of US adults are obese. Obesity can be monitored through body composition evaluation. Technological advances in body composition measurement have revealed more complete body composition information. An example is air displacement plethysmography (BodPod). Purpose: We evaluated BodPod usage in an inner-city Midwest fitness center, which was free of charge to members. This study evaluated health and fitness characteristics of members who used the BodPod (Users) compared to those who did not (Non-users) to determine if differences existed between each group. Methods: There were 468 Users and 179 Non-users, 46.2% self-reported being Non-Hispanic Black, 45.5% self-reported being Non-Hispanic White, and 8% self-reported being in the “other” race category. Each member received a free health assessment that measured their weight, height, systolic blood pressure (SBP), diastolic blood pressure (DBP), resting heart rate (RHR), body mass index (BMI) and performance on the Rikli and Jones Senior Fitness Test (SFT) that evaluated health-related fitness. Body composition was also measured. Analyses of this cross-sectional study included the use of ANOVA and T-tests. Results: Compared to Non-Users, Users were younger (45.22 + 15 vs. 48.30 + 15.4, p<.05), had lower RHR (73.15 + 10.8 vs. 76.76 + 13.2 p < 0.001), SBP (124.34 + 15.0 vs. 125.28 + 14.6, p=0.48), DBP (75.65 + 10.7 vs. 77.69 + 10.8, p < 0.05), and BMI (32.49 + 9.5 vs. 34.07 + 10.5, p=0.07). Users generally had higher SFT outcomes. All findings are included in the poster and report. Conclusion: Members with better health and fitness measures used the BodPod. Strategies to help less fit individuals understand the benefits of knowing their body composition should be identified.

Effects of ethanol exposure on epigenetic mechanisms in the Caenorhabditis elegans model

Melinda Song1, Yuanyuan Chen2,3, Feng C. Zhou, PhD2,3

1Carmel High School, Carmel, IN, and 2Department of Anatomy and Cell Biology, Indiana University School of

Medicine, Indianapolis, IN, and 3Stark Neuroscience Institute, Indiana University School of Medicine, Indianapolis, IN

Alcohol consumption has long been known for its adverse impact on the developing fetus, but only recently have its effects on epigenetic mechanisms come of interest. Previous studies have used mammalian models to analyze the mechanism of alcohol teratogenesis during early development. Instead, this study uses the nematode C. elegans model because of its completely mapped genome, rapid development with short life cycles, and easy manipulation. Epigenetics is the study of heritable changes in gene expression that are not accompanied by changes in the DNA sequence. Previous studies have identified the transcriptional activator H3K4me2 and transcriptional repression mechanism H3K9 methylation on different compartments of the chromosome. In combination with readers of these modifiers, they are thought to impact gene expression and various cellular functions. In this study, we are interested in understanding the histone modification patterns in and the role of ethanol exposure on C. elegans development. The N2 wild-type worms were exposed to ethanol for 2-7 generations, and larvae were harvested at each generation. Alcohol preference in the adults was measured using a conditioned cue assay, and the adult worms were then processed for immunohistochemistry with H3K4me2 and H3K9Me3 antibodies. We observed differential preference of ethanol after 7 generations of exposure. The expressions of these two histone markers were enriched in both neuronal and germ cells. Further investigation is needed to look into specific neuronal types, e.g. dopamine neurons, in correlation with these histone marks, and how ethanol exposure alters chromatin modification as well as cellular functions. These studies will help us to better understand the underlying molecular mechanisms in mammalian systems and in physiological processes leading to disease.

Modifying the synthesis of fluoxetine and duloxetine

Austin Stults1, Lucas Moore2, Robert E. Sammelson2, PhD

1Indiana Academy, Muncie, IN, and 2Department Chemistry, Ball State University, Muncie, IN

Selective serotonin re-uptake inhibitors (SSRIs) and serotonin-norepinephrine re-uptake inhibitors (SNRIs) are two classes of compounds that function as antidepressants by inhibiting the reuptake of serotonin and norepinephrine, neurotransmitters that are known to cause symptoms of depression in humans when present in low homeostatic levels. Fluoxetine, commonly known as Prozac, and duloxetine, with the brand name Cymbalta, are examples of SSRI and SNRI compounds, respectively. While many pathways exist to synthesize these compounds, some mechanisms require hazardous or expensive reagents that are not practical in a classroom setting. The goals of our project were to modify the published syntheses of these antidepressant compounds to improve efficiency during drug production, to develop a procedure that maintained safety and could easily be replicated in undergraduate laboratories, and also to understand the chemical processes used in drug development for future creation of antidepressant compounds. By adding solvents to the reactions at different times, doubling the amount of catalysts used in fluoxetine synthesis, purifying our products with column chromatography, and checking our reactions with NMR and IR spectroscopy, we were able to receive proportionately high yields throughout the reactions, while still maintaining a cost-effective and safe procedure that could be replicated by students. The knowledge gained from synthesizing existing SSRI and SNRI compounds can be used to create new compounds with antidepressant properties.

Stem-cell-enhanced nerve regeneration to the larynx

Amitha Sundaram1, Khadijeh Bijangi2, Caitlin Cavanaugh3, Stacey L. Halum, MD2

1Brebeuf Jesuit Preparatory School, Indianapolis, IN, 2Department of Otolaryngology, Indiana University School of Medicine, Indianapolis, IN, and 3Purdue University, West Lafayette, IN

Loss of vocal fold function due to neurological injury with incomplete recovery can result in dysphonia (disorders of the voice) and dysphagia (difficulty swallowing). Muscle stem cells (MSC) and placenta mesenchymal stem cells (PSC) can be readily incorporated into a tissue-engineered larynx, and, if programmed to facilitate reinnervation of the muscles within the engineered donor larynx, such a model may be a means of overcoming the existing hurdle of aberrant or inadequate reinnervation. The goal of this study was to determine if the spontaneous reinnervation that characteristically ensues after recurrent laryngeal nerve (RLN) injury could be selectively promoted and directed to the laryngeal abductor muscle with the use of stem cells, while antagonistic adductor reinnervation is inhibited with vincristine (VNC). A pilot study was performed involving five rats. Rats underwent left RLN transection injury, and at 3 weeks received injections of either MSC (n=1), PSC (n=1), MSC & PSC (n=2), or saline (n=1), into the left posterior cricoarytenoid (PCA) muscle; the animals simultaneously received left thyroarytenoid (TA) injections of VNC. One month after injection, reinnervation status based on laryngeal electromyography and the % motor endplates with nerve contact suggested enhanced early reinnervation of the MSC & PSC injected PCA, and absent reinnervation of the VNC-injected TA. These pilot data suggest that further investigations into stem cell-enhanced RLN regeneration approaches are warranted.

Pose model switching for robust face tracking

Naeem Tai1, S. Thomas Dunlap2, Gavriil Tsechpenakis, PhD2

1Ben Davis High School, Indianapolis, IN, 2Department of Computer Science, Purdue University School of

Science, Indianapolis, IN

Computer Vision is a rapidly growing field with the motivation of studying how computers process images and how to apply this knowledge to develop useful real-life applications. Face recognition from images and videos is emerging as an active research area with numerous possible commercial and law enforcement applications. These applications require robust algorithms for human face detection and recognition under varying, often non-optimal, situations. We present an approach for face detection and recognition under varying poses. Currently Active Shape Models are used for frontal or nearly frontal poses, but cannot detect/track faces under major off-plane rotation. A naive solution to this problem is to attempt to fit multiple models at different rotations/poses and pick the model with the best fit. However, we propose using statistical modeling methods for structured prediction (e.g. Hidden Markov Models and Conditional Random Fields) to provide more accurate, efficient, and robust model switching.

Amiodarone as an inhibitor of SK2 channel currents

Tianyu Tan1, Isik Turker2, and Tomohiko Ai, M.D., PhD2

1Carmel High School, Carmel, IN, and 2Krannert Institute of Cardiology, Indiana University School of Medicine, Indianapolis, IN

Heart failure (HF) is a major public health issue, affecting over 23 million people worldwide. A potential inducer of HF, cardiac arrhythmia can be caused by disruptions to the electrical conduction system of the heart. While antiarrhythmic agents used to treat such conditions exist, they are not always effective, and the mechanisms under which these agents perform are not fully understood. Cellular electric signals called action potentials are generated by ion channels, one of which is the SK2 channel, a member of the Small-conductance Calcium-activated Potassium channel family. An upregulation of the SK2 expression in failing ventricles coupled with the SK2 channel’s influence on the repolarization of cardiac action potentials suggests that the SK2 channel plays a role in cardiac arrhythmia. Therefore, the SK2 channel could be a potential drug target for treatment of HF, lending necessity to its research. Amiodarone, an effective drug in treating arrhythmia, is chosen to study the SK2 channel due to its known interference with many ion channels. Using whole-cell patch clamp techniques, potassium ion currents (IK) were analyzed in human embryonic kidney (HEK) cells transfected with the KCNN2 gene (coding for the expression of SK2 channel). In the presence of Amiodarone, the IK plot was significantly smaller compared to that in the absence of Amiodarone, which suggests that Amiodarone blocks SK2 currents. These results indicate the viability of SK2 channel as a therapeutic target. Further study on the interactions between Amiodarone and the SK2 channel and their relations to arrhythmia is underway.

Identifying perinatal risk factors for low birth weight using data mining

Ninad Thanawala1, Malika Mahoui, PhD2

1Fishers High School, Fishers, IN, and 2Polis Center, Indiana University—Purdue University, Indianapolis, IN

The occurrence of low birth weight poses known lifelong health issues, and health care providers require better models in order to design adequate surveillance procedures for populations units. Data mining provides a unique insight to the problem by applying a combination of statistical and machine learning methods; two types of models can be created: classification, which interprets risk nominally, and regression, which does so numerically. Such methods were employed in the study of all 212 census tracts of Marion County, Indiana, which each held 9 variables of birth risks as a percent (including low weight births). Raw data was aggregated from the Social Assets and Vulnerabilities Indicators database and processed using Waikato Environment for Knowledge Analysis and Statistical Package for the Social Sciences software. Methods such as Support Vector Machine, Multilayer Perceptron, and Stepwise Regression, among others, were employed. The resulting model for classification achieved a maximum accuracy of 0.781 (10-fold cross validation); the model for regression achieved a maximum coefficient of 0.721 (p < 0.001). The results support the high association of low weight birth with the mother’s alcohol use (p < 0.001), sexually transmitted disease (p < 0.001), and lack of prenatal care (p = 0.001). Such findings suggest that data at the census tract level contain can give a meaningful prediction model that concurs with widely accepted etiology, such as association of substance use and low birth weight. This study only examined the census tract level, but the same methods will allow more useful assessment and risk prediction for finer reporting levels.

Studies on new hydrazones as anti-cancer agents

Stephanie Tinsley1, Rachel Hawn2, Praveen Kusumanchi, PhD2, Hiremagalur N. Jayaram, PhD2

1Warren Central High School, Indianapolis, IN, 46229, 2Richard L. Roudebush Veterans Affair Medical Center,

Indianapolis, IN, 46202 In United States, cancer is a major health problem with an estimated 1,638,910 new cancer cases with 577,190 deaths in 2012. This disease is characterized by uncontrolled growth and spread of abnormal cells leading to death. Chemotherapy is a popular form of treatment for cancers, but the side effects are harsh and some treatments only last 12 to 16 weeks. Making chemotherapy more direct and effective would reduce harsh side effects. Ribonucleotide reductase (RR) is an enzyme involved in cell division, and when it is over-expressed in a cancer cell, its rate of cell division increases. However RR can be inhibited by purine nucleoside analogues when they bind to an active subunit of the enzyme. The compounds RPC038, RPC042, RPC046 are hydrazones, organic compounds that are believed to function similarly to purine nucleoside analogues, in the inhibition of RR. These hydrazones are found to have a low GI50 (the concentration needed to reduce the growth of treated cells to half that of untreated cells). So now that we know RPC038, RPC042, RPC046 are effective in killing a large number of cells with a very small concentration, the apoptosis pathway of RPC038 must be determined. This study aims to determine the pathway leading to apoptosis.

This pathway can be determined through Western Blotting, a method used to determine the concentration of protein in a sample.

SOST Deletion as a prophylaxis for disuse osteoporosis

Shreya Veera1, Lauren Deal2, Shana Bolt2, Raj Kedlaya, PhD2 ,and Alexander G. Robling, PhD3

1Carmel High School, Carmel, IN, and 2Department of Anatomy and Cell Biology, Indiana University School of

Medicine, Indianapolis, IN

Bone loss caused by reduced mechanical stimulation to the bone, known as disuse osteoporosis, is a common byproduct of numerous diseases and acquired disorders such as spinal cord injury, skeletal muscle paralysis, prolonged bedrest, and extended spaceflight (e.g. microgravity), among others. There is a growing need to identify new biological targets that can be manipulated pharmacologically to prevent disuse osteoporosis. The Wnt signaling pathway has been identified as one such pathway that might yield treatment solutions to disuse osteoporosis, due to its recently-elucidated role in bone growth and maintenance. Among the individual components of the Wnt pathway, the Sost gene has emerged as a mechanically sensitive gene that is also exclusively expressed by bone cells (osteocytes). Sost binds and inhibits the Wnt co-receptors Lrp5/6, and consequently, reduces bone mass. Previous studies indicate that enhanced bone loading leads to decreased levels of sclerostin, resulting in an increase of bone growth. Conversely, reduced bone loading results in an increase in Sost expression. We sought to determine whether preventing the normally-occurring increase in Sost expression that accompanies disuse, would mitigate the bone loss that normally occurs with disuse. To test this hypothesis, we subjected wild-type (Sost+/+) and Sost knockout (Sost-/-) mice to disuse of the hindlimbs via the tail suspension model. Sost knockout mice are incapable of upregulating Sost expression at the onset of disuse since the gene has been deleted. In the tail suspension model, the hindlimbs do not contact the cage bottom and therefore cannot generate ground reaction forces. 17-wk-old male Sost+/+ and Sost-/- mice were tail suspended or left unsuspended (ground controls) for 3 wks, and the change in hindlimb bone mass was measured radiographically via DEXA and pQCT. The wildtype (Sost+/+) mice subjected to tail suspension saw a greater decrease in bone mineral content than did the Sost knockout (Sost-/-) mice. The bone mineral content (BMC) in the ground control wildtype mice decreased while the BMC in the unsuspended knockout mice increased. The results show that through the deletion of the Sost gene, the bone loss resulting from disuse can be decreased.

Molecular mechanisms for the inhibition of metastatic behavior of breast cancer

cells by ganodermanontriol (GDNT)

Carmen Vernon1, Jagadish Loganathan2, Kayla Haffley3, Daniel Sliva2

1Ben Davis High School, Indianapolis, IN, 2Methodist Research Institute, Indianapolis, IN, and 3William Jewell

College, Liberty, MO

We have recently demonstrated anticancer effects of ganodermanontriol (GDNT), a biologically active

triterpene isolated from the Chinese medicinal mushroom Ganoderma lucidum on colon and breast cancer

cells. The gene expression DNA microarray analysis shows that GDNT down-regulates expression of genes

which are associated with metastatic characteristics of the highly invasive human breast cancer cells MDA-

MB-231. One of the genes down-regulated in MDA-MB-231 cells is coronin (CORO1A). When the breast

cancer cell line MDA-MB-231 is treated with GDNT, the invasive behavior (cell migration) of these cells is

markedly reduced. In addition, genetic silencing of CORO1A with siRNA also suppresses cell migration of

MDA-MB-231 cells. However, GDNT treatment does not affect protein expression of CORO1A in the whole

cell lysates. Since CORO1A is a membrane associated protein, additional studies are in progress to

determine if GDNT inhibits expression of CORO1A in the membrane fraction of MDA-MB-231 cells.

Computational modeling of neuron morphology in the Drosophila larval brain

Jonas Wang1, Xiao Chang2, Gavriil Tsechpenakis, PhD2

1Carmel High School, Carmel, IN, and 2Computer & Information Science Department, Indiana University School of Science, Indianapolis, IN

Even with recent advances in science and medicine, there is still relatively little information that has been concretely established about the brain and its function at single cell resolution. Our work aims at changing this as we take the first major step in determining how synaptic connectivity is established in vivo. The successful modeling of the C.elegans nervous system (302 neurons) motivates us to map at the single-cell level more complex brains such as the Drosophila, or fruit fly (estimated 100,000 neurons). In order to achieve this mapping, we use computer vision and machine learning methods to pattern neuron morphologies from single-neuron imagery. Using manual annotations of a large pool of motor neurons, we train our classifiers to automatically recognize the structure of the larval motor circuit. So far we have calculated twelve distinct morphologies among the 38 motor neurons in a single hemi-segment of the Drosophila ventral nerve cord.

Osteoblast migration is regulated by the tyrosine kinase Pyk2

Odette Zero1, Su Huang, PhD2, Pierre Eleniste, PhD2, and Angela Bruzzaniti, PhD2

1The International School of Indiana, Indianapolis, IN, and 2Department of Oral Biology, Indiana University School of Dentistry, Indianapolis, IN

Osteoporosis is a pathologic condition of bone caused by an imbalance between osteoclast bone resorption and osteoblast bone formation. It is characterized by low bone mass, deterioration of bone architecture, and increased fragility. Damaged or old bone is resorbed by osteoclasts and new bone is formed in the resulting cavity by osteoblasts. Osteoblast precursors migrate to sites of bone loss and form new bone. Osteoblast migration is controlled by a number of signaling proteins, many of which are still to be identified. Pyk2 is a proline-rich tyrosine kinase, which is important for osteoblast and osteoclast function. Genetically modified mice which lack Pyk2 (KO) were shown to be mildly osteopetrotic due to impairment in both osteoclast and osteoblast function. However, the role of Pyk2 on osteoblast migration is still unknown. The goal of this project is to identify the role of Pyk2 activity on osteoblast migration. The migration of osteoblasts from wild-type mice and Pyk2-KO mice will be examined by microscopy with photo imaging at the initial time (T=0 hours) and final time (T=12 hours). The role of Pyk2 kinase activity on osteoblast migration is being investigated. In addition, Pyk2 and kinase-defective mutants of Pyk2 (Y402F, K295M) will be expressed in Pyk2-KO osteoblasts and their role in osteoblast migration will be measured. Further knowledge in the role of Pyk2 in osteoblasts will lead to better understanding of the process of cellular migration. This may lead to the identification of novel proteins that can be targeted for therapeutic approaches to treat bone diseases such as osteoporosis.

Quantification of microglia cells in the hippocampus following TBI

Baizhen Zhu1, Matt Peters2, Xiang Gao, PhD3, Lin Xu3, Jinhui Chen, MD, PhD3

1Zionsville High School, Zionsville, IN, 2University of Notre Dame, Notre Dame, IN, and 3Stark Neuroscience Research Institute, Indiana University School of Medicine, Indianapolis, IN

mTBI (moderate traumatic brain injury) is an injury that affects a wide spectrum of people in various different fields from sporting activities to the military. Annually, it is estimated that 1.7 million people sustain traumatic brain injuries in the United States, and is responsible for one third of injury-related deaths. Currently, there are no effective treatments for mTBI. One of the areas in the brain affected by TBI is the hippocampus, which is responsible for memory and spatial navigation. We know that there is an impact on neurons in the hippocampus, but a type of non-neuronal cell that is activated following injury is the microglia cell. Microglia cells are a type of glia cell that act as an immune system within the brain, constantly scavenging for infections and dead cell debris. Following mTBI, the microglia cells are activated and respond to the damage. However, the response of the cells is still poorly understood; therefore, the effects of the cells can be quantified and used to modulate cell response to mTBI and lead to a better understanding of the effects of microglia cells in order to maximize the beneficial aspects while reducing the detrimental effects. We can assess microglial response by immunostaining a section of a mouse’s brain and use specific markers to quantify the result at various time-points. From this we can determine the time of highest microglia activation in order to determine the nature of the activity as well as the time point best suited for treatment.

Cholesterol binding to phospholipids

Van D. Zi1, Justin Williams2, Bruce D. Ray, PhD2, Stephen R. Wassal, PhD3

1Southport High School, Indianapolis, IN, and 2Department of Physics, Indiana University Purdue University Indianapolis, Indianapolis, IN

Cholesterol-lipid interactions are thought to play an intrinsic role in determining lateral organization within cellular membranes. Rafts enriched in cholesterol are held together by the high affinity that the sterol has for sphingolipids, whereas poor affinity for cholesterol is thought to drive the formation of polyunsaturated

fatty acid (PUFA)-containing phospholipid domains depleted in the sterol. In contrast to cholesterol -Tocopherol (vitamin E), a lipid-soluble antioxidant found in low concentration in plasma membranes, is proposed to have preferential affinity for PUFA-containing phospholipids in non-raft regions. Accurate quantitation of these affinities has been difficult to achieve. Prior methods to measure the affinity of cholesterol for model membranes have been criticized for potential interferences from probe size or potential for cholesterol depletion from filtration to separate vesicles from cyclodextrin carrier complex. As a probe, deuterium is not significantly larger than hydrogen. In addition, 2H NMR does not require separation of the lipid vesicles from the cyclodextrin cholesterol carrier. We compare preliminary results on 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) to prior attempts at cholesterol affinity quantitation. Although the change in chemical shift is minimal, linewidth for methyl deuterated cholesterol in POPC

vesicles is greater than in cyclodextrin complex. Ideally, a stable-isotope labeled -tocopherol would also be

used for NMR quantitation of -tocopherol in lipid vesicles versus in the cyclodextrin--tocopherol complex.

In the absence of such a label, spectrophotometric quantitation of -tocopherol in both components of a

centrifugal filter separation of cyclodextrin--tocopherol from lipid vesicles was investigated.

Inaugural class of 2012 SEED interns at Purdue University

Aneli Carrillo

Jennifer Pham

Kimberly Rios

Tania Sotelo

Thank you to our 2012 SEED preceptors at Purdue University

Suzanne C. Bart, PhD Stephen P. Beaudoin, PhD

Yue Wu, PhD Yu Xia, PhD

Development of a centrifuge-based adhesion force apparatus

Aneli Carrillo,1 Myles Thomas,2 and Stephen P. Beaudoin, PhD2

1Jefferson High School, Lafayette, IN 47905 and 2School of Chemical Engineering, Purdue University, West

Lafayette, IN 47907

When conducting experiments that require particles smaller than 50 µm, analyzing the particles can become very difficult for a variety of reasons. For instance, the smaller particles cluster closely together and become difficult to separate both physically and visually. When analyzing the images with high-throughput computational software, the clusters are often labeled as one large particle instead identifying the hundreds of smaller particles that make up that cluster. This can cause major problems during data interpretation, and in understanding fully the physics of important commercial processes (e.g., powder packing in active pharmaceutical ingredients) When using image analysis software, we have been able to write different computer scripts that analyze the images using different techniques. This provides a crucial research tool that results in particle size and distribution lists that are more accurate than the current state of the art.

Lanthanide complexes containing redox-active ligands for small molecule activation

Jennifer Pham,1 Nickolas Anderson,2 and Suzanne C. Bart, PhD2

1Twin Lakes High School, Monticello, IN, 47960 and 2Department of Chemistry, Purdue University, West Lafayette, IN 47097

The main focus of our work is to create ligands for different kinds of lanthanide-containing compounds. Ligands are organic molecules that donate the necessary electrons to form coordinate covalent bonds with metallic ions. Specifically, we are working with pyridine(diimine) ligands, which can give or take electrons as needed to the metal center. These ligands are studied with the rare earth elements cerium and lanthanum. By adding the pyridine(diimine) ligand to CeCl3 or LaCl3(dme) (dme = 1,2-dimethoxyethane), our aim is to make new coordination complexes. This is performed by stirring the ligand and metal salt in a good solvent for approximately 30 minutes, followed by addition of a poor solvent to initiate precipitation. In both metal-center cases, changes in color from a cloudy yellow solution to a clear orange solution were observed. Both compounds were characterized by 1H NMR spectroscopy. By performing this unique chemistry, we are able to explore new materials for a host of emerging chemical synthesis applications.

Evaluation of thermoelectric performance of PbTe nanocrystals

Kimberly Rios,1 Scott Finefrock,2 Anita Bhat,2 and Yue Wu, PhD2

1Twin Lakes High School, Monticello, IN 47960 and 2School of Chemical Engineering, Purdue University, West Lafayette, IN 47907

This research explored the thermoelectric properties of lead telluride (PbTe) nanocrystal films with the specific objective being to examine the effects of different annealing procedures on the thermoelectric response of PbTe thin films. In particular, PbTe nanocrystals were dip-coated on a glass sheet to form a thin film. The time, temperature, and atmospheric environment in which the films were annealed were then altered systematically. The atmospheres in which the annealing took place included forming gas (i.e., a mixture of hydrogen and nitrogen gases), nitrogen, and ambient conditions. We anticipate the annealing procedures to affect the Seebeck coefficient and the electrical conductivity, which will be measured using standard current-voltage-temperature techniques. Previously, dip-coated PbTe thin films have not been investigated in terms of their thermoelectric performance; furthermore, no studies have addressed annealing conditions in these materials for enhanced energy conversion. As such, our further evaluation of PbTe nanocrystals will expand the knowledge of thermoelectric materials and processing conditions which could lead to high thermoelectric performance and commercial implementation of these materials.

Identification of the stereochemistry and anomeric configuration for four unknown disaccharide samples by tandem mass spectrometry

Tania Sotelo,1 Chiharu Konda,2 and Yu Xia, PhD2

1Frankfort Senior High School, Frankfort, IN 46041 and 2Department of Chemistry, Purdue University, West

Lafayette, IN 47097

Collision-induced dissociation (CID) of deprotonated hexeose-containing disaccharides (m/z 341) with 1-2, 1-3, 1-4, and 1-6 linkages yields product ions at a mass-to-z- ratio (m/z) of 221, which have been identified as glycosyl-glycolaldehyde anions. From disaccharide with these linkages, CID of m/z 221 ions produces distinct fragmentation patterns that enable the stereochemistries and anomeric configurations of the non-reducing sugar units to be determined. Four unknown disaccharides samples were tested using mass spectrometry (QTRAP 4000) with the ElectroSpray Ionization method; the unknown components were analyzed and data was collected. Based on this methodology, CID of m/z 221 ions form four unknown disaccharide samples were collected. Stereochemistry and anomeric configuration for four unknown samples were identified, using a computer program developed to calculate the spectra similarity scan between unknowns and our standard data base. In this way, we have made a significant step in utilizing advanced mass spectroscopy techniques in identifying some of the most important biological molecules known to mankind.

Guest Presenters

Indiana University Bloomington

Taylor Hurst

Indiana University-Purdue University Indianapolis

Michael Blatchley

Steven X. Chen

The role of working memory in phonological abilities

Taylor Hurst, Roy Seo, Sharlene Newman, PhD

Indiana University, Bloomington, IN

Working memory works to hold, process, and retrieve information to help perform nonverbal and verbal tasks. These tasks are key helpers in learning and attentiveness. For example, working memory has been shown to help in learning to read. Phonology, the study of sounds and speech, is an essential part of working memory. In this study we explored how the phonological processing ability of children between the ages of 5 and 12 impacts working memory capacity. In this study, subjects (n-45) were asked to perform a series of tasks that looked at their phonological ability. The results showed that both forward and backward digit span was positively correlated with word attack, a phonological processing task where the subject sounded out pseudowords. Also functional MRI (fMRI) scans were taken as the subjects performed a phonological

processing task, rhyming, we will explore how working memory capacity relates to that activation.

Evidence of a paracrine crosstalk between the bone marrow and the lung

Michael Blatchley1, Kelly Schweitzer, PhD2, Irina Petrache, MD2

1Purdue University, West Lafayette, IN, and 2Department of Medicine, Indiana University School of Medicine, Indianapolis, IN

Emphysema is the third leading cause of death worldwide with the primary causative factor being cigarette smoking. Endothelial cell injury and apoptosis plays a central role in emphysema development. We have found that when mice are exposed to cigarette smoke (CS) the total number of bone marrow progenitor cells is reduced. It is increasingly evident that when used therapeutically, the majority of beneficial effects of progenitor cells are attributed to their paracrine activities. We hypothesized that lung structural cells will be protected by exposure to factors secreted by healthy bone marrow cells, but not by bone marrow cells isolated from animals exposed to CS. We isolated bone marrow and by centrifugation collected secreted factors named herein bone marrow conditioned media (BMCM) from adult mice that had been exposed to ambient air (BMCM-A) or to cigarette smoke (BMCM-CS). Cultured primary mouse lung endothelial cells were exposed to cigarette smoke extract (CSE; 4%) and treated with control media, BMCM-A, or BMCM-CS. Western blots were used to probe for LC3B and phosphorylated p38 MAPK and AKT, as markers of endothelial cell autophagy and activation of signal transduction, respectively. BMCM-A and BMCM-CS treatments markedly reduced CSE-induced lung endothelial cell autophagy, but did not affect p38 or AKT activation. Interestingly, in a complementary assay of endothelial monolayer injury measured by electrical cell impedance sensing (ECIS), both BMCM-A and BMCM-CS strengthened the lung endothelial monolayer barrier function in response to CSE. In conclusion, our data show a novel crosstalk between the bone marrow and the lung, based on secretion of bone marrow-derived paracrine factors that exert protective effects on primary lung endothelial cells. Understanding such crosstalk may help in designing optimal future therapeutic interventions for emphysema.

Nicotine alone induces lung endothelial barrier disruption by distinct mechanisms compared to soluble nicotine-containing cigarette smoke extracts

Steven X. Chen, Kelly S. Schweitzer PhD, Irina Petrache MD

Department of Medicine, Indiana University School of Medicine, Indianapolis, IN

Nicotine may play a role in the damaging effects caused by cigarette smoke (CS) on the lung structural cells. Our lab has shown that CS decreases both proliferation and the barrier function of lung endothelial cells. In addition, CS induces endothelial cell apoptosis through the dysregulation of ceramide biosynthesis, leading to ceramide accumulation. We hypothesized that nicotine may be sufficient to dose-dependently induce oxidative stress and endothelial cell MAPK signaling activation. Barrier function was measured using the electric cell substrate impedance system (ECIS) to measure relative changes in trans-endothelial cell monolayer resistance for 20 hours. Nicotine exposure caused a dose-dependent loss of endothelial barrier function, with increases in monolayer permeability noted as early as 3h, for concentrations of 10-15 mM. These changes are paralleled by an increase in ceramide accumulation and cell death at very high concentrations (20 mM). Inhibition of oxidative stress with the antioxidant N-acetyl cysteine (500µM) and inhibition of MAPK signaling through p38, unlike their effects on CS-induced permeability, failed to attenuate the nicotine-induced loss of barrier function. Inhibition of ceramide synthase (Fumonisin B1) or ceramidase (MAPP) did not attenuate nicotine-induced permeability. In conclusion, despite similar effects on cellular outcomes such as barrier dysfunction, apoptosis, and inhibition of cell proliferation, nicotine triggers distinct pathways to cause barrier dysfunction when administered alone, compared to when part of a mixture of other chemicals contained in the complex cigarette smoke extract. Future studies will characterize unique signaling pathways employed by nicotine to cause endothelial cell activation. With the emergence of electronic cigarettes, which contain nicotine vapors, it will be important to characterize and combat the potential detrimental effects of this increasingly utilized nicotine replacement approach.