The National Institutes of Health Short Term Training for Minority College Students Grant #5 T35 HL07807 NHLBI Basic and Translational Program Grant #1 U54 HL090511/CFDA #93.839

American Pediatric SocietyAmerican Heart AssociationElizabeth Nash FoundationWilliam Jenkins Fund

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2008 Summer StudentResearch Symposium

August 15, 2008

The program we have organized for students this summer celebrates our 15th year of providing research opportunities for high school, college, and medical school students. This year’s class was outstanding! Students participated in clinical (18) and laboratory (29) research settings throughout our medical center. The personal statements and research abstracts written by these students demonstrate the depth and value of our program and the commitment CHORI has to the education of young people who are considering careers in biomedical science or other health related fields. Students in this summer’s program came from a broad range of backgrounds and experiences and all received exciting opportunities to work with devoted clinical and laboratory based scientists. Students were provided opportunities and choices to participate in unique research projects, coordinated with mentors willing to mentor and counsel students. The goals were to have the students learn, enjoy the experience, and develop self-confidence in a biomedical research field.

We are grateful for the funding we received for the summer program from the NHLBI, as part of a Short Term Training to Increase Diversity in Research program, as well as from the American Heart Association, American Pediatric Society, Elizabeth Nash Foundation, and Williams Jenkins, M.D. We are currently submitting our competitive renewal application to the NIH and hope to obtain support from NIH so that we can continue to provide this unique scientific educational experience. We are actively seeking philanthropic support in the event the NIH grant is not awarded and to provide additional opportunities for students not covered by the NIH. In this regard, we would appreciate any support that family members of students could provide to help us continue this work.

I would like to thank the mentors who dedicated time and effort to train the students and to share the excitement and pleasure one gains by working in basic, translational, or clinical research. I would also like to thank those who participated in the seminar series and the Chief Residents in the hospital who provided students an opportunity to participate in a clinical setting involving patients. I am particularly grateful to Dr. Barbara Staggers who provided oversight to the students involved in clinical research, to Dr. Vasanthy Narayanaswami who provided similar support to students involved in laboratory research and served as my Co-Principal Investigator, to Diane Kohlman, who provided structure, administrative assistance to the program and emotional support to students, and to Phillip Bollinger, who provided computer technologic assistance and helped students prepare the sophisticated poster and oral presentations demonstrated at this symposium.

Again, congratulations and thanks to all members of the CHORI family for making the Summer Student Research Internship program a success in 2008.

Bertram H. Lubin, MDPresident, CHORIDirector of Medical ResearchChildren’s Hospital & Research Center at Oakland

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Co-Director:Vasanthy Narayanaswami, Ph.D.

Assistant Scientist at CHORI

Co-Director:Barbara Staggers, M.D.

Director, Adolescent Medicine at CHRCO

Director:Bertram H. Lubin, M.D.

President & Director of Medical Research at CHORI

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Janelle Noble, Ph.D.Michael Oda, Ph.D.Jennifer Olson, M.D.Babak Oskouian, Ph.D.James Policy, M.D.Robert Ryan, Ph.D.Julie Saba, M.D., Ph.D.Swapna Shenvi, Ph.D. Mark Shigenaga, Ph.D.Patty Siri, Ph.D.Stuart Smith, Ph.D., D.Sc.Po-Lin So, Ph.D.Barbara Staggers, M.D. Shiori Tamamizu-Kato, Ph.D.Jean Tang, M.D., Ph.D. June Tester, M.D.Lydia Tinajero-Deck, M.D.Elliott Vichinsky, M.D.Patsy Wakimoto, Ph.D.Gordon Watson, Ph.D.Jo Anne Welsch, Ph.D.Kasuen WongTony Xiao, Ph.D.Taichi Yamamoto, Ph.D.

Bruce Ames, Ph.D.Peter Beernink, Ph.D.James Betts, M.D.Cassandra Calloway, Ph.D.Sally Chiu, Ph.D.Ervin Epstein, M.D.Dejiang Feng, Ph.D.Horst Fischer, Ph.D.Ellen Fung, M.D.Henrik Fyrst, Ph.D.Dan Granoff, M.D.Paul Harmatz, M.D.Beate Illek, Ph.D.Lourdes Juarez, P.N.P.Hardeep Kaur, Ph.D.Janet King, Ph.D.Ronald Krauss, M.D. Edward Lammer, M.D.Bertram H. Lubin, M.D.Steve Mack, Ph.D.Gregory Moe, Ph.D.Rose Ellen Morrell, M.D.Vasanthy Narayanaswami, Ph.D.Lynne Neumayr, M.D.

MENTORS (Children’s Hospital & Research Center at Oakland)

SELECTION COMMITTEE

Bertram Lubin, M.D.Barbara Staggers, M.D.Vasanthy Narayanaswami, Ph.D.Caroline Kane, Ph.D.

Frans Kuypers, Ph.D.Tomas Magana, M.D.John Matsui, Ph.D.Laurie Schumacher, M.D.

PROGRAM ADMINISTRATION

Phillip BollingerDiane KohlmanAshley Blia Yang

Bertram Lubin, M.D.Barbara Staggers, M.D.Vasanthy Narayanaswami, Ph.D.

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2008 CHORI SUMMER RESEARCH PROGRAM CURRICULUM

Orientation, June 12, 2008There will be an all-day orientation for summer interns on Thursday, June 12, 2008, from 10:00 am until 4:00 pm. Lunch will be served. Agenda to include: Introduction and overview by Director and Co-Directors Keynote lecture Explanation of curriculum CHORI tour and badges

Safety Training, June 13, 2008The mandatory Safety Training with CHORI Safety Officer, Miriam Fang will be held on Friday, June 13 from 9:30 – 12:00. The students will be required to complete this training BEFOREbeginning their project.

Research Project: June 1� to Aug 1�, 2008The students will conduct research with assigned mentor. The details of project and research plan are left entirely to the individual mentor and each summer intern will follow the procedures, and schedule laid out by their respective labs.

Submit Written Research Plan: July 3, 2008Students must submit to the Summer Program Coordinator a written Research Plan outlining their project. The Research Plan should be 3-4 pages long and include:

(a) Statement of hypothesis (b) Specific aims (c) Background (d) Methods (e) Anticipated outcome of project

Students will work closely with their mentor in the preparation of these reports, and mentors should review and approve the reports before submission. Figures, flow charts and schematics may be used to illustrate the research plan. The written report will be sent to: summerstudent-program@chori.org, and must include student’s name, mentor’s name and the title of the project.

Presentations at weekly meetingsMeetings will be held every week, at which students are required to present their research plan in an informal setting to the Program Director, Co-Directors and peers. This is designed to aid them in discussing their projects with their peer group, and to help them to obtain an overall perspective of their progress. Each presentation should last approximately 10 minutes.

Weekly Lectures: Current Topics in Health and DiseaseStudents are required to attend weekly lectures delivered by CHORI and CHRCO faculty members. The lectures will cover various scientific topics, including women’s and minorities issues, career decisions, teen health issues and Responsible Conduct of Research.

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2008 CHORI Summer Student Symposium, August 1�, 2008A one-day symposium will be held on Aug 15, 2008 where all students are required to participate. The students will submit an abstract concisely summarizing their work, which will be considered for an oral or a poster session for the Symposium. Abstracts are due on August 1, 2008. A committee comprised of the Director, Co-Directors and other leading members of the CHORI scientific community will review the abstracts for the Symposium. Students will work closely with their mentors in the preparation of abstracts and presentations. Family members, teachers, lab members and friends are welcome to attend. Details of the Symposium and the scientific sessions will be available by Aug 4, 2008.

The Symposium will comprise of about 12 oral presentations (10 min each, with 5 min discussion), and a poster session during which the presenters will be on-hand to explain their research project. An abstract book, which will include the Symposium program, personal statements, and the research project abstracts, will be presented to each student. A catered breakfast and lunch will be provided for all attendees on the Symposium day.

A certificate of participation in the CHORI Summer Student Research Program will be awarded to those who successfully complete the program.

Summary of important dates:

June 12, 2008 Orientation: 10:00 A.M. – 4:00 P.M.June 13, 2008 Safety Training: 9:30 A.M. – 12:00 P.M.July 3, 2008 Written Research Plan due July 11, 2008 Personal Statement for Program Guide due by 5:00 P.M.August 1, 2008 Abstract for Program Guide due by 5:00 P.M.August 15, 2008 Summer Student Research Symposium

Please address any additional questions and concerns to: summerstudentprogram@chori.org

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2008 SCHEDULE OF WEEKLY MEETINGS:RESEARCH ETHICS AND PROJECT DISCUSSIONS

The lectures will be held in the Little Theater/Board Room at CHORI at 12:00 noonLunch will be provided

Wednesday, June 2� Carolyn KaneEthics in Research and Responsible Conduct of Research

Wednesday, July 2 John MatsuiEthics in Research and Responsible Conduct of Research

Wednesday, July �

Phillip BollingerCHORI IT Tips and TricksSummer Student Research Project Discussions

Wednesday, July 1�

Summer Student Research Project Discussions

Wednesday, July 23

Summer Student Research Project Discussions

Wednesday, July 30

Summer Student Research Project Discussions

Wednesday, August �

Carolyn KaneEthics in Research and Responsible Conduct of Research

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SYMPOSIUM SCHEDULE

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2008 Summer Student Research SymposiumFriday, August 1�, 2008

CHORI Little Theater

8:30 -- 9:00 Check-in and Continental Breakfast 9:00 -- 9:15 Introduction by Program Directors Bertram H. Lubin, M.D. - Director Barbara Staggers, M.D. - Co-Director Vasanthy Narayanaswami, Ph.D. - Co-Director

Oral Presentations

Session 1

Chairs: Jean Tang, M.D., Ph.D., Postdoctoral Fellow, Children’s Hospital Oakland Research Institute/ Assistant Professor, UCSF Stephanie Orth, Chapman University

9:15 -- 9:30 Bryan Arias, University of California Berkeley 22 Mentor: Dan Granoff, M.D., Peter Beernink, Ph.D., Jo Anne Welsch, Ph.D. Genetic and Antigenic Characterization of Epidemic Menigococcal Strains from Africa

9:30 -- 9:45 Tiffany Crouch, Grambling State University �2 Mentor: Jennifer Olson, M.D. Testing Outcomes for Congenital Adrenal Hyperplasia (CAH) at Children’s Hospital and Research Center Oakland in 2007

9:45 -- 10:00 Viviana Ruiz Barros, University of California 8� Berkeley Mentor: Janet King, Ph.D. Maternal Visceral Fat Thickness and Insulin Resistance in Obese Pregnant Women

10:00 -- 10:20 Mohammad Hajighasemi, St. Mary’s College �8 Regulation of Airway Proton Secretion John Cossette, Carleton College Living with Cystic Fibrosis Mentor: Horst Fischer, Ph.D., Beate Illek, Ph.D.

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10:20 -- 10:35 Deja Kono, Notre Dame de Namur �� Mentor: Ellen Fung, M.D. Dietary Intake Assessment in Patients with Thalassemia and Sickle Cell Disease

10:35 -- 10:50 Aemad Komarizadeh, University of California �� Berkeley Mentor: Barbara Staggers, M.D. Adolescent Resiliency

10:�0 -- 11:00 Break

Session 2

Chairs: Saloni Pasta, Ph.D., Postdoctoral Fellow, Children’s Hospital Oakland Research Institute Shantelle Nolen, University of California Berkeley

11:00 -- 11:15 Dana Collins, Spelman College 3� Mentor: Ronald Krauss, M.D., Patty Siri, Ph.D., Patsy Wakimoto, Ph.D. Self-reported Weight Gain is Significantly Associated with Plasma Triglycerides in African Americans

11:15 -- 11:30 Uchechukwu Eke, University of California �0 Berkeley Mentor: Steve Mack, Ph.D. Genetic Relations Between Siberian and Native American Populations Investigated by HLA Genotyping

11:30 -- 11:45 Joyce Nguyen, Presentation High School �8 Mentor: Lydia Tinajero-Deck, M.D. Perception of Acanthosis Nigricans & Reliability Amongst Medical Providers

11:45 -- 12:00 Ashley Moon, Johns Hopkins University �� Mentor: Ervin Epstein, M.D., Jean Tang, Ph.D. Tony Xiao, Ph.D. Exploring the Mechanism of Basal Cell Carcinoma Inhibition by Vitamin D3 and Statins

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12:00 -- 12:15 Andrew Lee, Duke University �0 Mentor: Julie Saba, M.D., Ph.D. Sphingadiene Effect on Akt Signaling in SW480 Colon Cancer Cells

12:15 -- 12:30 Tiffany Kim, University of Illinois at Chicago �2 Mentor: Paul Harmatz, M.D. The Role of Functional Assessments and Therapies for Mucopolysaccharidosis Patients with Carpal Tunnel Syndrome

12:30 -- 1:15 Lunch in the Senior Center Courtyards

1:15 -- 3:15 Poster Session in the Senior Center Courtyard

#1 Olayemi Akintunde, University of California 20 Berkeley Mentor: Vasanthy Narayanaswamio, Ph.D., Shiori Tamamizu-Kato, Ph.D. Effects of Environmental Tobacco Smoke on Apolipoproteins

#2 Nelson Bean, Claremont-McKenna College 2� Mentor: Lourdes Juarez, PNP Water Study

#3 Mayra Betancourt, University of California 2� Berkeley Mentor: Patsy Wakimoto, Ph.D. The Role of Religion or Spirituality and Other Factors Influencing Diabetes Management in Latinos

#4 Sophia Blachman-Biatch, College Preparatory 28 High School Mentor: Bruce Ames, PhD., Swapna Shenvi, Ph.D. Protective Effects of Estrogen are Mediated in Part Through the Transcription Factor Nrf2 in Human Hepatoma Cells #5 Benjamin Cedars, Bowdoin College 30 Mentor: Michael Oda, Ph.D. Bulk Flow Facilitates Nanodisk Translocation Across Calu-3 and A549 Lung Epithelia

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#6 Alex Chetkowski, Piedmont High School 32 Mentor: Stuart Smith, Ph.D., D.Sc., Dejiang Feng, Ph.D. Screening of the Effective Mouse Mitochondrial ACP shRNA Targeting Sequence #7 Stacie Collins, Howard University 3� Mentor: Gregory Moe, Ph.D., Hardeep Kaur, Ph.D. Does the Monoclonal Antibody SEAM 2 React With HSP60 or a Modified Form of HSP60?

#8 John Cossette, Carleton College 38 Mentor: Horst Fischer, Ph.D., Beate Illek, Ph.D. Stimulation of CFTR-mediated Cl- Transport by Vitamin C and SP600125 JNK-inhibitor

#9 Christopher Cox, Sacramento State University �0 Mentor: Julie Saba, M.D., Ph.D. Leveraging Sphingolipids in the Treatment of Leukemia

#10 Luiza Da Hora, Dominican University �� Mentor: Lourdes Juarez, PNP, June Tester, M.D. Street Vendors and Food Environment Influence on Kid’s After School Snacking #11 ` Leslie Dinh, University of California Berkeley �� Mentor: Lynne Neumayr, M.D. Restrictive and Obstructive Lung Disease in Pediatric Patients with Sickle Cell Anemia

#12 Caroline Dougherty, Miramonte High School �8 Mentor: Gordon Watson, Ph.D. Effect of Smith-Lemli-Opitz Syndrome on Erythrocytes #13 Edward Escamilla, San Francisco State �2 University Mentor: June Tester, M.D. After School Food Environment and Purchasing Patterns

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#14 Derek Fukumori, Berkeley High School �� Mentor: Mark Shigenaga, Ph.D. Modulation of Gut Bacteria on Apolipoprotein Expression and Plasma Lipoprotein Subclass Profiles in a Murine Model of Diet Induced Obesity #15 Kristen Glass, Bentley High School �� Mentor: Ellen Fung, M.D. Correlating Trace Element Status with Low Bone Mass in Patients with Thalassemia and Sickle Cell Disease

#16 Danielle Jackson, University of New Mexico �0 Mentor: Patsy Wakimoto, Ph.D. Sociocultural Factors Contributing to Native American Women’s Health in Northern California Greater Bay Area

#17 Adaira Landry, University of California �8 Los Angeles Mentor: Ervin Epstein, M.D., Po-Lin So, Ph.D. Tazarotene’s Chemoprevention Efficacy on Preclinical Models of Medulloblastoma and Basal Cell Carcinoma

#18 Sean Lindstedt, University of Nevada �2 Mentor: Lynne Neumayr, M.D. Prolonged QTc in Chronically Transfused Sickle Cell Disease (txSCD) Patients Compared to that of Non-iron Overloaded SCD Patients #19 Jennifer McLeod, George Washington University �� Mentor: Janelle Noble, Ph.D. Genetic Associations Between Type 1 Diabetes and HLA-DP Genes Among African Americans

#20 Shantell Nolen, University of California 80 Berkeley Mentor: Barbara Staggers, M.D. Pre- and Post-Behavior of High Risk Adolescents Exposed to Sexually Transmitted Diseases

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#21 Stephanie Orth, Chapman University 82 Mentor: James Betts, M.D. Rate of Infection in Pediatric Patients with Neurogenic Bladder Receiving Sterile Versus Clean Intermittent Catheterization

#22 Lauren Pallis, University of California 8� Berkeley Mentor: June Tester, M.D. Healthy Eating Research – Food Access and Environments Surrounding Elementary Schools #23 Timothy Ryan, University of Notre Dame 88 Mentor: Robert O. Ryan, Ph.D., Kasuen Wong Characterization of the C Terminus of Apolipoprotein A-V

#24 Georgia Sleeth, Head-Royce High School �0 Mentor: Barbara Staggers, M.D. Obesity and Stress Reduction Techniques

#25 Tara Streich-Tills, Yale University �2 Mentor: Horst Fischer, Ph.D., Beate Illek, Ph.D. Stimulation and Control of H2O2 Secretion in Lung Epithelial Cells

#26 Dagim Taddesse, University of California �� Berkeley Mentor: Edward Lammer, M.D., David Iovannisci, Ph.D. Maternal Smoking, Genetic Variation of N-Acetyltransferase-1 (NAT-1), and Risk of Gastroschisis

#27 Alex Vidal, University of California �� Los Angeles Mentor: James Policy, M.D. Trauma vs. Osteochondritis Dissecans Arthroscopies in the Pediatric Elbow

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#28 Sonja Washington-White, California State �8 University East Bay Mentor: Cassandra Calloway, Ph.D. Analysis of Caucasian and US Hispanic Population Samples Using a New Linear Array Probe Panel for Targeting Variation in the Mitochondrial Genome

#29 Julie Willems, St. Louis University 100 Mentor: Rose Ellen Morrell, M.D. 24 Hour Blood Pressure Monitoring in Pediatric Patients with a BMI Above the 95th Percentile and a Normal Clinic Blood Pressure

#30 Chris Wong, Acalanes High School 102 Mentor: Barbara Staggers, M.D. Improving Contraception and Condom Use in Adolescents

#31 Angela Wu, University of California 10� Berkeley Mentor: Ervin Epstein, M.D., Jean Tang, M.D., Ph.D. A Clinical Study of Serum Vitamin D Levels in Basal Cell Nevus Syndrome Patients

#32 Alex Wulff, University of Oregon 10� Mentor: Ronald Krauss, M.D., Sally Chiu, PhD. An Angiotensin II Receptor I Polymorphism is Associated with Multiple Metabolic Risk Factors

#33 Johnston Ye, Monte Vista High School 108 Mentor: Robert O. Ryan, Ph.D., Taichi Yamamoto, Ph.D. Study of the Interaction between the Receptor- Associated Protein and the Low-Density Lipoprotein Receptor

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#34 Maria Zizka, University of California 110 Berkeley Mentor: Julie Saba, M.D., Ph.D. The Effects of Resveratrol on Colon Cancer Cell Lines: Up-Regulation of Ceramide and Inhibition of the Wnt Signaling Pathway

3:15 -- 4:00 Certificate Presentation and Cake in the CHORI Main Courtyard

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Olayemi Akintunde

Funded by the NIH/NHLBIDiversity Student Program

University of California BerkeleyThird Year

Mentor: Vasanthy Narayanaswami, PhD Shiori Tamamizu-Kato, PhD

Whenever I reflect on my college experience, my first summer at CHORI always stands out as a defining moment. It was there that I not only came to understand how a research laboratory operates, but most importantly I saw the hard work and persistence that goes into creating experiments and returning to a problem after it fails. From that moment on, whenever things became too difficult, I would remember the strength and determination my mentors Shiori Tamamizu-Kato and Vasanthy Narayanaswami as inspiration to overcome any obstacle that came my way.

As I enter my last year at the University of California, Berkeley, I know that I will be involved in research in the future. Working in a research lab, combined with my personal relationships with people who have various illnesses, has shown me the necessity of research. Despite the fact I have always associated physicians with healing people, I realize that researchers are also working toward healing but their work transcends the individual patient. Scientists have the opportunity to help many people by pushing back the boundary of the unknown and developing new treatments for diseases.

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Effects of Environmental Tobacco Smoke on Apolipoproteins

O. Akintunde, S. Tamamizu-Kato & V. Narayanaswami Children’s Hospital Oakland Research Institute, Oakland, CA 94609, USA

Introduction. Apolipoprotein E (apoE), an anti-atherogenic protein, plays a crucial role in cardiovascular disease by regulating plasma cholesterol levels and lipoprotein metabolism. By assisting in the transportation of very low-density lipoproteins (VLDLs) and high density lipoproteins (HDL), apoE is able to remove excess amounts of cholesterol and triglyceride from the blood and into the liver for processing. ApoE is able to remove plasma lipoproteins by acting as a ligand for cell surface localized lipoprotein receptors and proteoglycans. Specific lysine residues on apoE play a crucial role in lipoprotein receptor and proteoglycan interaction. Acrolein, a chemical found in cigarette smoke and generated as a metabolite of age-related oxidative stress, is highly reactive with lysine residues on proteins. Objective. The objectives of my research were to isolate the lipoproteins from the plasma of rats which were exposed to environmental tobacco smoke (ETS) and filtered air (FA) and to examine if second hand smoke exposure leads to oxidative modification of plasma apoE by acrolein. Methods. 10-week old male rats, which were obtained from Dr. Kent Pinkerton of the University of California, Davis, were exposed to (i) FA and (ii) ETS (1 mg/m3 total suspended particulate matter) for six hours per day for three days. The lipoprotein fractions were isolated from the plasma using the density gradient ultracentrifuge method. In the western blot analysis, the membranes were respectively probed with a rabbit anti-rat apoE antibody (RAG-4) and a rabbit anti-mouse apolipoprotein A1 (apoA1) antibody and then exposed to a secondary antibody (HRP-conjugated anti-rabbit IgG). Using the chemiluminescent detection methods, the proteins were revealed on scientific imaging film. Results. We were successful in detecting apoE and apoA1 in the lipoprotein fractions of rats subjected to FA and ETS. The relative amounts of apoE were lower in the ETS group than the FA group whereas the amounts of apoA1 were higher in the ETS group compared to the FA group. In order to see if apoE was among the proteins modified by acrolein, we performed an immunoprecipitation assay. The samples were incubated with an anti-acrolein-lysine antibody (5F6) to allow the antibody-antigen complex to form. The desired complex was precipitated out of solution using Protein G Sepharose. The results show that acrolein modified apoE is present in the ETS fractions even though the ETS group had lower amounts of apoE in the lipoprotein fraction compared to the FA group. Conclusions. These results are consistent with earlier in vivo observations from our lab that acrolein modification from environmental tobacco smoke causes structural and functional impairment of apoE. Since acrolein alters the receptor binding ability of apoE by modifiying the lysine residues, this could lead to increased plasma cholesterol and triglycerides levels, which are risk factors for heart disease. Thus, second hand smoke exposure may predispose individuals towards developing heart disease.

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Bryan Arias

Funded by the NIH/NHLBIDiversity Student Program

University of California BerkeleySecond Year

Mentor: Dan Granoff, MD Peter Beernink, PhD Jo Anne Welsch, PhD

Growing up with my few pets helped me be interested in science. I always became intrigued by the way life-forms live and function. My curiosity also fueled my interest in science to find answers to questions that I did not know or we do not know. As a rising junior at Berkeley, I still am interested in learning more about chemistry and biology and how to apply that knowledge in the real-world. Though it is a difficult track to follow, my determination will eventually guide me to my goal.

CHORI introduced me to the research field and the scientific community. Prior to CHORI I had no experience to research; I only had a medical internship two years ago but it was not as excited as I thought it would be. Medicine is a great option and field, but I wanted to explore my options and hopefully find something I enjoy. I also enjoyed how I apply my “textbook” knowledge to solve real-life biology problems—something that I have enjoyed all my life. There is not doubt that I will take great experiences from CHORI.

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Genetic and Antigenic Characterization of Epidemic Meningococcal Strains from Africa

B. Arias, P. Beernink, J. Welsch & D. GranoffChildren’s Hospital Oakland Research Institute, Oakland, CA 94609

Background: Neisseria meningitidis (N.m.) is a gram-negative bacterium that causes endemic meningococcal disease worldwide, and devastating epidemics in sub-Saharan Africa and other parts in the world. Five capsular groups—A, B, C, W-135 and X—cause the majority of disease. An immunogenic outer membrane protein, Genome-derived Neisserial Antigen (GNA) 1870, was identified and was subsequently renamed factor H-binding protein (fHbp) to indicate its function in binding factor H, which is a regulator of the alternative complement pathway. Another potential vaccine candidate surface protein that has been discovered is NadA. These antigens, as well as others, could be part of a safe, effective outer membrane vesicle (OMV) vaccine to prevent disease in Africa. Objective: African epidemic meningococcal isolates were characterized with respect to both fHbp and NadA gene and protein expression. This information may be useful in developing a vaccine against regionally prevalent variants of N.m. Methods: Heat-killed cells from epidemic strains collected in various parts of sub-Saharan Africa were obtained and suspended in Phosphate Buffered Saline solution. At the DNA level, quantitative polymerase chain reaction (qPCR) was used to determine whether the isolates contained the specific genes for fHbp and NadA. At the protein level, an Enzyme-Linked ImmunoSorbent Assay (ELISA) and Western blotting were used to determine antibody binding and protein expression. Dried bacterial suspensions were immobilized as the antigens on ELISA plates. Primary antibodies against fHbp and secondary alkaline phosphatase-conjugated anti-mouse Immunoglobulin Gamma (IgG) antibodies were used to detect the antigens. In a Western blot, the proteins from the outer membrane are first separated by polyacrylamide gel electrophoresis. Western blots were detected with horseradish peroxidase-conjugated secondary antibody and chemiluminescent detection. Results: All of the meningococcal group A or X isolates were positive for the fHbp variant 1 gene, whereas sixty-four percent of the group W-135 isolates had the fHbp variant 2 gene. One hundred percent of the group W-135 isolates, thirty-six percent of group A isolates and zero percent of group X isolates had the NadA gene. The Western blotting results confirmed that the respective proteins were expressed on the surface of the isolates. The results suggested that one vaccine strategy to protect against the majority of meningococcal disease in Africa is to develop an OMV vaccine that contains over-expressed fHbp variant 1 and naturally high levels of the NadA protein.

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Nelson Bean

Volunteer

Claremont-McKenna CollegeFirst Year

Mentor: Lourdes Juarez, PNP

Born and raised in Oakland, Nelson Bean is a college student currently pursuing a double major in biology and Literature at Claremont-McKenna college in Los Angeles, California. Since high school, Nelson has entertained an interest in medicine and the biological sciences, participating in the Children’s Hospital Faces for the Future program in 2005 hoping to get as much hands on experience as possible. He hopes that through participation in programs such as CHORI, he may further focus his efforts to practice the sciences in a professional setting. Also a multi-instru-mentalist, Nelson has furthered his efforts in the student music scene in both Oakland and Los Angeles.

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Water Study

N. Bean & L. JuarezChildren’s Hospital & Research Center at Oakland, Oakland, CA 94609

Several biological factors affect the manner in which the human body metabolizes resources. Indeed some of these factors are well known. Diet, genetics and age all can impart change in chemical mechanisms which influence basic metabolic rate and function. Recently, there has been an increase in the number of overweight individuals in developed nations. Although there are many parts to the issue, one of primary focus is the diet of those who are over-weight. The study was conducted on subjects between ages 9 and 12 years to determine the effect of water consumption on urine, serum and saliva osmolality. Each subject was ran-domly assigned a given treatment (consumption of orange juice carbonated mineral water or normal mineral water ad libitum). Each subject then submitted to regular visits at which time urine, serum and saliva osmolality were determined through measurement after break-fast in which the subject consumed either 300 or 600 mL arrowhead water, 300 or 600 mL arrowhead carbonated water, or 300mL orange juice. From the 20 subjects currently in the study there was an observed increase in saliva osmolality when subjects consumed orange juice and carbonated mineral water as compared to water. There was an additional similar correlation in urine osmolality and serum osmolality.

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Mayra Betancourt

Funded by the NIH/NHLBIDiversity Student Program

University of California BerkeleyThird Year

Mentor: Patsy Wakimoto, PhD

My interest in medicine and the biological sciences began when I was very young. As a child, I found that healthcare was an area that needed change, especially for those families that are living in poverty. Ever since I was young, my mother has struggled to provide for my family. This is because my mother is a single parent who often has to work more than one job at a time to support a family of five. My family’s difficult economic situation appeared to be magnified whenever any family member was in need of any type of medical services. Even though we had Medical, my mother was often left with the difficult choice of taking any of us to the physician or having food on the table. However, when we would seek medical attention, the experience was never pleasant for my family and I. The physicians seemed to show no interest for the welfare of my family and instead seemed to only be concerned with finishing up our consultation to get the next patient into the room. We had many issues with communicating with physicians and very often I was always the interpreter. As a child, I was frustrated with the language barrier because I knew that I couldn’t communicate what the doctor was saying and I couldn’t sufficiently express my mother’s concerns. It was during these stressful moments that I would realize that there were no doctors that were of color in our community and they really couldn’t relate to our cultural background. This was an area that affected the entire community and needed to be changed.

It is because of my personal experiences that I am passionate about pursuing medicine and the biological sciences. I want to be able to bridge the gap between underserved communities and medical professionals that was created through under representation of minority groups in biological and medical sciences. The CHORI Summer Research Program is a wonderful opportunity to expand my interests particularly in community based research. By participating in the program, I am able to gain valuable experience in the realm of research and learn how research translates into helping individuals and communities. This upcoming fall I will be entering my final year at UC Berkeley, majoring in Molecular Environmental Biology. After I graduate I plan to continue my education and apply to medical school.

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The Role of Religion or Spirituality and Other Factors Influencing Diabetes Management in Latinos

M.Betancourt & P.WakimotoChildren’s Hospital Oakland Research Institute, Oakland, Ca 94609

Introduction: Diabetes is a chronic disease that affects 23.6 million people in the United States. According to the Center for Disease Control, Latinos are 1.7 times more likely to have diabetes than non-Hispanic whites. Religion or spirituality may represent a coping mechanism that promotes positive behaviors in diabetes management. Objective: The objectives are to: 1) examine the relationship between faith and positive diabetes management and 2) explore other factors which influence diabetes management. Methods: Latinos with diabetes were recruited by Alameda County Public Health Department Diabetes Program staff (ACPHDDP). Participants were limited resource men and women over the age of 18. Four focus groups were conducted with 6 to 8 participants per group. The focus groups lasted approximately 60 minutes and were conducted in Spanish. A series of neutral open-ended questions were developed to address religion and diabetes management. Participants were asked to discuss factors that affect their management including family members with diabetes and any other factors that promote positive or negative practices. Focus groups were audiotaped. The data was transcribed and analyzed; common themes were identified. Results: Subjects (n=25) ranged in age from 33 to 81, with a mean of 54.2 years. Eighteen participants were from Mexico, 3 from El Salvador, 3 from the U.S. and 1 from Argentina. Residence in the U.S. ranged from 7 to 81 years. Twenty-one of the participants were Catholic and 4 were Christian. The results indicated that for the majority of the participants, religion or spirituality played a major role in dealing with diabetes both emotionally and mentally. Family support and other support groups such as the ACPHDDP were positive influences in diabetes management. However, family was described as also having negative influence. For example, family requesting or expecting that some of the less healthy, traditional foods be served at family meals caused difficulties for participants trying to make changes to manage their diabetes. Also, stress and financial burdens cause discouragement and negative outlook on diabetes management. Conclusion: Having a strong sense of faith in Latinos promoted positive behaviors in diabetes management. Other factors that influenced diabetes management included social support, access to more information, food/diet and family.

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Sophia Blachman-Biatch

Volunteer

College Preparatory High SchoolJunior

Mentor: Bruce Ames, PhD Swapna Shenvi, PhD

Hello my name is Sophia Blachman-Biatch. I am a rising senior at the College Preparatory School in Oakland, California. I love learning biology, creative writing and performing on aerial silks. Ever since I was young I have always cared about taking care of my health and the health of those around me. Whether it meant working out, actively encouraging my peers to take care of their health, or interrogating my doctor at my annualcheck up, I’ve always been curious and passionate. As the Sergeant of Arms and co-chair of Health and Human Needs Subcommittee of the Berkeley Youth Commission, I have been able to support programs encouraging youth to quit smoking and deal with obesity. So when I got the opportunity to work at CHORI for a second summer, I was overjoyed.

Last summer I enjoyed being introduced to lab sciences in the Smith lab. So I knew that this summer I would get another opportunity to learn more and have lots of fun and I am very pleased. I’m working with Swapna Shenvi in Bruce Ames’ lab detecting the changes in Nrf2 protein levels when both Selective Estrogen Receptor Modulators (SERMs) and lipoic acid are added to HepG2 cells.

I attribute my wonderful summer at CHORI to the members of the Ames lab, especially Swapna, so I want to thank everyone. Also I want to thank Dr. Bert Lubin, Diane Kohlman, and VasanthyNarayanaswami for creating such a unique and wonderful program. I feel so lucky to have taken part in it.

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Protective Effects of Estrogen Are Mediated in Part Through The Transcription Factor Nrf2 in Human Hepatoma Cells

S.B. Blachman-Biatch, S.V. Shenvi & B.N. AmesChildren’s Hospital Oakland Research Institute, Oakland, CA 94609

Introduction. Over 200 protective genes encoding for antioxidant and detoxification enzymes are regulated by a transcription factor, NFE2 related factor 2 (Nrf2) that binds to a DNA sequence called the Antioxidant Response Element (ARE), are present in their promoter region. A small amount of Nrf2 constitutively translocates into the nucleus for transcriptional activity and its nuclear levels are significantly increased under various exogenous and endogenous stresses. The female sex hormone, estrogen has been shown to be protective against many insults. Whether the protective effects of estrogen are partly mediated through the Nrf2-ARE system, however is unknown. Objective. To determine whether 17β-estradiol, a major estrogen, enhances expression of a panel of defensive genes through an Nrf2-mediated mechanism. Methods. We used human hepatoma (HepG2) cells as a model system since these cells constitutively express Nrf2 and are sensitive to stimulation by estradiol. HepG2 cells were treated with varying concentrations of estradiol up to 100 nM for up to 24 hours to determine at what concentration and time-point estradiol up-regulated the Nrf2-ARE system. To measure nuclear levels of Nrf2, we isolated nuclear protein by the Dignam method and performed immunoblot analyses using an anti-Nrf2 antibody. We assessed transcriptional activity of the ARE under estradiol treatment using a luciferase reporter containing the ARE of an Nrf2-regulated gene. Finally we measured the expression of a panel of Nrf2 target genes by isolating RNA for quantitative RT-PCR. Results. Results obtained from our experiments revealed that 10 nM estradiol treatment for 4 hours induced the maximum increase in nuclear Nrf2 levels (213% of vehicle), demonstrating that estradiol partly acts through an Nrf2-mediated mechanism. We anticipate that estradiol will also increase ARE transcriptional activity as measured by luminescence produced by the luciferase reporter. Lastly, we anticipate that the expression of the six Nrf2-regulated genes represented in our mRNA panel will be boosted by estradiol treatment. Conclusion. Based on these results we conclude that estradiol exerts its protective effects in part by a mechanism that involves the Nrf2-ARE network of defense genes.

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Benjamin Cedars

Volunteer

Bowdoin CollegeFirst Year

Mentor: Michael Oda, PhD

Forgive the cliché, but I have always harbored the desire to help people. My passion for science began my junior year of high school when I first took biology, due to both the inherently interest-ing subject matter and the enthusiastic and caring teacher. I realized that a career in science or medicine doing research could meld my two main interests. Don’t get me wrong, I have also entertained the notion of becoming a writer, an actor, or perhaps even a director (all of them pipe-dreams, I was told). Actually, I have not given up on any of these possibilities, but they remain side projects. The truth is I grew up in a family of doctors: parents, grandparents, aunts and uncles, etc. For courtesy’s sake I should probably attribute my love of science to them. After all, nothing captures the attention of a child as much as hearing incomprehensible medical jargon thrown about in dinner conversation as casually and frequently as the word “the.” But I think I would attribute it more to a childlike sense of wonder and an insatiable curiosity. When most kids enter the teenage years they tend to develop a sense of ennui and adopt a jaded attitude toward even the most extraordinary phenomena. Yet for some reason I never lost my ability to be fascinated. For example, I often lose myself in the night sky, searching for constellations and waxing philosophical. I also learned to appreciate watches, and I maintain the desire to become a watchmaker. I have a similar sense of wonder when I learn about new biological processes and mechanisms. I have become especially interested in neuroscience (my intended major) due in part to the many unknowns that still abound. College courses like “Intro to the Brain and Behav-ior” and “Physiological Psychology” have served to reinforce my pursuit of research. And CHORI has allowed me the opportunity to get a sense of the lab environment with hands-on experience that I know will be of great help to me in the future.

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Bulk Flow Facilitates Nanodisk Translocation Across Calu-3 and A��� Lung Epithelia

B.E Cedars, B.L. Burgess, R.O. Ryan, T.M. Forte, B. Illek & M.N. OdaChildren’s Hospital Oakland Research Institute, Oakland, CA 94609

Approximately 33% of the drugs currently approved by the FDA are hydrophobic. However, the use of these compounds is often limited by toxicity and the requirement for intravenous administration. To overcome these challenges, we have developed Nanodisk, a drug delivery platform for hydrophobic compounds. Structurally, ND consists of an apolipoprotein A-I (apoA-I) protein scaffold surrounding a central phospholipid bilayer to form an 8-10nm discoidal particle. A native function of apoA-I is to solubilize lipids for transport as the core protein of high-density-lipoprotein. ApoA-I as part of HDL is reported to cross vascular epithelium by a transport mechanism involving ATP-Binding Cassette-A1 (ABCA1). Similarly, ND applied to the lungs of mice enters the circulation, suggesting that ND may be transported across lung epithelium by a similar pathway. Understanding this pathway is important for the development of inhalable applications for ND. Objective: To develop an assay to study ND translocation across lung epithelium and evaluate the contribution of ABCA1. Methods: The assay measures the translocation of fluorescently labeled ND across cellular models of lung epithelium. Calu-3 cells, a model of bronchial epithelium, or alveolar-derived A549 cells, were used. Cells were cultured on semi-permeable transwell supports with distinct apical and basolateral chambers and the translocation of fluorescence from the apical to basolateral chamber over 24 hours was measured. To control for bulk-flow, results were compared to separate transwells containing the polysaccharide Inulin, which cannot enter cells or cross membranes. To evaluate the contribution of ABCA1, cells were treated with either Liver-X-Receptor activator TO 901317 (0.5µM) or cyclic AMP (0.5mM), to increase expression of ABCA1. Results: The translocation of ND across Calu3 and A549 cells was determined between 50 and 350µg/mL of apoA-I protein and was not saturable within that range. The rate of transit across Calu-3 cells was determined for ND (148.2 +/- 34 pmol/day), apoA-I (408 +/- 228 pmol/day) and Inulin (2426 +/- 964 pmol/day) for samples containing 100µg/mL protein or equimolar quantity of Inulin. The rate of translocation across A549 cells was determined for ND (548 +/-0.15pmol/day), apoAI (749 +/- 54 pmol/day) and Inulin (6472 +/- 96 pmol/day). Neither TO901317 (0.5µM) or cAMP (0.5mM) were observed to increase the translocation of ND or apoA-I alone (p>0.05). Conclusion: ND and apoAI were observed to undergo apical to basolateral translocation across both Calu-3 and A549 monolayers. However, Inulin transport was approximately 17-fold greater than ND across Calu-3 monolayers and 12-fold greater in A549 experiments, suggesting that bulk flow may account for some or all of the transport observed.

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Alex Chetkowski

Volunteer

Piedmont High SchoolJunior

Mentor: Stuart Smith, PhD, DSc Dejiang Feng, PhD

My interest in science began at an early age, searching for animals in our backyard and visiting my father’s hospital laboratory. As I grew older, my involvement with nature kept increasing through various summer camps and ecological trips to Costa Rica, Mexico and Fiji. I was always the one to find the camouflaged or unusual animals like a black boa constrictor or a blue starfish. Next year, I will be entering my senior year at Piedmont High School. Every year I have taken two science classes, including regular and AP biology, all of which have helped me understand and maximize my experience at CHORI.

Applying to the CHORI Summer Program, I was interested in getting a more hands-on view of science and in seeing how it is actually conducted in the lab. I was fortunate to be paired with my mentors, Dr. Stuart Smith and Dr. Dejiang Feng, who have shown me how intricate and elaborate biomedical research is. I now possess a clearer understanding of and admiration for science. I don’t know exactly where my scientific interests will lead me in the future, but it is very clear that my experience at CHORI has enriched me enormously. Doing experiments with no pre-determined outcomes has really helped me discover many things, both about science and about myself. I am extremely grateful to the faculty at CHORI for giving me such a phenomenal opportunity to grow and explore new areas in such depth.

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Screening of the Effective Mouse Mitochondrial ACP shRNA Targeting Se�uenceMouse Mitochondrial ACP shRNA Targeting Se�uenceMitochondrial ACP shRNA Targeting Se�uence

A. Chetkowski, D. Feng & S. SmtihChildren’s Hospital Oakland Research Institute, Oakland, CA 94609

Introduction. There is strong experimental evidence for the existence of a mitochondrial pathway for de novo fatty acid biosynthesis in both fungi and plants that is essential for mitochondrial function. Recently, several of the enzymes required for the de novo fatty acid synthesis also have been identified and characterized in humans. These discrete, nuclear-encoded, mitochondrially-targeted enzymes are quite distinct from the type I multifunctional polypeptide fatty acid synthase system found in the cytosol. The question arises why animals require a second system for fatty acid synthesis located exclusively in the mitochondria? Using extracts of bovine heart mitochondria, we showed that octanoyl-ACP (acyl carrier protein) is a major product of the pathway and the (acyl carrier protein) is a major product of the pathway and the is a major product of the pathway and the octanoyl moieties can be translocated to lipoylation sites on an acceptor protein. Down-regulation of mitochondrial ACP, one of the important enzymes in the mitochondrial fatty acid synthesis pathway, in HEK293 cells leaded to decreased protein lipoylation staining by anti lipoic acid staining by anti lipoic acid antibodies. So the mitochondrial fatty acid biosynthesis pathway might produce octanoyl moietieso the mitochondrial fatty acid biosynthesis pathway might produce octanoyl moieties that could be utilized for the synthesis of lipoyl moieties. To elucidate the physiological importance of the mitochondrial fatty acid synthesis pathway in whole animal may aid in understanding the etiology of diseases in which mitochondrial defects have been implicated. Objective. The specific aim for this project was to evaluate the most effective targeting sequence (short hair RNA) againstagainst mouse mitochondrial ACP in C2C12 mouse myoblast cells.mitochondrial ACP in C2C12 mouse myoblast cells. Methods. As the first step of the final goal of the project we, initially, evaluated the effectiveness of a panel of shRNAs available fromnitially, evaluated the effectiveness of a panel of shRNAs available from Open Biosystems in mouse C2C12 myoblast cells, since muscle mitochondria express relatively in mouse C2C12 myoblast cells, since muscle mitochondria express relativelyells, since muscle mitochondria express relatively high levels of the mitochondrial FAS enzymes. Assessment of the efficiency of knockdown was made by screening for defective protein lipoylation, which provides an indication of the availability screening for defective protein lipoylation, which provides an indication of the availabilityfor defective protein lipoylation, which provides an indication of the availability, which provides an indication of the availabilitywhich provides an indication of the availabilitys an indication of the availability an indication of the availability of de novo synthesized octanoyl moieties for the lipoylation pathway. shRNA plasmid DNAs weres were werewere isolated using High-pure endotoxin-free NucleoBond Xtra Midi EF kit from Clontech. 5 shRNA High-pure endotoxin-free NucleoBond Xtra Midi EF kit from Clontech. 5 shRNAigh-pure endotoxin-free NucleoBond Xtra Midi EF kit from Clontech. 5 shRNA endotoxin-free NucleoBond Xtra Midi EF kit from Clontech. 5 shRNA kit from Clontech. 5 shRNA. 5 shRNA 5 shRNA constructs against mouse mitochondrial ACP. A negative control construct also transfected C2C12. A negative control construct also transfected C2C12negative control construct also transfected C2C12also transfected C2C12 myoblast cells by using Arrest-in transfection reagent from Open Biosystems. Finally, calcium by using Arrest-in transfection reagent from Open Biosystems. Finally, calciumby using Arrest-in transfection reagent from Open Biosystems. Finally, calciumusing Arrest-in transfection reagent from Open Biosystems. Finally, calcium Finally, calcium phosphate transfection method was used to transfect C2C12 cells. Results. The transfection efficiency of Arrest-in method is very low (1%) estimated by microscopy. Transfection efficiency with calcium phosphate transfection method is around 90%. Conclusions. The transfection efficiency is the key step in transcient knockdown experiment. The Arrest-in transfection reagent, which was claimed for efficient-delivery of shRNA plasmid DNA, does not work on C2C12 cell line. Screening of the effective mouse mitochondrial ACP shRNA targeting sequence will bemouse mitochondrial ACP shRNA targeting sequence will bemitochondrial ACP shRNA targeting sequence will be investigated in mitACP shRNA transfected C2C12 cell line.

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Dana Collins

Funded by the William Jenkins Fund

Spelman CollegeFourth Year

Mentor: Ronald Krauss, MD Patty Siri, PhD Patsy Wakimoto, PhD

When I began pursuing my degree in psychology, I wanted nothing to do with research. It seemed laborious, technical, and generally was something I didn’t want to do. I didn’t begin to understand just how important research is to psychology until my last year of college. I want to earn my PhD in Clinical psychology, and I learned that research experience is an essential for gaining admittance into a graduate program. I completed my B.A at Spelman College a semester early, and decided to devote time to doing research, mainly because it is a requirement for being accepted into graduate school. I submitted my resume to CHORI, and Dr. Patty Siri, who is now one of my mentors, offered me a chance to volunteer in the Krauss lab. As a psychology major, I was skeptical about working in a biological science lab, but I decided that any research experience is good research experience. Dr. Siri and I are conducting a post hoc analysis to determine whether lifestyle impacts African Americans’ developing dyslipidemia. Dr. Siri has also introduced me to Robin Rawlings, RN, who allows me to work in the Cholesterol Research Center, a subset of CHORI, and to Dr. Patricia Wakimoto, the director of outreach at CHORI, who has allowed me to become involved in a project whose aims are to determine the underlying causes of ethnic minorities’ infrequent involvement in scientific research and identify ways to increase their involvement.

I still believe that clinical practice is important in that it assists individual people, butn realize that it isn’t really designed to assist whole populations. Research, however, can effect change on a much larger scale, and so I plan to divide my time between clinical work and research. This Fall I will pursue my M.S in counseling psychology at Indiana University, Bloomington, and I will some time to conducting research. Spending my summer at CHORI has allowed me to get some of the research experience I need for success as a graduate student but more importantly, has shaped my career choice by showing me that I should consider more seriously making research a part of my professional life plan.

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Self-reported Weight Gain is Significantly Associated with Plasma Triglycerides in African Americans

D. Collins, P. Siri Tarino, P. Wakimoto & R.M. KraussChildren’s Hospital Oakland Research Institute, Oakland, CA 94609

Introduction: Dyslipidemia, characterized by elevated triglycerides (TG), low HDL cholesterol (HDLC), and small dense LDL cholesterol, has been associated with cardiovascular disease, and obese African Americans may be less likely than obese Caucasians to develop it. A genetic polymorphism has been identified as potentially protective against dyslipidemia (CAP study). Sixty-six obese but otherwise healthy African Americans were included in a study to confirm this association GOLD-A study, Krauss, R.M, 2008, unpublished data). No association was observed between the polymorphism and dyslipidemia . Objective: To determine whether lifestyle variables including demographics, family history of disease, socioeconomic status, diet, exercise, and menstrual status were associated with plasma triglycerides, HDLC and LDL size. Methods: Data were collected from a questionnaire that GOLD A participants completed. Distributions of the lifestyle variables were evaluated. Univariate and multiple logistic regression analyses were conducted to consider relationships between lifestyle variables and biochemistries. Results: Self-reported weight gain since the age of 25 was associated with elevated levels of TG (n=44; p=0.002) by univariate analysis and after adjustments for age and sex (p<0.01). When 25-year or current BMI or weight was included in the model, weight gain remained significantly associated with elevated plasma TG (p≤0.01 for all models). Family history of disease was associated with TG and LDL size by univariate analysis (n=59; p=0.03 and p<0.05, respectively), but was no longer significant with adjustments for age and sex (p=0.45 and p=0.33, respectively). Menstruation status was associated with TG, HDLC, and LDL size by univariate analysis only (n=47; p<0.01, p=0.01, and p=0.03, respectively). Coffee drinkers had elevated levels of HDLC, even when adjusted for age and sex (n=65; p<0.01). Conclusion: Weight gain since the age of 25 may be a more important predictor of current TG concentrations than are previous or current weight or BMI. This discovery makes a strong argument for recommending weight maintenance as a public health measure for preventing dyslipidemia, even in overweight individuals.

* * *African Americans face the problem of a number of health disparities; among other things, they are more likely to develop ailments, such as high blood pressure, obesity, and cardiovascular disease. Despite these disparities, it is often difficult to engage participation for studies designed to examine these health disparities due to factors such as mistrust and a lack of understanding on the wide spread impact that research can have on health. The National Center for Minority Health Disparities (NCMHD) has launched an initiative designed to further explore this problem, and to formulate ways to encourage greater research participation from African American and other minority communities. CHORI’s community outreach division has taken part in this initiative by starting its own investigation, which consists of conducting focus groups with community members and interviews with members of the science community. The findings will reveal ways in which scientists can elicit greater participation in research from minority communities.

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Stacie Collins

Funded by the NIH/NHLBI Diversity Student Program

Howard UniversitySecond Year

Mentor: Gregory Moe, PhD Hardeep Kaur, PhD

I grew up in a household where the importance of giving back to those who are less fortunate was always stressed. My summers were spent volunteering at various free dining halls, convalescent homes, and health care facilities, where I had the opportunity to work closely with bay area residents from a wide variety of backgrounds and walks of life. As a community volunteer, I observed that many of the people encountered suffered from varying degrees of mental illness. Seeing first-hand how poor mental health can affect a person’s life peaked my interest in psychology and the study of the human brain. When I entered my freshman year at Howard University in Washington DC in 2006, I declared myself a psychology major with the intention of pursing a career in the mental health field as an adolescent psychologist or psychiatrist. My long-term goal is to serve those who do not have the resources to obtain the mental health care that they need, and also promote a message emphasizing the importance of acknowledging, destigmatizing, and actively treating mental illnesses that are prevalent in our communities.

As I have pursued my studies in psychology, I have learned that there is a very strong connection between the brain, the body, and an individual’s emotional state. I have also surmised that to become intimately familiar with the relationship between psychology and the human mind requires a strong background in biology and physiology. As a result, I have chosen biology as my minor and am currently learning about the intricacies of the human body and their physiological and emotional affects on the human psyche.

I chose to come to CHORI to strengthen my science background, and learn more about research processes and techniques. Working in Dr. Greg Moe’s lab, I have gained valuable hands-on laboratory experience that I would not have been able to get in a large college classroom setting. I know the knowledge and experience that I take away from my summer at CHORI will be vital to my development as I continue to pursue my academic studies and eventually my professional career.

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Does the Monoclonal Antibody SEAM 2 React with HSP60 or a Modified Form of HSP60?

S. Collins, H. Kaur & G.R. MoeChildren’s Hospital Oakland Research Institute, Oakland, CA 94609

Introduction. The monoclonal antibody (mAb) SEAM 2 was produced using a vaccine containing the N-propionyl derivative of Neisseria meningitidis group B capsular polysaccharide (N-Pr NmB PS) conjugated to tetanus toxoid. Surprisingly, SEAM 2 is poorly reactive with N-Pr NmB PS but binds to antigens expressed by several Nm capsular groups including A, B, C, and W135. SEAM 2 also activates protective responses against strains from all four groups. The identity of the naturally expressed antigen recognized by SEAM 2, however, is unknown. On Western blots of meningococcal membranes, SEAM 2 is reactive with two proteins having apparent masses of 67 kDa and 47 kDa, which were identified by MALDI-TOF tryptic peptide mass fingerprinting as the chaperone protein HSP60 and the alpha subunit of DNA polymerase III, respectively. The two proteins have no direct relationship to each other and there is no significant amino acid sequence homology between them. Our hypothesis is that the antigen recognized by SEAM 2, which likely is a polysialic acid derivative, is either associated with or covalently modifies the proteins. Objective. The goal of the project is to clone the gene encoding HSP60 from Nm, express the recombinant protein in E. coli, and determine the reactivity of recombinant Nm HSP60 protein with SEAM 2 by Western blot. Methods. Genomic DNA was prepared from NmB strain MC58. The gene encoding HSP60 (nmb1972) excluding the 5’-ATG and 3’-TAA, was amplified by PCR and ligated into the pGEM-Teasy vector (Promega). After transforming JM109 cells with the ligation reaction, plasmid DNA was isolated from 3 transformants identified by blue/white selection on amplicillin plates containing IPTG and XGal. All 3 contained an insert of the expected size when digested with BamHI and HindIII (New England BioLabs). The presence of the complete gene was confirmed by DNA sequencing of three isolates. A BamHI/HindIII fragment was prepared from the plasmid DNA and purified by agarose gel electrophoresis using Qiagen gel extraction kit. The fragment was ligated into the pQE30 (Qiagen) expression vector and the ligation reaction used to transform M15 [pREP4] cells (Qiagen). 22 transformants were obtained and the plasmid DNA from the 16 tested contained an insert of the expected size. The resulting plasmid was designated pNmHSP60ex. Results. By SDS-PAGE, M15 [pREP4] cells transformed with NmHSP60ex expressed very high levels of recombinant HSP60 when induced with IPTG. Whole cells containing the recombinant Nm HSP60 were compared to membranes prepared from Nm strain NMB on a Western blot probed with SEAM 2 or an anti-HisTag mAb as the primary antibodies. SEAM 2 reacts with 67 kDa and 47 kDa proteins in the NMB membrane preparation but shows no reactivity with recombinant HSP60, which was identified by the anti-HisTag mAb. Conclusions. The results clearly demonstrate that SEAM 2 is not reactive with a trivial linear peptide epitope present in HSP60 and suggests that HSP60 naturally expressed in Nm is modified with or is associated with another molecule that is recognized by SEAM 2. Experiments testing whether the antigen can be reconstituted by combining the purified recombinant protein with cell extracts from NMB are in progress.

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John Cossette

Funded by the Elizabeth Nash Foundation

Carleton CollegeFirst Year

Mentor: Horst Fischer, PhD Beate Illek, PhD

Since my parents first explained to me that I had the genetic disease cystic fibrosis (CF), I have always done everything I possibly can to educate other people about my disease and to help the CF community. And since my first day of AP Biology in 11th grade, I have been captivated by the science of life. So, it’s only natural that I want to pursue a career that combines these two passions—which is why I jumped at the opportunity to apply for the Elizabeth Nash Foundation award to do CF research as a CHORI Summer Student. I was ecstatic when I was chosen, even though it meant spending the summer far from my home in Minnetonka, MN. By working under premier CF researchers like Dr. Illek and Dr. Fischer, I’ve gained invaluable insight into how my disease works and experiences that will serve me well as a future CF researcher or CF doctor. After this amazing summer, I’ll return to MN for my sophomore year at Carleton College.

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Stimulation of CFTR-mediated Cl- Transport by Vitamin C and SP�0012� JNK-inhibitor

J. Cossette, B. Illek & H. FischerChildren’s Hospital Oakland Research Institute, Oakland, CA 94609

Introduction: In cystic fibrosis (CF), a mutation in the CF transmembrane conductance regulator (CFTR) gene results in faulty or absent expression of this vital chloride channel. Vitamin C (L-ascorbate), normally a water-soluble antioxidant, and SP600125, an anti-inflammatory compound known to inhibit c-Jun N-terminal kinases (JNKs), have been shown to exhibit the novel effect of stimulating CFTR-mediated Cl- transport in cells with working CFTR channels. Preliminary data also suggested the normally lipid-soluble Vitamin E in the form of water-soluble g-tocopherol may have had a similar stimulating effect. Objective: We aimed to further investigate the stimulating effect of L-ascorbate, SP600125, and g-tocopherol on Cl- transport, both separately and in combination, in CFTR-corrected CFBE 41o- cells. We expected all three compounds to exhibit some stimulating effect, with the strongest response to L-ascorbate. Methods: Ussing chamber assays were employed to measure the transepithelial short-circuit current on CFTR-corrected CF bronchial epithelial cells (wtCFBE 41o-) subjected to a 120mM serosal-to-mucosal Cl- gradient, as well as ΔF508 homozygous CFBE 41o- cells (parent, control). Cells were stimulated with the experimental variables, then administered 30 µM GlyH101, a CFTR-specific blocker, to verify the validity of observed differences in transcellular Cl- transport. Results: Preliminary results showed stimulating effects of individual supplementation with both L-ascorbate and SP600125 on CFTR-mediated Cl- transport in CFTR-corrected cells, as well as distinguishable, complementary effects from concerted supplementation with both substances. A selected number of trials (n=6) showed a mean I

SC response of +102.4 µA/cm2 to L-ascorbate (100µM) supplementation, with an additional

(n=5) response of +31.0 µA/cm2 when SP600125 (10µM) was then added. Trials of SP600125 alone have yielded similar visual results to L-ascorbate (more raw data are needed for these effects to be quantified). In contrast, trials with g-tocopherol, both individually and in combination with L-ascorbate, displayed negligible or possibly even inhibitory effects. Conclusions: L-ascorbate and SP600125 both seem to be promising stimulators of CFTR function in normal epithelial cells, especially when used in combination; when added to cells stimulated to a 116.0% mean increase in I

SC by L-ascorbate (n=6), SP600125 provided an additional 35.1% mean increase over the

baseline current (n=5). The fact that the effects of each compound were visually and quantifiably distinguishable from each other when added together seems to indicate that they may stimulate via different intracellular mechanisms – more research is needed to verify this hypothesis. In contrast, g-tocopherol would seem to have negligible or inconsistent effects on CFTR function from our data.

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Christopher Cox

Volunteer

Sacramento State UniversityPostbaccalaureate

Mentor: Julie Saba, MD, PhD Babak Oskouian, PhD

Hello everyone! My name is Christoper Cox and I am a 26 year old second bachelors student at Sacramento State University majoring in Chemistry while taking classes to gain acceptance to a medical school program. I first came to Oakland in 1992 as a pediatric oncology patientand was treated for my leukemiz by the hospital staff at Children’s. I finished treatment in 1994 and have been healthy ever since. Upon deciding to enter the medical field after earning a degree in business I called up Dr. Feusner at Children’s and he suggested that I do some research at CHORI. So here I am in my second year at te Institutes’s summer program working to help find less invasive treatments for cancer with some of the greatest scientists in the world. Thank you all for the opportunity!

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Leveraging Sphingolipids in the Treatment of Leukemia

C. Cox, B. Oskouian & J. SabaChildren’s Hospital Oakland Research Institute, Oakland, CA 94609

Introduction: About 33 percent of cancers in children ages 0-14 years old are leukemia. Current treatment for leukemia is invasive and non-specific. Sphingolipids are natural lipids found in soy products and the fruit fly model as well as in the endoplasmic reticulum of all human cells. They contribute to the function of the endoplasmic reticulum and give rise to lipid signaling molecules. Some of these signaling molecules produce cell death and account for the main mechanism by which chemotherapy and radiation kill cancer cells. In contrast other molecules have been shown to stimulate the proliferation and survival of cancer cells. Our lab is currently focused on levels of the sphingosine 1 phosphate (S1P) signaling molecule in cancerous cells. We have found that S1P levels are greater in colon cancer polyps than in other areas of the colon, affirming the hypothesis that colon cancer cells use elevated levels of S1P to multiply and grow. Similarly, many studies link S1P accumulation to leukemia cell development, survival and resistance to chemotherapy. These findings suggest the balance of growth-inhibitory versus growth-stimulating sphingolipids could be responsible for determining whether a leukemia cell lives or dies. Last summer some interesting findings further supported the hypothesis that lipid treatment of leukemia cells causes cell death and aides in the effectiveness of an AKT phosphoralation pathway inhibitor. Hypothesis: Altering intracellular sphingolipids could be leveraged to improve leukemia responses to chemotherapy. This could be accomplished by either adding cytotoxic sphingolipids or removing growth-stimulating sphingolipids such as S1P. Specific Aims: 1) Characterize the sphingolipid pathway in leukemia cells; 2) Evaluate the effect of different sphingolipid compounds on leukemia cells alone and in conjunction with chemotherapy. Experimental Design: We intend to propagate several kinds of lymphocytic and myelocytic leukemia cell lines, treat the cells with different concentrations of sphingolipids, and evaluate the effect on cell survival, apoptosis, and autophagy. Next, we will harvest the cells from the culture and determine the amount of S1P lyase in these cells, which requires separating the endoplasmic reticulum (where the lyase resides) from the cytoplasm. This allows us to probe specifically the S1P-Lyase in the endoplasmic reticulum without any background interference. We will then use a western blot technique to identify S1P-Lyase levels in the cells. These results can then be analyzed to determine relative levels of S1P-Lyase. Similar methods are used to explore AKT activity and cell death proteins in the leukemia cells. MTS assay and cell counts will also be used as a determinant of cell viability. Anticipated Outcome of Project: We wish to gain a clearer understanding of the ways in which leukemia cells proliferate; this idea critical to the development of better, less invasive treatments for leukemia. Results that affirm our hypothesis would be a major step in using a new signaling pathway to treat leukemia.

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Tiffany Crouch

Funded by the NIH/NHLBIDiversity Student Program

Grambling State UniversityThird Year

Mentor: Jennifer Olson, MD

My goal has been to develop a successful career in the health sciences. Initially, my love for children and my interest in anatomy and physiology were the motivational factors behind my wanting to become a pediatrician. Pursuing my bachelor degree in biology at Grambling State University has given me an even greater appreciation for science in general. Communicating with professionals, while participating in other health-related programs at schools like Meharry Medical College and Howard University, has allowed me to realize that there is so much more that I want to know and many more things that I can pursue in health-related sciences. I chose to attend the summer research program at CHORI to further explore some of the many opportunities that are available.

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Testing Outcomes for Congenital Adrenal Hyperplasia (CAH) at Children’s Hospital and Research Center Oakland in 200�

T. Crouch & J. Olson Children’s Hospital & Research Center at Oakland, Oakland, CA 94609

Introduction. Congenital adrenal hyperplasia is an autosomal recessive disease that an infant is born with where the adrenal glands are enlarged and, because of a missing or deficient enzyme, 21-hydroxylase, cannot produce the hormone, cortisol. Females with CAH are usually easy to identify at birth because of their enlarged genital area, but male newborns are not as easily noticed. Untreated CAH can also cause low blood pressure, vomiting, shock, and even death. Due to the severity of this disease if it goes unrecognized, beginning in July 2005, the California Department of Health Sciences required the state’s hospitals to screen newborns for the disease by testing levels of 17-OHP before they were released from hospitals. The screening brought about many false positive results for the disease. Since cortisol is the hormone that is released to protect the body from infection, injury, and disease, underweight babies tend to have an excess amount of cortisol’s precursor product, 17-hydroxypergesterone (17-OHP). Because of the high levels of 17-OHP, the screening is often misled to believe that these infants are affected by CAH when they are not. The California Department of Health Sciences recognized this amount of false positives to be unnecessary and decided to revise the cutoff level of 17-OHP needed to test positive for CAH. This revision occurred in March 2006. Objective. The purpose of this study is to determine whether or not the cutoff level of 17-OHP should be revised again in order to cut back on the number of false positives in the two lowest birth weight classes. We will also try to determine the optimal age at which repeat levels should be drawn in very premature babies. Methods. The methods for our research included collecting data from patients who were screened for CAH through the California screening program at CHRCO in 2007. Some of the information we collected include birth weight, 17-OHP levels, and gestation. We noted all of the patients that tested positive in the initial screening. After we collected the initial positives, we went on to look up the patients’ rescreening results to determine how many of the initial positives were actually true positives. This information was entered into a database and analyzed. Result. Out of 280 newborns screened in 2007, 15 had an initial positive result for CAH. 25 out of the 280 newborns weighed less than 1000 grams. Out of these 25, there were 2 that had positive results. No newborn in the <1000g weight class were actual true positives. 24 of the 280 newborns had birth weights of 1000 grams to 1499 grams. Out of these 24, there were 2 that had positive results. None of these were actual true positives. In the 1500g- 2499g weight class, there were 70 newborns with 3 testing positive. None of these 3 were actual true positives. In the last weight class, 2500g+, there were 70 newborns. 8 of these 70 tested positive in the initial screening. 4 out of the 8 were true positives. Conclusion. Based on these studies, we concluded that the state screening was more accurate when screening newborns weighing at least 2500g. None of the newborns in the two lowest weight classes that had a positive result in the initial screening had a positive result during the rescreening. It was not concluded that there is a better cut-off 17-OHP level. Collecting data from a much larger sample would have aided in coming to this conclusion. Therefore, further studies including more results should be conducted.

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Luiza Da Hora

Volunteer

Dominican UniversitySecond Year

Mentor: Lourdes Juarez, PNP June Tester, MD

My name is Luiza Da Hora. I am currently a Junior at Dominican University of California pursuing a BSN in Nursing with minors in psychology and religion. I am the eldest of three and currently the only one in my family to be in pursuit of a college education. My family is from Brazil and I am fortunate to be able to speak English, Portuguese and a little Spanish.

Once I graduate I want to be able to work in pediatrics. Ever since I was thirteen I have always been involved with working with kids; anywhere from tutoring after school to teaching swim lessons to kids as young as three. I have also been honored to have worked with children with a variety of learning and developing disabilities. My passion for working with children, especially in the health field, also stems from raising a younger brother who is autistic.

I am interested in a variety of ailments and conditions which affect children; from hematological disorders to obesity. I am still not quite sure which area I would like to fully specialize in; however I am using exposures granted at the university and other opportunities such as CHORI to narrow my areas of interest.

This is my first summer participating in CHORI’s summer research program. I am excited to be working with a childhood obesity clinic and hope to be able to help make a difference in understanding and curbing this epidemic. My project this summer will be looking at children’s after school eating habits, from what items they are purchasing to how much on average they are spending. Are they alone, buying with their own money or with their parents?

I am grateful for the opportunity to be interning at CHORI and look forward to getting as much as I can out of the experience. I hope to be back next summer with another topic to research!

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Street Vendors and Food Environment Influence on Kid’s After School Snacking

L. Da Hora, J. Tester & L. JuarezChildren’s Hospital & Research Center at Oakland, Oakland, CA 94609

Background: A small time frame such as from the time the children are exiting from school until they arrive home sometimes is all the time needed for bad food choices to be made; thus aggravating the ever worsening childhood obesity epidemic. From what children eat during school lunch hours to the different types of stores available and their cost of food, it all demonstrates how hard it is for children to make good nutritious choices when there aren’t many healthy options available to them. Objectives: I looked at children’s after school purchasing pattern and the range of items that were commonly and frequently purchased. Methods: I performed an observational study through secondary data research of transactions at five different elementary and one middle school located in Oakland, California from April 28 through June 12 in the spring of 2008. It was an observational study of transactions of mobile vendors to people within ¼ mile of the school which occurred during the hour after school was let out for the day. The population was people exiting the schools on the outside and included school aged children, teenagers and adults. Results: 1018 transactions were observed. Of that number, 372 transactions were made by school aged children without adult supervision. The items frequently purchased were grouped into 12 different categories: from the healthy fruit (cherry, watermelon, mango, orange) and water to the unhealthy ice cream, chips, frozen treats, burrito_taco, sugary drinks, elote, paleta, candy, and tamale. Of the 372 children who made purchases without adult supervision, 56.4% were male and 41.9% were female. Of the 372 transactions made by children without adult supervision, 29.3% of the items bought were fruits, 13.9% was ice cream, 13.4% was chips, 12.6% was frozen treats, 9.9% was candy, 5.3% were paleta, 4.8% were elote, 4.5% were burrito_tacos, 4% were sugary drinks, 0.80% was water and 0.26% was tamale. I was very surprised to find out that fruits were the most frequently purchased items by children with their own money without adult supervision. Conclusion: Many things were sold and some of the items were healthy. I believe this warrants further study in the nutritional value of items and more exploration on the expansion of providing healthier food options. As shown above, children did react and purchase the healthier items more frequently an opposed to the unhealthier choices.

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Leslie Dinh

Funded by the NIH/NHLBIDiversity Student Program

University of California BerkeleySecond Year

Mentor: Lynne Neumayr, MD

For as long as I can remember, I have always been curious about many things in life, from the tiniest details to broader views. I began studying animals at a young age, owning anything that may be considered a “pet” from insects to fishes to puppies. Although I am an animal lover, I was amazed by how much we can learn about ourselves from studying and dissecting pigs and insects even though they could have been added to my collection of “pets.” As I came to UC Berkeley, I became more active and aware of the issues in my communities and its surroundings through mentoring, volunteering, facilitating, and listening and speaking to others. My grandmother’s passing due cancer, the issues and inequalities of healthcare, my passion for the well-being of pets and people, curiosity about life, and life experiences have driven me to become more interested in medicine, public health, research, and biology, the study of life, itself.

Curious about research, my feet led me to CHORI. Here, I learned that research is important to answer life’s incessant questions and paves way for the combination of both “bench work and bed work.” This program can be summarized in three words- short, sweet, and simple. The conciseness of this program gave me a glimpse of the importance of sickle cell research and snapshots of clinical and lab work, the sweetness made me want to crave for more knowledge and experience, and the simplicity yet complexity of the program encouraged me to read and research on my own, to simply ask questions, and to explore outside of my boundaries or what Dr. Lynne suggests “to get the most out of this experience.” Her breadth of knowledge and support inspired me to grow not only as a summer student researcher but also as a person. I hope to continue to follow my aspirations in research and medicine and positively impact the communities that I grew up in (Oakland, San Jose, and Stockton), whether it is to provide affordable and qualitative health care, active support, or to simply learn from them as well as let them learn from me.

Thank you BSP, Reach!, TAL!, Xuan and Jennifer Dinh, Kathi, and Nurredina for their mental and emotional support, patience, and motivation to follow my aspirations, Dr. Lynne Neumayr for guidance and support, and Janice E., Dr. Keith Quirolo, Sean L., Ashley H., Eileen M., CHRCO & CHORI staff, and other student interns for making my experience a great one.

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Restrictive and Obstructive Lung Disease in Pediatric Patients with Sickle Cell Anemia

L. Dinh, L. Neumayr, C. Morris, S. Robertson, M. Newaskar & E. VichinskyChildren’s Hospital & Research Center at Oakland, Oakland, CA 94609

Introduction: Abnormal pulmonary function tests (PFTs) consistent with restrictive physiology have been reported in 90% of adult sickle cell disease (SCD) patients. Pulmonary complications that predispose SCD patients to respiratory compromise are common in SCD and include acute chest syndrome (ACS), asthma, and pulmonary hypertension (PH). At CHRCO, echocardiograms (ECHOs) are obtained routinely on patients starting at age 10 and annually as PHT become recognized as a risk factor for early demise in SCD. Asthma occurs in significantly higher rates in SCD than in the general population. Objective: The purpose of this study is to review lung function patterns in pediatric SCD patients with PH. Methods: Medical records of SCD patients with hemoglobin SS disease between the ages of 5 and 21 from 2003-2008, who had both an ECHO and PFT, were reviewed. All lung volumes were recorded as percent predicted for age, gender, and height. At CHRCO, an FEV

1/FVC ratio

of < 85% in pediatric patients is considered an indicative of obstructive disease. Restrictive physiology was defined as total lung capacity (TLC) of < 80%. On ECHOs, PH was defined as a tricuspid regurgitant jet velocity (TRJV) of ≥ 2.5 m/s. Results: 41 patients had both PFTs and ECHOs within 4 months of each other (median 63 days). The average age of the patients was 16.4 ± 3.3 years; 56% were male. 87% had an episode of ACS, 64% had a history of PH, 63% had a clinical diagnosis of asthma, 23% had femoral avascular necrosis (AVN), and 5% had a history of stroke. 54% were chronically transfused. The average hemoglobin (Hb) was 9.3 ± 1.4, WBC 12.1 ± 3.4, platelets 378 ± 108, LDH 441 ± 172, and total bilirubin 3.9 ± 2.3. The mean TLC was 82 ± 13%, predicted. The FEV

1 of 77 ± 5%, FVC 82% ± 13%, FEV

1/

FVC 85% ± 4.6, and FEF25-75

78 ± 20% predicted were low normal. PFTs were abnormal in 74% of patients: restrictive physiology was seen in 29%, an obstructive pattern in 29%, and mixed obstructive and restrictive in 16%. Values for DL

CO corrected for alveolar volume were

available in 32 patients, and the mean was 77 ± 14% (median 77%); DLCO

and DLCO

corrected for hemoglobin were rarely reported. 74% patients with a history of PH had a clinical diagnosis of asthma compared to 43% in patients without PH (p=0.05). The majority of patients had an episode of ACS and was not more frequent in PH. TRJV was reported in 37/41 (90%) ECHOs in this study, and 18/37 (49%) had PH. PH was associated with a lower mean FVC (86 ± 16 vs. 95 ± 14%, p=0.05) and trended to lower FEV

1 (80 ± 12 vs. 88 ± 14%, p=0.08).

TLC, FEV1/FVC, FEF

25-75, and DL

CO corrected for alveolar volume were not different between

patients with PH and those without. Markers of hemolysis were higher PH (total bilirubin: 4.8 vs. 3.0, p=0.03, and LDH: 499 vs. 362, p=0.05). Also, WBC was higher in PH: 13.0 vs. 10.7, p=0.04. PFTs were not correlated with TRJV or lab values. Conclusion: Abnormal PFTs are common in pediatric patients with SCD with both restrictive physiology and obstructive disease. Studies in adult SCD patients suggest that these abnormalities are progressive. Patients with PH are more likely to have asthma and spirometry volumes trended lower in PH. Longitudinal studies of PFTs and ECHOs are imperative to study the progression of lung function and PH in SCD.

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Caroline Dougherty

Volunteer

Miramonte High SchoolSenior

Mentor: Gordon Watson, PhD

Caroline Dougherty will be attending Wellesley College next year in Boston, Massachusetts. She has a wide variety of academic interests ranging from the sciences to social sciences to literature. Although her current major is International Relations, a triple major of history, political science, and economics, she plans to continue her scientific inquiry at Wellesley.

In January of her senior year, Caroline was asked to be a debutant as a part of a Hill Branch fundraiser for the Children’s Hospital of Oakland. At an informational meeting about the 50 year old charitable organization, she was introduced to Dr. Lubin who inspired her to volunteer at CHORI this summer. She has greatly enjoyed connecting her debutant experience on a personal level through her project at CHORI.

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Effect of Smith-Lemli-Opitz Syndrome on Erythrocytes

C. Dougherty & G. WatsonChildren’s Hospital Oakland Research Institute, Oakland, CA 94609

Introduction: Smith-Lemli-Opitz syndrome (SLOS) is a genetic disease in which cholesterol production is inhibited as a result of deficient amounts of 7-dehydrocholesterol reductase. In SLOS patients, 7-dehydrocholesterol (7DHC), the precursor to cholesterol, is unable to be reduced to cholesterol. Thus, a patient with SLOS has increased amounts of 7DHC and insufficient amounts of cholesterol. In severe cases, for example when the 7-dehydrocholesterol reductase gene is deleted in a homozygous mutant carrier, death is the result. In missense mutations such as the T93M methionine substitution for threonine, the phenotype is still serve, but not fatal. Common characteristics include mental retardation, physical deformity, and numerous internal medical complications. Cholesterol is a compound of central importance in the mammalian body and is essential to the production of steroid hormones, myelin sheaths, bile, and cell membranes. Red blood cells are of particular interest to the study of SLOS because of their phospholipid bilayer membranes and lack of organelles. Cholesterol plays a special role in cell membrane function by controlling the fluidity of the phospholipid bilayer and maintaining cell membrane equilibrium at varying temperatures. In SLOS patients and in mouse models of SLOS, 7DHC is used as a substitute for cholesterol in the brain and liver, and the ratio of 7DHC to cholesterol has been found to be abnormally high. Thus, it is likely that the ratio in red blood cells will also be irregular. Learning how SLOS affects membrane structure and function in red blood cells could lead to a better understanding of the toxicity of 7DHC. It is thus far unknown whether or not the symptoms of SLOS are caused by a deficiency of cholesterol, an increase in 7DHC, or a combination of the two. Objective: The aim of the project is to compare the erythrocytes in mice that are affected by the T93M missense mutation to erythrocytes that are considered normal. Both the physiological characteristics and biochemical composition will be measured. Experimental Procedures: Five pairs of T93M/+ heterozygous female mice and T93M/T93M homozygous male mice and a T93M/T93M homozygous female mouse and a T93M/+ heterozygous male mouse were bred to produce a projected progeny of 50% T93M/T93M with a SLOS phenotype and 50% T93M/+ with a normal phenotype. The pups were then decapitated within twenty-four hours of birth, and their blood and remaining tissues were collected. Approximately 50 micro liters of blood were obtained from each sample. DNA from tails was used to genotype the newborn pups, and the blood was used in both hematology and mass spectrometry. To determine the physiological effects of SLOS on red blood cells, the blood was tested for red blood cell count, cell volume, and hematocrit analysis using an Advia 120 Hematology System. The mass spectrometer was used to determine the ratio of 7DHC to Cholesterol, which can be derived from quantitative measurements of 7-dehydrocholesterol and cholesterol following chemical extraction of sterols from the mice’s red blood cells. Results: The six pairs of breeders produced twenty five mutant mice and sixteen control mice. Two more were born dead but were not genotyped. Blood collection proved to be more challenging than expected, yielding less blood than anticipated. Smaller samples of RBCs had to be used for the hematology lab, and the results concluded that the physiological characteristics of normal and mutant red blood cells did not differ substantially. The ratio of 7DHC to cholesterol was detectable but not drastically high. Conclusion: The erythrocytes of mice affected by SLOS do not differ substantially from the erythrocytes of normal mice. This implies that, unlike liver and brain cells, red blood cells selectively exclude 7DHC.

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Uchechukwu Eke

Funded by the NIH/NHLBIDiversity Student Program

University of California BerkeleySecond Year

Mentor: Steve Mack, PhD

While growing up I always knew that whatever I did when I got older I wanted to help serve others. Seeing my parents pack up all my siblings old things on their trips back to Nigeria and explaining that they were taking them to an orphanage for children who had lost their parents to disease was great, but what was more influential was reading the thank you notes they brought back with them. Reading the words of the children they had helped; their hopes, their dreams, and their gratitude helped me to understand that by serving others my parents were serving themselves. Their good works not only helped these childrens’ immediate future but helped inspire them that a brighter future was possible, they inspired me too. As I went through high school and started college, different experiences led me to pursue a career in healthcare serving others. I am still on my journey pursuing a place in healthcare and my internship in the CHORI Summer Medical Student Program has without a doubt helped to broaden my horizons on what a career in healthcare can be. The wonderful people I have met in the Erlich Lab have taught me so much about what a career in biomedical research is like and it has especially been great to actually see some of the science that I have just memorized and learned about in the classroom applied in a real world setting. For me, this experience has been a priceless one, the researchers here at CHORI definitely serve their community in ways that are vital and of great importance and I can only hope to one day be as good a servant for my own community.

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Genetic Relations Between Siberian and Native American Populations Investigated by HLA Genotyping

U.R. Eke (1), M. Schanfield (2), H.A. Erlich (1,2) & S.J. Mack (1)(1) Children’s Hospital & Research Center at Oakland, Oakland, CA; (2) George Washington University, Washington D.C.; (3) Roche Molecular Systems, Pleasanton, CA

Background. Modern Native Americans populations are theorized to be descended from peoples who migrated to the Americas from Asia over 12,000 years ago; modern Siberian populations could represent the original gene-pool from which Native groups arose. Objective. We generated high resolution HLA genotype data at the DRB1, DQA1, and DQB1 loci, and used these data to estimate DRB1:DQA1:DQB1 (DRDQ) haplotypes in two Siberian populations. We compared this allele and haplotype data to published data for Native American and Asian populations in search of similarities in the allele distributions between Native American and Siberian populations (e.g., DR4, DRB1*0802, *1402, *1602, and *0901 are the principle DR alleles observed in Native Americans). Methods. Purified genomic DNA from 35 individuals from the Siberian Ket and Evenki groups were PCR amplified for the HLA-DRB1, DQA1, and DQB1 loci and genotyped using reverse-format SSOP reagents and StripScan software (version 5.9). DRB1 allele frequencies, and DRDQ haplotype frequencies for Native American, East Asian and other populations were obtained from NCBI’s dbMHC database, and the Pypop.org population database. PyPop (Python for Populations genetics, v0.7.0rc3) was used to generate DRDQ haplotypes, and PHYLIP (v3.67) was used to generate phylogenetic trees comparing Ket and Evenki DRDQ haplotypes with 221 other populations from around the world. Results. The Ket/Evenki populations displayed increased DRB1 diversity relative to Native American populations, with little overlap with common Amerindian DRB1 alleles; several DR4 alleles as well as DRB1*0901 and *1402 were observed in the Evenki. Twenty distinct haplotypes were observed for the Ket and 11 for the Evenki; of these, 6 were common to both groups. Phylogenetic analyses revealed the Ket and Evenki groups to be in the same clade but related to different groups. The Kets are close to indigenous groups in South America (Ecuador) and Mexico (Mixtec), whereas the Evenki were closer to Siberian (Chukchi, Nivkh, and Koryak), Central Asian (Khots and Nganas), and northern American (Eskimo) groups. A distinction between the Ket and Evenki clade and a clade including many of the Native American groups (Sioux, Ticuna, Colombia, etc) was noted. Conclusions. The identification of common DRDQ haplotypes validates the HLA-typing and the haplotype estimations. The Evenki and Ket groups are related to other Siberian/North Asian populations, again validating the genotyping. Although, relations between the Kets and indigenous groups in South/Central America and relations between the Evenki and indigenous groups within northern North America show support for the hypothesis of genetic relations between Siberians and Native Americans, the pattern of DRB1 allelic diversity seen in Native American populations is absent from these Siberian groups. Finally, the notable distinction between the Siberian/Central Asian clade and the clade of native North and South Americans indicates that the overall reduction in DRB1 diversity seen in Native Americans is not present in the Siberians. Native American groups may be descended from a different historical Asian population than the one that gave rise to modern Siberians or they may represent a small group that broke off from the ancestors of modern Siberian groups, with a distinct reduction in HLA-DRB1 diversity.

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Edward Escamilla

Funded by the NIH/NHLBIDiversity Student Program

San Francisco State UniversityFirst Year

Mentor: June Tester, MD

I am privileged to be attending a University and want to thank CHORI for giving me the opportunity to do research that will benefit the community. I am a Chicano born in Stockton CA, raised in a farm in Holt CA as a day laborer. I am the only individual in my immediate family that was born in the United States my family members are immigrants from Guanajuato Mexico.

As far back as I can remember my favorite subject has always been science. I am blessed to have always known what my passion was but recently I have developed another passion which is empowering my Raza/Latino community. I am a part of La Raza Student Organization on my campus which was established in 1968. Thankfully CHORI is giving me the opportunity to do research in Fruitvale which is part of Oakland CA where there is a large Raza/Latino population. I am majoring in Cell and Molecular Biology and Raza Studies.

I have many long-term goals but there is not one specific way to reach them. The opportunities available for me after I receive my degree are many and I would like to keep them that way for now so I will not limit myself to a certain aspect of science or my Raza/Latino community. The research I am doing with CHORI is certainly an eye-opening experience, one which I will benefit from for the rest of my life as I intend to be a life-long learner.

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After School Food Environment and Purchasing Patterns

E. Escamilla & J. TesterChildren’s Hospital & Research Center at Oakland, Oakland, CA 94609

Background: Childhood obesity and related maladies like diabetes are soaring, especially among Latinos. Pushcart vendors selling fruit, ice cream, and paletas (fruit bars) are a common sight in primarily Latino areas. Objective: To determine the purchasing habits of children afterschool, as well as to assess the children’s willingness towards purchasing healthy items and snacks in a mobile food vending setting. Methods: Intercept interviews were conducted upon the children’s exit of class, or after a mobile food cart purchase. The interviews were conducted alongside mobile food vendors with their permission, and a selection of healthier choices were presented to the children completing the interview for them to select from, as a “hands on” approach. Results: Their were seventeen interviews conducted with children. Eighty-two percent of the children stated that they consumed fast food at least once a week, on average two and a half times per week. However there is enthusiasm for a healthier change, all children selected at least two items of the sixteen in the selection and on average four and a half selections, with the most popular being orange juice selected by fifty-nine percent of the children. Conclusion: Based on these findings, along with the alliances of the mobile food vendors an intervention will take place during the children’s next fall school year, stocking a mobile food vendor with the most favored items by children within their average price range.

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Derek Fukumori

Volunteer

Berkeley High SchoolJunior

Mentor: Mark Shigenaga, PhD

The CHORI program has been a great opportunity to bring my interest in science, which until now has been confined primarily to the classroom, into an environment in which results are produced that resonate in the world outside of textbooks. I am largely uncertain as to what I want to study once I leave high school, and my intent going into this program was to try my hand at something that might be a large part of my academic future in college and beyond. Working with Dr. Shigenaga and everyone in the lab has given me a unique and amazing insight into what an eventual pursuit of biology could entail. I am thankful for the opportunity to have been a part of research that could potentially have important implications in general health. This is definitely not something I could have ever accomplished in a high school classroom. For me, taking part in the CHORI program has served a double purpose: It has both gratified my interest for the sciences and provided an opportunity to explore a possible future academic and career path.

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Modulation of Gut Bacteria on Apolipoprotein Expression and Plasma Lipoprotein

Subclass Profiles in a Murine Model of Diet Induced Obesity

D. Fukumori & M. Shigenaga

Children’s Hospital Oakland Research Institute, Oakland, CA 94609

Introduction: Ongoing studies in our lab identify gut bacteria as a possible cause of the systemic chronic inflammation associated with cardiovascular disease. There is evidence suggesting that exposure of the gut to high fat irritates the epithelial barrier of the small intestine and results in increased intestinal permeability, which may lead to a rise in systemic endotoxin levels, and an increase in protective immune responses at various extraintestinal sites. Apolipoprotein expression may be affected as part of a heightened immune response since many of these proteins that circulate in the form of lipoprotein particles protect the body from excessive immune activation, in part, through binding and neutralization of bacterial endotoxin. An increased expression of apolipoproteins may be connected to cardiovascular risks, as certain of these apolipoproteins are linked to atherogenic lipoprotein subclasses. Connecting gut bacteria to apolipoprotein expression, lipoprotein subclass levels, and plasma biomarkers of disease risk may improve our understanding of the relationship between diet and disease risk. This effort may also help identify agents in our diet that minimize endotoxin penetration and the chronic inflammation increasingly linked to diseases common in western societies. Objectives: 1. To quantitate the expression of endotoxin binding apolipoproteins E, A-I, A-IV, and C-I in various mouse tissues from samples acquired in a study designed to evaluate the effect of a high fat diet and interventions that may reduce the metabolic impact. 2. To quantitate the HDL and LDL subclasses from the above mouse study using a newly developed ion mobility technique established by Dr. Ronald Krauss and colleagues. 3. To perform correlation analysis comparing apolipoprotein expression to the amount of HDL and LDL lipoprotein subclasses present in the mouse plasma samples and to previously acquired clinical measures (i.e. glucose, insulin, leptin, insulin resistance, etc.) in an attempt to identify relationships among these host responses. Methods: We assessed the expression of apolipoproteins in various tissues via quantitative PCR using a set of apolipoprotein primers targeted to mouse Apolipoprotein E, A-I, A-IV, and C-I. In ion mobility analysis of lipoprotein subfractions, mouse EDTA-plasma samples underwent sequential ultracentrifugation in order to isolate lipoprotein classes VLDL, LDL, and HDL. Dialyzed LDL lipoproteins were further subfractionated into seven classes and particle size and number characterized by ion mobility by and compared to that of human plasma . Results: For many of the observed apolipoproteins, expression dropped with exposure to a high fat diet. This observation contradicted our original hypothesis, which predicted a rise in apolipoprotein expression as part of the host immune response to endotoxin. There was a notable positive correlation between the expression many of the apolipoproteins and that of Ppargc1a, a protein associated with mitochondrial biogenesis. We speculate that long term exposure to a high fat diet impairs mitochondrial function, leading to, among other things, a decreased capacity for apolipoprotein expression, an outcome that may render the host more vulnerable to the deleterious metabolic effects of bacterial endotoxin. The expression of several apolipoproteins in various tissues was brought to normal or near-normal levels in animals treated with gut bacteria-inhibiting agents in the diet, supporting the claim of endotoxin as a source of inflammation and potential mitochondrial impairment.

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Kristen Glass

Volunteer

Bentley High SchoolJunior

Mentor: Ellen Fung, MD

For as long as I can remember, science has been a passion of mine. My interest in science be-gan as a child while attempting to classify certain species of birds and plants while on hiking trips in Yosemite National Park. Ever since then, i have been devouring scientific magazines such as Discover and Popular Science. My passion has continued through my high school education, learn-ing about esthers, the functions of the human body, genetics, and interactions between different chemicals.

I am honored to participate in the Children’s Hospital Oakland Research Institute (CHORI) summer internship program. I know that it will prepare me for my senior year at Bentley High School as well as my dream to become more involved in the field of medical research. Under the guidance of Dr. Ellen Fung, I have learned about clinical research.

My project on the impacts of trace element deficiencies in individuals with hemoglobinopathies (specifically Thalyssemia and Sickle Cell Disease) is fascinating and important for determining con-nections between trace elements and symptoms in patients. I want to thank Dr. Fung for teaching me how to work with clinical machinery, interpret scientific data, utilize medical resources, and for patiently answering all my questions. I would also like to take this time to thank Dr. Aenor Sawyer who inspired me to pursue a career in the medical field as well as Dr. Lubin, Dr. Staggers, and Dr. Narayanaswami for providing this wonderful opportunity.

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Correlating Trace Element Status with Low Bone Mass in Patients with Thalassemia and Sickle Cell Disease

K.N. Glass & E.B. FungChildren’s Hospital & Research Center at Oakland, Oakland, CA 94609

Introduction. Individuals with trace element deficiencies and patients with hemoglobinopathies often present with similar co-morbidities such as low bone mass, hypogonadism, growth deficits and hypothyroidism. Previous research conducted primarily in the Middle East has shown that many patients with Thalassemia (Thal) have reduced serum zinc (Zn) and elevated copper (Cu). Similar work conducted in Jamaica has purported that patients with Sickle Cell Disease (SCD) are also at risk for trace element deficiencies. Furthermore, it is known that individuals who are severely deficient in Zn present with growth retardation, delayed maturation, and hypothyroidism. Severe Cu deficiency results in osteopenia and growth failure, and selenium (Se) deficiency is related to muscle weakness. However, there is a paucity of studies conducted in contemporary samples of patients in the U.S. with hemoglobinopathies that focus on trace element status and its relationship to the specific co-morbidities mentioned above. Objective. The objective of this project was to test whether patients with Thal and SCD at CHRCO have an in creased prevalence of trace element deficiencies (in specific zinc, copper, and selenium) and if abnormal levels of these nutrients are related to low bone mass, hypogonadism, growth deficits and hypothyroidism. Methods. Data previously collected from research studies of Zn and iron status at CHRCO were used. Fasting morning blood samples (Zn, Cu, ferritin), bone density by DXA (spine, hip, wb) and liver iron concentration by SQUID were collected in patients with Thal and SCD. Clinical data regarding iron status, fracture and medication history and endocrine function were obtained by chart review. A healthy control group was also recruited and similar assessments made. Results. 85 patients with Thal (Mean age: 19.3 ± 10.6, 48 Male), 32 with SCD (age 23.2 ± 16.8, 18 M), and 21 Controls (age 20.2 ±5.9, 9 M) were evaluated in this study. Patients with Thal had slightly though not significantly lower serum ferritin (2814 ± 2784) compared to SCD (3404 ± 2450), whereas LIC was similar (2269 ± 1456 vs. 2246 ± 1558). Mean plasma zinc was not significantly different between Thal (88.4 ± 13.9 ug/dL) and SCD (95.3 ± 114), however a much higher proportion of patients with SCD had levels that were considered deficient: 7% of Thal vs. 44% of SCD (p<0.001). Mean serum copper was significantly higher in SCD (108.9 ± 28.5) compared to Thal (90.0 ± 26.4, p=0.002) and Controls (86.1 ± 30.1, p=0.018). Only 6% of SCD had Cu levels that were considered deficient, compared with 21% of Thal (p=0.06) and 24% of controls (p=0.1). Whereas, 23% of SCD, 14% of Thal, and 33% of controls had elevated Cu levels. As for selenium, approximately 15% of Thalassemia and 13% of Sickle Cell subjects were low. Low bone mass was extremely prevalent in both Thal and SCD, 62% and 41% respectively compared to l ess than 10% of the healthy controls. Diabetes (n=8), hypothyroidism (n=12) and growth hormone deficiency (n=9) were only found in the Thal group. Neither circulating Zn, Cu nor Se were significantly correlated with BMD Z-scores. Conclusion. These results suggest that there is a high prevalence of zinc deficiency, particularly in SCD. Low copper levels are surprisingly pervasive in Thal, but also in the healthy cohort. As anticipated, the majority of the patients with Thal and SCD have low bone mass, however, in this small cross-sectional study, we did not find any relationship between circulating zinc, copper or selenium and low bone mass. The strongest predictors of low bone mass in Thal were growth hormone deficiency, diabetes and hypothyroidism (all p<0.01).

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Mohammad Hajighasemi

Funded by the NIH/NHLBIDiversity Student Program

St. Mary’s CollegeSecond Year

Mentor: Horst Fischer, PhD Beate Illek, PhD

My passion for science and medicine did not develop when I was a young child. Like many children I spent hours exploring and hiking through my backyard while avoiding my mother to not be called back into the house to study. Even though I had a desire to learn I did not enjoy sitting at a desk to study. My mother developed an ingenious plan to deal with my desire to play and my desire to learn. My mother allowed me to climb on the living room furniture while I recited my multiplication tables and my weekly vocabulary words. Thanks to my mother’s plan I succeeded in my elementary school studies. I don’t climb on the living room furniture anymore, but my passion to explore and learn still lives within.

My academic interest in the sciences developed during my freshman year at Saint Mary’s College of California. For the first time in my life I was not asked to memorize facts to show my competency in the sciences. I quickly discovered that Saint Mary’s was different; I had to learn fundamental principles and apply them to specific cases. The application of principles and the practice of critical thinking inspired me to become a Biology major. It also allowed me to expand my interests and minor in Theology and Religious Studies. My personal interest in airway diseases coupled with my interest in human physiology inspired me to participate in the CHORI summer research program.

Researching with Drs. Horst Fischer and Beate Illek has been an amazing experience. Both have challenged me to think critically and apply the concepts I have learned in my science courses. Most importantly I now understand the connection between basic science research and the practice of clinical medicine. It is gratifying and remarkable to know that my research is contributing to the understanding of the airway disease Cystic Fibrosis. I would like to thank Dr. Horst Fischer and Dr. Beate Illek for answering my list of daily questions and challenging me to research with the same intensity and dedication as they do.

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Regulation of Airway Proton Secretion

M. Hajighasemi, H. Fischer & B. IllekChildren’s Hospital Oakland Research Institute, Oakland, CA 94609

Introduction. The human airways are continuously under attack by infectious particles. With every inhaled breath of air thousands of microbes viciously attempt to infect the lung. The airway epithelium contains an innate defense system to prevent lung infections. The airway epithelium is covered by an airway surface liquid (ASL), which is composed of a watery sol layer and a mucus blanket. Inhaled particles stick to the mucus blanket and are propelled by the cilia; ultimately the particles are swallowed or expectorated. It is believed that an acidic ASL is required for the innate defense system to properly function. However the ASL pH changes when the human body has a disease. For example, people with cystic fibrosis (CF) have an estimated ASL pH of 6.0, while people without CF have an estimated ASL pH of 6.6. CF is a hereditary disease caused by a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. This mutation adversely affects the functioning and existence of the CFTR ion transporting protein. The hyperacidic ASL of people with CF causes the lung’s defense system to improperly function, which makes people with CF more prone to lung infections. The mechanisms of ASL pH regulation are currently not well understood. Specific transmembrane transport proteins have been proposed as contributors to ASL pH regulation, however the characteristics of each protein are also not well understood. Objective. The objective of this research project was to investigate the characteristics of ASL pH regulation. The specific goals of the research project were to 1) identify the ASL pH at which the airway epithelium starts to secrete protons, 2) investigate the pH dependence of the airway epithelium’s H+ secretion and the role of the HVCN1 proton channels, and 3) determine the effect of H

2O

2 on the airway epithelium’s H+ secretion. Understanding the

characteristics of ASL pH regulation will aid in understanding illness in which the ASL pH is not properly regulated, such as CF. Methods. A human bronchial epithelial cell line (CFBE41o-) and primary human tracheal epithelial cells (HTE) were investigated with Ussing chambers. Ussing chambers simulate the lumen and blood side of the airways. The rate of protons secreted by each cell type was measured on the lumen side with a pH electrode. The pH of the lumen solution was adjusted from the pH equilibrium to pH 7.4, 8.0, and 8.5 at specific time intervals. The rate of H+ secretion was recorded for each pH value. Subsequently 10 µM of ZnCl

2 was added to the

lumen side and the pH was increased in the same manner at specific time intervals. ZnCl2 is a

known HVCN1 H+ channel blocker, its use allows for the identification of the HVCN1 protein in the recordings. In a separate experiment the lumen H

2O

2 concentration was adjusted by adding 1 µM,

10 µM, 100 µM and 1000 µM of H2O

2 at specific time intervals. The rate of H+ secretion at each

H2O

2 concentration was recorded. Results. We found that the airway epithelium starts to secrete

protons at pH 7.190 (range 7.106 – 7.274) for the CFBE41o- cells, and at pH 5.70 (range 5.65 – 5.75) for the HTE cells. It was discovered that the rate of H+ secretion by the airway epithelium and the ASL pH displayed a positive correlation for both cell types. On the other hand, a negative correlation was discovered between the rate of H+ secretion by the airway epithelium and the ASL H

2O

2 concentration for both cell types. We found that the HVCN1 H+ channel contributes 32.8% to

the total cell H+ secretion for the CFBE41o- cells, and 30.4% for the HTE cells. In both cell types a positive correlation was discovered between the rate of H+ secretion by the HVCN1 H+ channel and the ASL pH. Conclusion We found the ASL pH at which the airway epithelium starts secreting protons and discovered that all epithelial cells do not secrete protons at the same ASL pH. We discovered the pH and H

2O

2 dependence of the airway epithelium. Also, we have determined the

quantitative contribution of the HVCN1 H+ channel to the total cell H+ secretion.

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Danielle Jackson

Funded by the NIH/NHLBIDiversity Student Program

University of New MexicoFourth Year

Mentor: Patsy Wakimoto, PhD

I am Danielle Jackson and just finished my fourth year at the University of New Mexico. This upcoming year I hope to graduate with a degree in Biology and Psychology. Future goals include going to medical school and eventually coming back to my Native American community. Until this summer I had never heard of CHORI but thanks to a great friend who had previously done the program, I gained valuable knowledge and experience at this great institution. This program has enabled me to step closer to my goals and for that I would like to thank all the people who have helped me along the way this summer. Thank you, Patsy, Mary, Lisa and Sua for welcoming me into the lab and teaching me about this unknown world of community-based participatory research.

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Sociocultural Factors Contributing to Native American Women’s Health in Northern

California Greater Bay Area

D. Jackson & P. WakimotoChildren’s Hospital Oakland Research Institute, Oakland, CA 94609

Introduction: The 4.3 million Native American and Alaska Natives residing in the United States constitute approximately 1.5% of the total population and about 67% live in urban areas. Native Americans report significantly greater stress (37%) than non-Hispanic whites (30%). Native American women have higher death rates due to diabetes (45.4 vs. 22.4 per 100,000), all types of accidents combined (38.1 vs. 22.0), motor vehicle accidents (22.7 vs. 10.5), chronic liver disease and cirrhosis (20.5 vs. 6.1), alcohol abuse (20.3 vs. 3.5) and suicide (5.2 vs. 4.4). Native communities have some of the highest levels of cardiovascular risk factors of any ethnic group. Lifestyle factors tied to socioeconomic status clearly play a role in many of the higher disease burdens. Objective: The specific aim of this project was to examine and identify stressors that impact on health in Native American women in the urban settings. Methods: The study sample consisted of self-identified Native American women from Northern California Greater Bay Area. The women were recruited from the Oakland Native American Health Center who were accessing services or were currently employed at the center. Individual interviews were conducted to identify stressors and the interviews lasted approximately 45 minutes. A questionnaire was developed based on stress-related issues identified in the research literature, including topics such as discrimination, racism, and sexism. The participants shared personal experiences, feelings and viewpoints. Transcripts and notes were reviewed and analyzed; common themes, major points and notable comments were identified. Results: Subjects (n=15) ranged in age from 20 to 67 years old, with a mean age of 43.7 years. The length of residency in the Greater Bay Area was 12 years to life and the average number of people in the household was 4. The mean level of education attained was 12th grade. Of the 15 women interviewed, 7 were currently unemployed, retired or on disability. Preliminary results indicated that for the majority of the urban native women, the top two stressors were (1) worrying, caring, and providing for children and family and (2) lack of adequate finances. Other identified stressors included isolation, sexism, identity issues and oppression. There is an underlying continuing cycle of poverty, stress and poor health. Conclusions: The results indicated that more adequate urban health care, with comprehensive services including mental health services, is critical to the survival of urban Native Americans. More research is needed to better understand the underlying sociocultural factors that impact on Native American women’s health and contribute to health inequities.

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Tiffany Kim

Funded by the American Pediatric Society

University of Illinois at ChicagoMedical Student

Mentor: Paul Harmatz, MD

My name is Tiffany Kim and I will be a second year medical student at the University of Illinois at Chicago. However, my heart will always remain in Northern California and where I grew up in Santa Rosa. I attended UC Berkeley as an undergraduate and my major was molecular cell biology with a minor in music. Berkeley was where I first discovered my interest in research. The summer after my sophomore year, I worked at a state health laboratory and I learned how molecular biology could be used to help the public. For the next two and a half years, I worked at the Lawrence Berkeley laboratory studying DNA repair proteins. My senior thesis evaluated the use an unusual model organism called Alvinella pompejana to study a difficult, but important DNA repair protein called XPD. Although the project was very challenging, I ran my own experiments, solved unexpected problems and wrote a paper about the results with much help from a wonderful mentor. It was a rewarding and enlightening experience in which I learned how science is conducted firsthand. I decided to apply to the CHORI summer program because I wanted to continue research but a clinical setting, I enjoyed volunteering at the hospital as an undergraduate, and I love working with kids.

Besides studying all the time, I also play the violin and enjoy all forms of music. I would like to thank everyone involved in the CHORI summer program for giving me the opportunity to do clinical research. I would especially like to thank Dr. Harmatz for being a wonderful mentor, Ellen Fung for her help on the MPS bone project and Jo Ann Johnson, Jacqueline Madden and Jennifer Shue for being great coworkers.

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The Role of Functional Assessments and Therapies for Mucopolysaccharidosis Patients

with Carpal Tunnel Syndrome

T. Kim, K. Gibson & P. HarmatzChildren’s Hospital & Research Center at Oakland, California, 94609

Introduction: The mucopolysaccharidoses (MPS) are a group of disorders resulting from missing enzymes in glycosaminoglycan (GAG) degradation. Intralysosomal GAG accumulation in connective tissue develops into symptoms such as contractures, stiff joints and carpal tunnel syndrome. Traditionally, clinicians treat MPS patients with hand complications by close monitoring via nerve conduction studies. The purpose of this study is to demonstrate the use of new measurements and treatments for patients with MPS. Clinical presentation: We will report on an 11-year-old male patient with MPS II who has finger contractions and suspected carpal tunnel syndrome. We began following our patient a year and a half ago, by evaluating motor and sensory function of his hand by nerve conduction studies. Additional treatment was performed by a certified hand therapist. The therapist evaluated the patient using the Weinstein Enhanced Sensory Test (WEST), which determines light touch-deep pressure tactile cutaneous thresholds. Active joint range of motion was also measured to follow changes in joint flexibility. Therapy consisted of neutral and dynamic splinting, a paraffin unit, a writing aid and iontophoresis, which uses electrical currents to deliver corticosteriods to the wrist. Since beginning treatment, our patient showed increases in degrees of flexion in his metacarpophalangeal joints, or knuckles, by an average of 36% in the right hand and 44% in the left hand. Although the WEST showed slight improvements in sensory thresholds, it was more faithful in reflecting the patient’s sensory status than the nerve conduction study that showed no sensory function in the median nerve. Discussion: For our MPS patient, therapy has improved joint range of motion and provided tools like the writing aid, both of which increase functionality of the hands. We have seen WEST to be a reliable, simple and consistent test which carries almost no risk to the patient compared to the potential anesthesia complications associated with difficult patients undergoing a nerve conduction study. Based on our results in this case study, we recommend evaluation of these techniques and therapies in a larger population for monitoring and treating MPS patients with hand complications.

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Aemed Komarizadeh

Funded by the NIH/NHLBIDiversity Student Program

University of California BerkeleyFourth Year

Mentor: Barbara Staggers, MD

I initially applied to the program because I wanted to see if medicine was the right career choice for me. I was also interested in biomedical research so naturally the CHORI summer program caught my attention. I was excited about being given the opportunity to work with medical doctors who also did research. During the summer program I have learned so much about different aspects of medicine and also about myself. My time working along side Dr. Barbara Staggers at the teen clinic has left my eyes wide open to adolescent medicine. I have come to realize how important different factors play in a child’s mental, social, and physical health. I am so excited to realize how much there is to learn and it has been very empowering for me to realize that I’m med school material. Thank you so much to the CHORI Program for giving me this wonderful opportunity.

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Adolescent Resiliency

A. Komarizadeh & B. StaggersChildren’s Hospital & Research Center at Oakland, Oakland, CA 94609

Introduction: Resiliency is the ability to bounce back in the face of adversity. The purpose of my project is to analyze teenage resiliency and the factors which are involved. A little background about why I chose this topic was because in the US the three leading causes of death among teenagers are Motor vehicle accidents, Homicide, and Suicide. 70% of the deaths of teenagers in this country are caused by one of the above mentioned. The interesting aspect about adolescent medicine in dealing with this issue is that these causes of death are all preventable. The interesting question is how do we prevent it? We have to realize that teenagers who are recognized as “at risk” are increasingly more likely to participate in behaviors leading to increased sexual activity, gang activity, drug use, and poor performance in school and such behaviors have shown to increase the likelihood of falling into one of the above mentioned categories. Youth development programs have been established as a response to this growing statistic. Objective: The objective of my project deals with determining factors involved in adolescent resiliency. Understanding these factors will allow physicians in adolescent medicine to help prevent their patient from becoming “at risk” youth but instead assist them in getting back on their feet. Methods: A survey was developed looking at age, gender, academic performance, parental income levels, and factors affecting personal resiliency. The sample was taken from CHO Teen Clinic and to a lesser extent UC Berkeley BSP program. These responses were analyzed and similar responses were tallied. Results: Approximately, 54% Friends/Family. 20% Money. 15% Mentors. 7% Exercise. 3% Other. These were honest responses from teens which they felt kept them resilient. In order to develop a successful program whether it is a youth development program or a teen clinic, these factors need to be put as a priority when dealing with teens. There needs to be first and foremost a family atmosphere where teens feel safe. Next there needs to be some sort of academic stipend to help ease financial strain and of course good mentors. A paradigm already in place is Youth Uprising located in East Oakland. Conclusion: Further research and investigation is necessary if youth development programs are to incorporate our findings with aims of developing resilient teens. I plan to further develop my ideas and research in adolescent resiliency in hopes to develop already existing programs more effective and perhaps develop a youth center of my own.

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Deja Kono

Funded by the NIH/NHLBIDiversity Student Program

Notre Dame de NamurSecond Year

Mentor: Ellen Fung, MD

My name is Deja Kono and I will be entering my junior year of college as biology major. For six years, I have had two “interests” which have each developed into genuine passions: (in no particular order) one is my interest in medicine/biology and the other is my commitment to social justice issues. From my freshman high school course in biology I learned about genetics and gene research. Since then I have had an interest in medical research. For the next three years I participated in an internship called Faces for the Future, here at Children’s Hospital in Oakland. This combined both of my passions since “Faces” not only exposed me to medicine, it was designed to encourage minority/disadvantaged students to not only enter medical fields but also to return to inner urban cities after graduation.

While the Faces program gave me a broad overview of the fields of medicine it did not expose me to medical research. Now, with two years more of biology and science, I felt this summer’s CHORI program would give me a first hand chance to apply what I have learned in the classroom. But I’m getting much more from CHORI than I anticipated! It may appear that I have my “act together”…actually I don’t! CHORI is invaluable in giving me the chance to ask many, many professionals in science and medicine to help me re-set my career goals. Together they are demonstrating to me that there are many paths that I can take to fulfill my passions listed above.

I want to thank Dr. Ellen Fung for her mentorship in both academia and personal life. On one hand, she answers all of my “medical” questions and concerns about Thalassemia and Sickle Cell Disease. More importantly though, she demonstrates living “social justice” as she equally treats all of her diverse patients with such dignity, compassion and nurturing care.

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Dietary Intake Assessment in Patients with Thalassemia and Sickle Cell Disease

D. Kono & E.B. FungChildren’s Hospital & Research Center at Oakland, Oakland, CA 94609

Introduction: Patients with Thalassemia (Thal) and Sickle Cell Disease (SCD) are at increased risk for low bone mass and growth deficits. The majority of clinical care for both Thal and SCD is focused on these issues, with little emphasis on nutrition. Individuals with poor nutritional status have also been shown to have reduced bone mineralization and lean body mass deficits. Therefore, it might be assumed that poor nutritional status would be associated with the presence of these co-morbidities in patients with hemoglobinopathies. However, there have been few studies focused on nutritional status in these patient groups and even fewer, which have made the link between nutrition and organ dysfunction in patients with Thal or SCD. Objective: The aims of this project were 1) to determine the adequacy of growth and bone forming nutrients in the diet of patients with Thal and SCD as compared to the Dietary Reference Intakes (DRI) and healthy control subjects, 2) to correlate the adequacy of nutrient intake with circulating levels of these nutrients, and 3) to relate dietary intake and circulating levels of these nutrients with bone density Z-scores as assessed by Dual Energy x-ray absorptiometry (DXA). Methods: Patients with Thal (n=41) and SCD (n=10) currently enrolled in research studies at CHRCO completed a comprehensive nutrition evaluation, in addition to having their bone density assessed and blood drawn. A healthy control group (n=21) was recruited, and similar assessments made. Dietary intake was assessed using a standardized, validated food frequency questionnaire (Block ©2005 and Block Kids ©2004). Data were analyzed using STATA, version 9.0. Results: There were a total of 72 patients in the study including: 41 patients with Thal (Mean age 17.4 ± 5.2 y, 20 Male), 10 patients with SCD (14.9 ± 3.0 y, 6 M) and 21 healthy controls (20.2 ± 5.9, 9 M). Dietary assessment is summarized below:

Thal SCD Control p-valueCalories, %RDA 121±63 167±69 100±35 NSFat, %kcal 36±6 ** 32±7 * 32±5 * 0.02Protein, %kcal 15±3 ** 13±3 * 16±3 ** 0.03Vitamin A, %EAR 124±87 * 338±190 ** 116±43 ** <0.001Vitamin C, %EAR 183±117 * 338±181 ** 196±120 * 0.002Vitamin D, %AI 73±54 67±41 99±67 NSVitamin E, %EAR 66±36 93±55 56±20 NSCalcium, %AI 68±45 71±37 80±38 NSMagnesium, %EAR 83±45 94±35 90±31 NSCopper, %EAR 197±96 243±101 198±77 NSZinc, %EAR 136±81 154±66 122±49 NS

AI: Adequate intake; EAR: Estimated average requirement

Intake for vitamins D, E and calcium were inadequate as compared to the recommended intake for all three groups: Thal, SCD as well as the healthy controls. Despite seemingly adequate intake of copper, we found surprisingly low circulating levels: 39% of Thal and 23% of controls had low serum copper. Additionally, despite a significant relationship between dietary intake of vitamin D and serum 25-OH vitamin D (r=0.3, p=0.02), 90% of SCD, 63% of Thal and 38% of the controls had 25-OH vitamin D levels that were considered insufficient (<30 ng/ml). We found no relationship between any of these nutrients and bone density Z-scores or fracture prevalence. Conclusion: Despite adequate calorie and protein intake, vitamin D and calcium were particularly limiting in the diets of patients with Thal and SCD. Though we did not observe any relationship between intake and low bone mass, our sample size was small, and our assessment of bone health was limited to DXA. Further attention should be given to micronutrient adequacy in this population, and its relationship to co-morbidities, in particular low bone mass.

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first student abstract Adaira Landry

Funded by the NIH/NHLBIDiversity Student Program

University of California Los AngelesMedical Student

Mentor: Ervin Epstein, MD Po-Lin So, PhD

I have always been the type of person to make big decisions at the last minute. The suspense and uncertainty are oddly both stressors and stimulators. After graduating from Berkeley, I figured that I would study something related to science, in either graduate or medical school. I was not confident enough to decide since I never participated in research or shadowing as an undergraduate. However, a few days before the deadline, I signed up for the MCAT and figured, why not? It’s just a 10-year route that I may or may not enjoy. I figured the clinical aspect of medicine would be more relevant to my interests. Turns out, my intuition was right. I just finished my first year of medical school at UCLA School of Medicine and I have no regrets. The first year has seriously and strangely been the best year of my life. The large amounts of information that will never be learned in time, the cold and eerie anatomy lab, and the 2am study sessions the night before the practical are all worth it. I know most of the students in CHORI are thinking about applying to either medical or graduate school. While you may hear advice that you need to know your career plans early, I want to serve as an exception. Be patient and wait for the decisions to be clear. There is no need to be anxious and choose prematurely. I figure we have the rest of our lives to think quickly and concretely. So for now, enjoy the anticipation of not knowing where we will be in the next 10 years.

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Tazarotene’s Chemoprevention Efficacy on Preclinical Models of Medulloblastoma and Basal Cell Carcinoma

A. Landry, P.-L. So, Y. Gelfand & E. EpsteinChildren’s Hospital Oakland Research Institute, Oakland, CA 94609

Hyperactivation of Hedgehog (HH) signaling causes both sporadic and inherited cutaneous basal cell carcinoma (BCC). The inherited form of BCC, Basal Cell Nevus Syndrome (BCNS, or Gorlin Syndrome) is an autosomal dominant genetic disease in which patients have only one functional allele of PATCHED 1 (PTCH1). PTCH1 protein is a major repressor of HH signaling and loss of function leads to BCC and the childhood brain cancer, medulloblastoma. BCNS patients develop multiple BCCs and a subset (5%) develops medulloblastoma. Similarly, all Ptch1 heterozygous (+/-) mice develop BCC and approximately 10% develop medulloblastoma. Our lab has recently shown that the retinoid, tazarotene, effectively inhibits the development of BCC in the Ptch1+/- murine model of BCC. Tazarotene is a retinoid that is metabolized to tazarotenic acid and specifically activates retinoic acid receptors (RAR)β and g. RARs heterodimerize with retinoid X receptors (RXRs) to regulate retinoid target genes. Other labs have shown that tazarotene is effective against medulloblastoma using xenograft models. Therefore we tested whether tazarotene could inhibit medulloblastoma using the Ptch1+/- murine model in which all mice develop the cancer. If effective, tazarotene could be a potential therapy for both medulloblastoma and BCC for BCNS patients and for patients with sporadic disease. Ptch1+/- mice were exposed to a single dose of X-rays at birth and tazarotene (10 mg/Kg) or vehicle was administered daily (5x/week) from age 2.5 months, the time when medulloblastoma starts to develop in these mice. After 7 weeks of tazarotene treatment, compared to vehicle treated mice, no significant difference in survival was observed. Also, we analyzed RAR and RXR expression by immunohistochemistry. In skin, BCC and medulloblastoma samples, we found that RXRa was highly expressed; RARa was expressed at lower levels, while RARg and β were slightly expressed in some samples but not others. Gli1, a HH target gene was also expressed as expected. From these data, it is not clear whether tazarotene’s ineffectiveness against medulloblastoma was due to 1) unresponsiveness of the cancer to the drug in our model; 2) drug penetration problems across the blood-brain barrier; 3) drug dosage; or 4) whether the RARs and RXRs are present in all medulloblastomas (due to inconsistencies in the staining results). In addition, we have tested whether tazarotene therapy combined with a HH pathway inhibitor cyclopamine could have a synergistic effect against BCC carcinogenesis. Both agents can inhibit BCC as single agents. A BCC cell line, ASZ001, was treated with tazarotene or cyclopamine alone, or in combination. After 48h, using the WST1 Cell Proliferation assay, the combined treatment at the highest suboptimal concentrations (5 mM) resulted in the greatest reduction of cell proliferation, suggesting that a combined treatment of a retinoid and a HH pathway inhibitor may have greater effect than as single agents alone. Further experiments are necessary to confirm these findings.

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Andrew Lee

Volunteer

Duke UniversityThird Year

Mentor: Julie Saba, MD, PhD

I will be entering my senior year at Duke University in Durham, North Carolina, majoring in biology and minoring in chemistry and economics. This is my second summer in Dr. Saba’s lab at CHORI, and I am extremely grateful for the opportunity to continue doing research here for another summer. This summer, I am able to build upon the concepts and techniques which I learned last summer as well as the understanding of how to develop a hypothesis and design the experiments to test it.

My time here at CHORI has brought much greater meaning to the sometimes theoretical concepts I have been learning for the past several years in my science courses at Duke. I am currently applying to medical school, and the chance to do research at CHORI has shown me the incredible importance of research in medicine. In our lab, there is a constant focus on the clinical applications of our research studying the effect of sphingolipids on colon cancer cells. This focus ensures that our research is always of importance to the public at large; it also keeps us captivated.

I would like to thank Dr. Saba, Dr. Fyrst, and all of the other scientists and students in our lab who have made my last two summers so influential and enjoyable.

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Sphingadiene Effect on Akt Signaling in SW�80 Colon Cancer Cells

A. Lee, J. Saba & H. FyrstChildren’s Hospital Oakland Research Institute, Oakland, CA 94609

Introduction: Sphingolipids are extremely important molecules which affect a host of cellular processes, including cell survival, migration, and proliferation. One novel sphingolipid, named the C14-sphingadiene, has been discovered in the common fruit fly, Drosophila melanogaster. In HT29 human colon cancer cells, the sphingadiene has been shown to inhibit cell growth and induce apoptosis, programmed cell death. It does so by preventing the phosphorylation of the Akt protein, thereby inactivating the Akt pathway, a prominent intracellular pathway which controls protein synthesis, cell growth, and apoptosis. Because of the sphingadiene’s ability to inactivate the Akt pathway in HT29 cells, its effect on other cancer cell lines is of great interest in developing anti-cancer treatments. Objective: The objective of my research was to determine the mechanism by which the C14-sphingadiene inhibits growth and causes cell death in SW480 human colon cancer cells. More specifically, I examined whether or not the C14-sphingadiene deactivates the Akt pathway in SW480 cells by preventing the phosphorylation of the Akt protein. Methods: SW480 cells were cultured in Dulbecco’s Modified Eagle Medium with 10% Fetal Bovine Serum and 1% antibiotic. Cells were treated with 5 µM of the C14-sphingadiene in 6-well plates for durations of 5 min, 15 min, 30 min, 1 hr, 2 hr, 2.5 hr, 3 hr, 3.5 hr, 4 hr, 4.5 hr, 5 hr, 6 hr. Western blot analysis was used to probe for Akt, pAkt, phospho-4E-BP1 (eukaryotic translation initiation factor 4E binding protein 1), with signals normalized to Actin. In the Caspase3 assay, SW480 cells were treated in 6-well with 10 µM LY294002, 1 µM Wortmannin, or 10 µM Rapamycin, both with and without 5 µM C14-sphingadiene. Cells were harvested after 18 hours of treatment, lysed and treated with caspase reagent. Light absorption at 405 nm was measured. For mass spectrometry analysis, one 25cm2 flask of SW480 cells was treated for 3 hours with 5 μM C14-sphingadiene; one flask was left as an untreated control. Results: Western blot analysis of protein extracts from SW480 cells revealed no differences in levels of pAkt between samples treated with and without the C14-sphingadiene. However, levels of p-4E-BP1 were found to decrease over time with sphingadiene treatment, with a maximum decrease of 85% at 3.5 hours as compared to control. Results for Caspase3 assay and mass spectroscopy are pending. Conclusion: Unlike in HT29 cells, the sphingadiene does not appear to significantly decrease levels of Akt phosphorylation in SW480 human colon cancer cells. In fact, research has shown that Akt is constitutively activated in SW480 cells. Instead, my data suggests that the sphingadiene affects SW480 cells by decreasing phosphorylation of 4E-BP1, allowing 4E-BP1 to bind to and inactivate eukaryotic translation initiation factor 1, thereby preventing protein translation and inhibiting cell growth. Further research should focus on the exact mechanism by which 4E-BP1 phosphorylation is inhibited as well as other targets of the C14-sphingadiene in SW480 cell growth pathways.

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Sean Lindstedt

Funded by the AmericanPediatric Society

University of NevadaMedical Student

Mentor: Lynne Neumayr, MD

Life is a curious adventure that seems to have a surprise around every new corner you come to. If you had asked me several years ago when I was beginning my high school career what field I could see myself going into, it would have been atmospheric science. For some crazy reason, the atmosphere and the environment it provides for us on this planet has always been a source of fascination and utter humility. However, in my junior year of high school, I took A.P. chemistry which was taught by a teacher that was both thrilling to be around and forthright. I had considerable interest in biology and chemistry secondary to my love of atmospheric science before the start of this course, but by the time it had finished my interest in these fields was solidified.

College came around and I suddenly had the enormous task of selecting a major. Biochemistry seemed like a perfect fit as it allowed me to blend the two areas of science I loved. So, I selected this major and began taking the slew of courses necessary to achieve my dearly coveted bachelor’s degree. My undergraduate journey contained many experiences both inside and outside the classroom that shaped the person I am now. I was fortunate enough to volunteer a good portion of my free time in a pediatric oncology/hematology clinic in Reno, Nevada and I absolutely loved it. At the same time, a good friend of mine was diagnosed with a 4th stage glioblastoma multiforme, essentially a death sentence disguised in indecipherable medical terminology. Seeing her go through the bouts of chemotherapy and clinical trials, organizing a concert to raise money for her exorbitant medical costs and seeing all the amazing children come through the clinic, my focus shifted to medicine.

I am in now in medical school and just finished my first year. I love it and each day that I get to see the work everyone does down here is truly inspiring. I don’t know exactly where I will be for residency and which field I will go into, but I will forever cherish this opportunity given to me in the summer of 2008. Lastly, and surely not least, I must extend my thankfulness and gratitude to Dr. Neumayr, Dr. Kurio, Diane, Shanda, Dr. Vas and Dr. Lubin for this opportunity.

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Prolonged QTc in Chronically Transfused Sickle Cell Disease (txSCD) Patients Compared to that of Non-iron Overloaded SCD Patients

S. Lindstedt, L. Neumayr, G. Kurio, C. Morris, S. Robertson, E. Vichinsky & the Multi Center Study of Iron Overload (MCSIO)Children’s Hospital & Research Center at Oakland, Oakland, CA 94609

Background: QTc prolongation has been associated with sudden death and its prevalence ranges from 1% to 18% in the general population. QTc prolongation has not been well studied in sickle cell patients (SCD). Recently QTc prolongation was found in 11% of SCD patients and also found to be associated with pulmonary hypertension. In this study, ECGs in chronically transfused and iron overloaded SCD patients (txSCD) were compared to a control group of SCD patients. Iron overload has become increasingly more frequent in patients with SCD due to transfusion regimens aimed at stroke prevention and treatment of stroke, pulmonary hypertension (PHT) and recurrent vaso-occlusive crisis. Excessive iron overload can lead to left ventricular dysfunction (LVD) and cardiac arrhythmias. QTc interval prolongation has also been documented in iron-overloaded patients with thalassemia and associated with sudden death. Methods: In this study, ECGs from the Multicenter Study of Iron Overload (MCSIO), a five-year prospective study of the complications in severely iron-overloaded patients with SCD and thalassemia, were analyzed for QTc prolongation. A subset of 96 EKGs from 199 txSCD and 64 control SCD patients were analyzed for QTc prolongation, defined as ≥.450 seconds and borderline as .440 to .449 seconds and was calculated using Bazett’s formula. Echocardiograms (ECHO) obtained in the same year were reviewed for left ventricular dysfunction (LVD was defined as an EF <55% or SF < 28%) and the presence of PHT, defined as a tricuspid regurgitant jet velocity (TRJV) of >2.5 cm/second. Results: There was no difference in mean age (25.3 ± 13.6) or gender (64% female) between the 65 txSCD and 31 control SCD patients. The average ferritin was 3107 in the txSCD with a mean transfusion exposure of 8.5 +/- 5.8 years, compared to 116 in the controls. QTc prolongation was present in 32% of the txSCD and 29% of the control group (n.s.); borderline prolongation was seen in an additional 17% and 13%, respectively. Mean QTc in txSCD was also not significantly different from the controls: .45±.04 vs. 43±.03 (ns). Sinus arrhythmia was common in both groups (11%) and LAE was found in 20%. None of the 68 patients with ECHOs had LVD. 36 patients had TRJV measured and the average QTc in the patients with PHT was .45 ± .04 compared to .43 ± .04 in those without (p=.09). However, the frequency of either prolonged or borderline QTc was significantly higher in patients with PHT (72% vs. 22%, p= .003). No correlation between QTc and age, gender, hemoglobin, WBC, or platelet count was found. In the txSCD, QTc was not correlated to ferritin or years of transfusion. Conclusions: QTc prolongation was found in 31 % of SCD patients and borderline prolongation in an additional 16%. QTc intervals ≥ .44 were more common in patients with PHT. Despite reports in thalassemia, transfusion exposure and iron overload were not associated with QTc prolongation in this group of SCD patients. The frequency of QTc prolongation is concerning since SCD patients are often treated with medications known to cause QTc prolongation such as hydroxyurea, erythromycin, and methadone. Further investigation and serial ECGs in SCD patients are needed to confirm these findings.

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Jennifer McLeod

Volunteer

George Washington UniversityThird Year

Mentor: Janelle Noble, PhD

Children’s Hospital Oakland Research Institute is an esteemed institution that has and will continue to pave the way for biomedical research and discovery. As a current undergraduate at the George Washington University, with a major in Biology and a minor in Chemistry, I am glad to have the opportunity to work alongside the talented and motivated research team at CHORI, particularly the Noble Lab, in efforts to reveal the medical secrets of tomorrow. Under Dr. Noble’s mentorship, I am exploring the genetic associations of HLA with Type 1 diabetes, particularly in African Americans, hoping to reveal susceptibility loci. While at CHORI, I’ve gained ample experience in genetic research and am able to contribute Dr. Noble meaningful research efforts.

I have always been interested in the biological aspect of health, looking to understand the vast possibilities in medicine. Also, I enjoy the interactive cooperation and collaborative development of research. My summer with CHORI, has allowed me to not only contribute to the progress of medicine but also continue exercising my passion for research.

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Genetic Associations Between Type 1 Diabetes and HLA-DP Genes Among African Americans

J. McLeod, J. Lane, D. Nguyen, R. Kathail, B. Flores & J. Noble Children’s Hospital Oakland Research Institute, Oakland CA 94609

Currently the Noble lab is using molecular genotyping techniques to uncover susceptibility allele and haplotypic combinations among the HLA gene region that display an association with the type 1 diabetes, particularly in understudied populations such as African Americans. The HLA genes are highly polymorphic, showing a high degree of variance. However, different ethnic groups show patterns of particular allele combinations so it is essential to study these genes with respect to ethnic backgrounds in order to better understand their distribution. During my internship, I aided with both the biological and clinical aspect of this research. For the biological portion of my project, I genotyped the class II HLA-DP genes from population-based control samples. We obtained the control genetic samples from anonymous newborn African-American bloodspot cards, which we were given permission by the state to use. From these samples, I genotyped the DPA1 and DPB1 genes, whose gene products combine to form the DP molecule. From my work I was able to identify the genotype of 192 samples at the DPA1 and DPB1 loci, determining the allele frequencies among the sample set, which will later be compared to the frequencies of the case samples from the same locus. For the clinical portion, I worked with the pediatric nurse practitioner, Betty Flores, at Children’s Hospital and Research Center Oakland to help introduce the research study to potential participants, particularly African Americans and other understudied populations, and facilitate consent to use their genetic samples. Currently there are currently 95 patients enrolled, of which 17 are African Americans. In addition to helping enroll subjects into the study, I also helped establish an electronic database to better track current samples and find new ones. Overall, my work this summer with the Noble lab contributed to the final goal of genotyping the population based African-American cohort and aided with the recruitment of more case samples to expand Dr. Noble’s research. Although I was able to help genotype about 20% of the control set at the DP locus, remaining control samples need to be genotyped, along with the case samples, at the various HLA loci. From my work at CHORI, I have added to the depth of this research, which will hopefully lead to the better understanding and possible treatment of T1D in the future.

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Ashley Moon

Volunteer

Johns Hopkins UniversityMedical Student

Mentor: Ervin Epstein, MD Jean Tang, PhD Tony Xiao, PhD

I would say that my fascination with the biological sciences began in freshman biology laboratory when we had to dissect fetal pigs. I had even come prepared with my very own protective mask, foreshadowing my future desire to become a doctor. During high school, my science interest matured as I took as many AP science classes as I could and then eventually went on to start my undergraduate studies at Johns Hopkins University. I am currently a rising senior, majoring in Molecular and Cellular Biology with a minor in French Cultural Studies. In addition to completing my pre-med requirements, I began my research experience at the Alzheimer’s Research Center at the Johns Hopkins School of Medicine. With a one-year laboratory experience under my belt, I was able to spend my last semester in Paris conducting research at the Institut Pasteur. While developing the strategy for determining the crystal structure of an integrase protein, I was able to pursue my greatest academic interests—science and French—in a very unique environment.

My valuable training at the Institut Pasteur has allowed me to integrate well at CHORI in Dr. Epstein’s lab with my mentors Dr. Jean Tang and Dr. Tony Xiao. My project consists of understanding vitamin D’s effect on inhibiting basal cell carcinoma, the most common type of skin cancer. Oncology has been one of the medicals fields of interest to me ever since members of my family had been diagnosed with ovarian cancer. My experience this summer has given me the chance to learn more about a specific cancer and to help contribute creating a possible treatment for a disease that affects so many.

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Exploring the Mechanism of Basal Cell Carcinoma Inhibition by Vitamin D3 and Statins

A. Moon, T. Xiao, E. Epstein & J. Tang Children’s Hospital Oakland Research Institute, Oakland, CA 94609

Introduction. Basal cell carcinoma (BCC) is the most common type of skin cancer, affecting close to 1 million Americans a year. BCC carcinogenesis occurs due to inappropriate activation of the Hedgehog (HH) signaling pathway, commonly a result of mutational inactivation of the tumor suppressor, the Patched1 (Ptch1) gene. Ptch1 inhibits HH signaling by repressing Smoothened (Smo), preventing the Smo transmembrane receptor from activating downstream hedgehog effectors, the glioma-associated (Gli) family of transcription factors. Gli mRNA is thus a read-out for HH signaling. Our lab has found that vitamin D3 inhibited BCC proliferation in murine cell lines and inhibited BCC tumors in Ptch1+/- mice. Objective. To determine whether the in vitro and in vivo inhibition of BCCs by vitamin D3 and atorvastatin correlates to decreases in Gli1 mRNA. Methods. In order to specifically determine how and when the HH pathway is inhibited, the concentration of Gli mRNA and vitamin D receptor (VDR) mRNA are kinetically measured under different drug applications. Ptch1+/- murine cell lines are established from BCC tumors and are screened with varying vitamin D precursor and derivative drugs, which are removed at different time intervals (12h, 24h, 48h). RNA is extracted and purified from the harvested cells using TRIzol® reagent with the PureLink ™ RNA Mini Kit. Complementary DNA (cDNA) is then produced by reverse transcription (Taqman®, USA). Quantitative PCR (qPCR) subsequently allows for simultaneous amplification and quantification of the cDNA sequence of interest. Due to the primers containing fluorescent tags, relative concentrations of DNA present during the exponential phase of the reaction are determined by plotting fluorescence against cycle number on a logarithmic scale. Housekeeping genes such as 18S rRNA and GAPDH normalize any possible variation in the amount and quality of RNA between different samples.Results. Murine ASZ cell lines were screened with cyclopamine (a known inhibitor of HH signaling), vitamin D3, 1,25(OH)

2 vitamin D3 and atorvastatin (an HMG-CoA reductase inhibitor). The first

three drugs showed significant decrease of Gli mRNA expression relative to controls (DMSO or ethanol) as follows: CPN and vitamin D3 had a sixteen-fold decrease and 1,25 (OH)

2 vitamin D3

had a four-fold decrease during the 24h – 48h time period. Atorvastatin did not show any reduction of Gli mRNA expression. To serve as a positive control, kinetic profiles of vitamin D3 receptor (VDR) target genes and HMG-CoA reductase mRNA indicated high levels of expression when vitamin D3 or atorvastatin were applied to the cell lines. Conclusion. Gli mRNA repression by vitamin D3 and various vitamin D derivative agents correlates with in vitro and in vivo inhibition of BCCs. Most importantly, vitamin D3 is able to suppress Gli mRNA expression that is comparable to cyclopamine. By repressing Gli and HH signaling, vitamin D3 may be a novel chemopreventive agent against BCCs.

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Joyce Nguyen

Volunteer

Presentation High SchoolJunior

Mentor: Lydia Tinajero-Deck, MD

My name is Joyce Nguyen, and I will be a senior at Presentation High School in San Jose this fall. Aside from concentrating on my academic work, I am on my school swim team and am involved in various activities in my community both on and off campus. I have organized blood drives and holiday food drives on campus and participated in hepatitis B screenings and vaccinations in my local community. I also tutor at an all-girls middle school, Our Lady of Grace School, in downtown San Jose. On Saturdays, I usually volunteer at Pacific Free Clinic, which offers free healthcare to individuals with no insurance in San Jose. Seeing all the people who work to make the clinic run smoothly, especially the researchers and physicians, has inspired me to pursue a career in the health field. By researching at CHORI this summer, I am learning more than I ever could in a classroom environment. I feel that this knowledge will bring me one step closer to my career goal of becoming a medical doctor.

I want to thank Dr. Tinajero-Deck, Dr. Tester, Lourdes Juarez, PNP., along with all the staff at the Healthy Eating Active Living (HEAL) clinic where I have been working ever since the summer started. Their dedication to patients and commitment to the HEAL clinic mission never ceases to amaze me. The opportunity to work with each different clinician and to interact with their patients has not only taught me about the problems of obesity in children but also the complexities that accompany it. The disparity in socio-economic classes is evident in healthcare. Patients may come in wanting change their lifestyle, but they lack the proper means to do it. Perhaps they are unable to pay for the program and are forced to dropout. Other times, they do not have access to the same things we may have taken for granted as children – a safe environment to play in, a pool, or fresh fruits and vegetables every day. Seeing all this, I aspire to be a physician who can help patients no matter what barriers they face.

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Perception of Acanthosis Nigricans & Reliability amongst Medical Providers

J. Nguyen, L. Tinajero-Deck, J. Tester & L. JuarezChildren’s Hospital & Research Center at Oakland, Oakland, CA 94609

Introduction: Childhood obesity has become a serious problem in America. Along with juvenile obesity come many other health risks such as cardiovascular diseases and type 2 diabetes. Type 2 diabetes, also known as non-insulin-dependent diabetes mellitus (NIDDM), is an illness that should not be occurring in children and adolescents. This is when the body does not produce enough insulin or the body’s cells ignore the insulin. The more common case in pediatric diabetes is that the body ignores insulin levels. Insulin production in these patients is normal, but the body does not use the insulin properly. Therefore, there is a disparity between insulin production and need. Eventually, the pancreas loses its ability to properly produce insulin. The highest-risk ethnicities of this disease are African Americans, Hispanic/Latino Americans, American Indians, and some Asian Americans and Native Hawaiians or Other Pacific Islanders patients. A condition called acanthosis nigricans that is characterized by papillomatosis and hyperkeratosis of the skin are seen in obese kids at risk for diabetes. This darkening of the skin worsens with an increase in insulin level. It is seen in obese kids, particularly those who are at risk for diabetes. It is important that doctors know where to look for acanthosis nigricans and to recognize it; furthermore, it is important to properly rate the severity of it since there is a correlation between the increase in severity of acanthosis nigricans and an increase in the negative health risks in children. Severe acanthosis nigricans is strongly associated with an elevated BMI, fasting glucose and insulin, systolic blood pressure, and total cholesterol. Acanthosis nigricans is a legitimate screening tool for type 2 diabetes in obese children. For this project, the neck will be singled out to see if there is inter-rater reliability between medical providers on how to classify acanthosis nigricans. Objective: Determine internal consistency of neck severity ratings of acanthosis nigricans between doctors. Find out if more experienced doctors get better scores on the survey, implying that they better understand the dermatologic condition. Educate providers on how to properly diagnose acanthosis nigricans. Methods: A survey was created to be taken by 30 medical providers, ranging from medical residents to attending doctors and nurse practitioners including endocrinologists, cardiologists, gastroenterologists, pulmonologists, and general pediatricians at the Children’s Hospital Oakland and Claremont Clinic. The answers to the surveys were compiled on Microsoft Excel along with the doctor’s level of expertise and patient population information but not his/her name. Extrapolate from these results about how providers are doing with regards to properly diagnosing. An educational component was provided to those who participated in the form of a handout, poster, or email. Anticipated Results & Conclusion: Many medical providers are unfamiliar with how to classify acanthosis nigricans. Previously, some doctors had simply marked if it was present or not on a child’s physical exam, but this project drew their attention to the importance of acanthosis nigricans so that follow-up action could be taken if needed. Some medical providers were unsure as to how to rate acanthosis nigricans severity or underestimated the implications of the condition. The survey created in this project determined if there is reliability between medical providers about acanthosis nigricans severity rating and the dermatologic condition in general. The data shows that there is no significant correlation between years of medical training or experience and the correct rating of acanthosis nigricans. It is projected that those who have a large percentage of obese children and/or a large percentage of patients of the ethnicities that are of highest risk for diabetes in their patient population are able to better rate acanthosis nigricans and point out the places where the condition would occur. The project should ultimately establish consistency in the diagnosis of acanthosis amongst the doctors of the Claremont clinic and Children’s Hospital & Research Center Oakland who are included in the study.

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Shantell Nolen

Funded by the NIH/NHLBIDiversity Student Program

University of California BerkeleyFirst Year

Mentor: Barbara Staggers, MD

My name is Shantell Nolen and I will be a second year at UC Berkeley this fall. I remember the day that my love for science started. It was Christmas 1998, my mother had gotten me a lab set with different chemicals and elements as well as a book of experiments I could try. Ever since then my passion for research has only grown. I took as many AP and honors science classes that I possibly could throughout high school and middle school and I am continuing to do so now as I pursue my degree in Molecular and Cell Biology.

This program has been an amazing opportunity and experience for me. I have been introduced to so many areas of research and other clinical medicine. Being able to work under someone who is as accomplished as Dr. Barbara Staggers and seeing her commitment and passion for her work has been inspiring. Working with adolescents at the Teen Clinic has given me a different outlook on medicine and a whole new perspective on what it takes to make a significant difference with people my own age.

I want to thank CHORI for giving me this chance. I hope I can be involved again next year. I also want to appreciate Dr. Barbara Staggers again for being such a huge influence to me this summer, the environment surrounding the teen clinic and the other CHORI affiliated clinics is more than amazing. Also much thanks to the attendings, 1st year residents and medical students I have met this summer for being so lively and welcoming as well as supportive and willing to teach me.

I hope to go on to get my PhD and possibly a Masters in Public Health. One thing this program has instilled into my head is that “Professional is Personal,” and my great grandmother lived over 10 years with Breast Cancer and she is the sole reason that I hope to get into a career involving Cancer research. I asked for a lab coat every year following that Christmas in 1998, but unfortunately it never came. I see now that wearing that coat is an honor and a privilege, it is proof that you are making a difference and I hope that one day I’ll wear one and make my own difference. .

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Pre- and Post-Behavior of High Risk Adolescents Exposed to Sexually Transmitted Infections

S. Nolen & B. StaggersChildren’s Hospital & Research Center at Oakland, Oakland CA 94609

Introduction. STI’s have become a raging epidemic among people in the US. Adolescents ages fourteen to twenty-four have the highest rates of infection. Of the nineteen million people reported each year with a STI, 3.2 million (26%) are women between the ages fourteen and nineteen. Although 20% of those women are white, more than half of those women are of color, 48% being African American. In Oakland, where the population is mostly Black and Latino, STI rates are regularly high. The Teen Clinic is one of three locations provided by the Children’s Hospital Oakland spread throughout Oakland that is available for adolescents needing STI treatment. Still despite their efforts to encourage contraception, adolescents continue risky sexual behavior and catch STI’s from multiple partners on numerous occasions. Objective. Understand why adolescents who have gotten an STI chose to partake in risky sexual behavior and why some, even after first time exposure, continue to ignore contraception methods and re-infect themselves. The goal is to decrease rates of re-infection amongst adolescents in Oakland and to help develop strategies for better quality of health care. Methods. Orally conduct a survey with adolescents with a history of an STI. The survey will address whether or not they’ve had sex education, their goals, social experiences, home life, and feelings about sex, themselves, and others. Results. 25 oral surveys were conducted. Ratio of women to men that came in for STI treatment is 10:1. 90% of heterosexual and bisexual women report being monogamous at the time they caught their STI. 39 % of those women said they maintained a sexual relationship with that partner and 2/3 managed to catch a second STI from that same person. 55% of women had a history of 2 or more STI’s. More than 75% of women are not sure who gave them a STI. Certain things that trigger having unprotected sex are inexperience, curiosity, convenience, feelings of love and trust, alcohol and sometimes drugs. More importantly 60% of people said they don’t take treatable STI’s or Pregnancy as serious as HIV/Aids or HSV-1 and HSV-2. Discussion. Ratio of women to men makes sense given that the client population at the clinic is 64% women and 34% men. The diversity of the people interviewed caused scattered answers, concluding that a person’s specific lifestyle or age cannot solely predict whether or not they will get an STI. Whether one person is better off than another is, doesn’t play a big role in connection to sexual behavior. However, their reasoning behind their decisions is more closely connected to sexual behavior. Besides lack of judgement during intoxication, most women who continue to sleep with partners they have gotten an STI from say that it is not their partners fault because either one of them might have gotten it from a previous relationship. Both men and women ignore coming in for treatment because adolescents aren’t scared of STI’s. They associate them with being temporary and associate pregnancy with abortions. They fail to realize that treatable STI’s make you more susceptible to HIV/Aids, which they associate with death, and multiple abortions can have side effects on the reproductive system. Possible suggestions to improve �uality of clinical healthcare. Suggest teens get tested before changing partners. Mandate that women with an STI bring their current partner and anyone they’ve been with since they were last tested, in for treatment. This will decrease the likelihood of passing infection from partner to partner and increase the amount of men coming in for treatment. Protocol for talking to adolescents about STI’s should be redirected to link STI’s to HIV/AIDS, birth defects and complications.

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Stephanie Orth

Volunteer

Chapman UniversityThird Year

Mentor: James Betts, MD

Until my junior year of high school I was anything but a scientist. I spent the bulk of my time doing back tucks on floor, basket tosses on Friday nights, and dancing in the ballet studio whenever possible. I planned on going to college simply because my sister went and was sure that I wanted to be an artist partly because I loved to paint, but mostly because I come from a family of artists.

Out of necessity for high school graduation I enrolled in sports medicine and was surprised to discover that I actually had an interest in science. I learned not only the way the body works, but also the impact that pediatric medicine can have on young lives. It was en route to a football game when I decided to inform my mother of my new found career aspirations. Upon hearing the news the car came to a halt on the side of the road and my mother said, “what?!?” In hindsight I suppose the jump from artist to physician may have come as a bit of a shock to her, but at the time the change in career goals seemed completely normal to me.

Although the change was drastic at first, today, a little over four years later my goals for the future have changed, but only slightly. This fall will mark my senior year as biology major with a double minor in chemistry and departmental honors at Chapman University and my second year as a CHORI summer student. For me, the CHORI summer student program is about so much more than the research, it is a chance to become immersed in the medical field and a chance to discover where you fit in. I am nervous about life after college, but at the same time I feel that CHORI and Chapman could not have made me more prepared. Chapman has been a perfect match for me as the small university has allowed me to truly become a leader and mentor on my campus. In the coming year I will be the president of AMSA and SSS, vice president and co-founder of GMB, and an on-campus tutor, just to name a few. I am looking forward to my future as a nurse practitioner and am grateful to CHORI and Chapman for guiding me and opening the door to a future that I never would have imagined a mere four years ago.

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Rate of Infection in Pediatric Patients with Neurogenic Bladder Receiving Sterile Versus Clean Intermittent Catheterization

J. Betts, L. Baskin, A. Hinds, P. Jones, S. OrthChildren’s Hospital & Research Center at Oakland, Oakland, CA 94609

Introduction: Patients with neurogenic bladder experience sensory deficits and loss of voiding control. Intermittent catheterization, IC, which is utilized by this population several times per day can lead to urinary tract infections, UTI. Sterile intermittent catheterization, SIC, involves the use of a new sterile catheter before each use, while clean intermittent catheterization, CIC, involves washing and reusing urethral catheters. While much research has been done to examine overall infection rates during IC for long-term patients, little has been done to compare infection rates between those using SIC versus CIC in a home rather than hospital setting. It is hypothesized that the likelihood of infection will be equal whether the sterile or clean intermittent catheter is used. It is also hypothesized that CIC will be more cost effective and biologically friendly than SIC. Methods: One hundred children with neurogenic bladder who currently use IC to void will be evaluated. Upon enrollment in the study, children will be asked to complete a survey describing; 1) Their diagnosis 2) History of UTI 3) Current intermittent catheterization method 4) How often they catheterize 5) How they clean and store their catheters and 6) Whether they catheterize themselves or are catheterized by their parents. Participants will be randomly assigned to use either the clean or sterile method of catheterization for six months at a time, with enrollment in the study lasting a total of one year. Once every three months, participants will give a urine sample which will undergo a bacterial count. Changes in bacterial count will be noted. If there is a symptomatic infection, participants will be asked to report it and submit a urine sample. Participants will also be asked to complete a daily calendar describing how often they catheterize themselves, how they clean their catheters (if applicable), and ease of comfort with using the study method compared to their usual method of catheterization. Interim conclusions: As the study protocol is in the process of being submitted to internal review board, IRB, no subjects have been admitted to the study at this time. Literary review suggests that incidence of UTI is linked to the number of times a person voids per day. Infrequent urination causes bacteria to grow while urine is still in the bladder. The infected urine is then passed to the urethra which then results in a UTI. As the current recommendation for individuals with chronic UTI is to urinate more often, it is logical that the same recommendation be made to individuals using IC. Significance: Recent MediCal policy states that patients must use SIC, a procedure which will cost $429 per patient per month. The use of this large amount of money to cover the cost of catheters provides no benefit to the patient and money would be better spent on a product or procedure that would actually be beneficial rather than insignificant. In addition, the use of a new catheter for each catheterization results in an unnecessarily large amount of non-biodegradable waste.

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Lauren Pallis

Volunteer

University of California BerkeleySecond Year

Mentor: June Tester, MD

My name is Lauren Pallis and I will be entering my third year at the University of California, Berkeley in the fall. My passion for science, particularly for human anatomy and biology, began during my sophomore year of high school, and since then has only strengthened. I injured my knee that year in school and ended up getting reconstructive arthroscopic surgery on my ACL and meniscus right at Children’s Hospital Oakland. After meeting with my doctor several times, I learned exactly what was going to be done to my knee and hearing about it fascinated me. I was hooked. I took all the biology and anatomy classes I could at my high school and after graduating I knew I wanted to pursue a pre-medical and biology degree at Cal.

The CHORI summer research program was the perfect opportunity to explore the aspect of the scientific realm that I was not as familiar with: research. About halfway through with my pre-med pre-requisites, I have grown accustomed to the classroom aspects of biology and medicine, but aside from my Chemistry labs, I have never worked in a lab or participated with any sort of research team on a project. I am so happy that I have this opportunity to become part of a project because it is a fascinating learning experience regarding what I may have to come in my future career. Additionally, the experience of being a part of a research team and working with others is highly exciting and rewarding in itself.

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Healthy Eating Research – Food Access and Environments Surrounding Elementary Schools

L. Pallis & J. TesterChildren’s Hospital & Research Center at Oakland, Oakland, CA 94609

Introduction. Access to healthy and fresh foods varies with different neighborhoods. Supermarket-type stores are the most likely to have fresh produce and healthful foods available at reasonable prices, however supermarkets are not found in every type of neighborhood. Supermarkets are predominant in wealthier, suburban areas, while smaller groceries and corner stores tend to be seen in lower-income regions. These smaller stores have a smaller variety and tend to carry less healthy options. Objective. This project aims to examine the areas surrounding the poorer elementary schools in Oakland and the access to food that children from these schools have on their walk home. I enter all of the corner/convenient stores I encounter around the school and evaluate the food environment. This information will be used to evaluate the prevalence of healthy foods in these small stores. These are the types of stores children frequent the most for snacks, typically on their way home from school, and corner stores tend to stock less healthy, junk foods. Methods. I visited the neighborhoods of thirteen elementary schools in Oakland, CA, and these schools represented the poorest ones in the city. We determined the list of specific schools by taking the top quartile of schools with the most students who are on the free or reduced price lunch program, part of the national school lunch program. The study is an observational one, I walk through each corner store I come across within a quarter-mile radius of the school and make notes of foods to buy. I have a pre-made checklist, one developed by the members of a nationwide working group, participating in the project, that surveys specific types of foods we would like to find available. Results. Thus far, I have visited eleven of the thirteen elementary school neighborhoods, and I have examined ten corner stores. Some of the most commonly found healthy items are beans/chickpeas/lentils, bagged nuts, and canned fruits and vegetables. Fresh produce is not entirely rare, but I have only come across it in four of ten stores, and every time the selection is limited by about 4-5 kinds of produce, maximum. High fiber bread and low-fat/non-fat milk are virtually never found; I have only seen low-fat milk in one store and it was chocolate milk. Often, some of the healthier items are not available in single serving, making it less likely for children to purchase. Conclusion. Based on the data collected from the stores, it is apparent that there are some healthful snacks available for children at corner stores, but there is not very much variety from store to store. There are still some healthy foods that can be easily stored that are constantly missing at these stores – brown rice, high fiber breads, low sugar drinks, and nonfat or 1% milk to name a few examples. We could use the collected information to implement policy change providing incentives for corner storeowners to stock fresh produce and other healthy snacks for kids.

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Viviana Ruiz Barros

Funded by the NIH/NHLBIDiversity Student Program

University of California BerkeleyThird Year

Mentor: Janet King, PhD

I was born and raised in Colombia and migrated to Los Angeles in 1999. It was shortly thereafter that I enrolled in mainstream English courses and earned a high school diploma from Warren High School in Downey. I began my educational journey at Cerritos Community College and transferred to the University of California at Berkeley as a Molecular and Cell Biology major in the Fall 2007.

In the last two summers, I have carried out basic research in molecular and cell developmental biology using fruit flies as a study model. This summer, I am conducting clinical research with my mentor, Dr. Janet King. In the King lab, I study the relationship between maternal visceral fat thickness and insulin resistance among obese pregnant women. Carrying out clinical research in a hospital setting, interacting with patients, and observing the relationship between research findings as applicable to the human population, are only some of the reasons why this experience has helped me prepare for a career as a physician. In addition, this opportunity has allowed me to gain a better understanding of how both clinical and basic research are carried out- ideal training to becoming a critical thinker and making wiser assessments about the quality and validity of data and results.

Thanks to the King lab, my desire to explore reproductive issues among women from disadvantaged backgrounds has become a reality. I work with pregnant women, most of whom are monolingual Spanish-speakers and at risk of developing gestational diabetes. Working in the King lab, I have realized the importance of a healthy life style and adequate nutrition. I have learned that metabolic disorders, such as gestational diabetes, can be prevented with a healthy diet and active lifestyle; yet women continue to develop the disease. Furthermore, CHORI’s summer student research program has been instrumental to helping me meet my professional development goals. The experience has been awakening, as I have experienced first-hand the power of preventing, rather than curing, diseases. I now understand the need to reform our current health care system so that its main focus is prevention and early intervention, and I aspire to contribute to such reforms in the near future.

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Maternal Visceral Fat Thickness and Insulin Resistance in Obese Pregnant Women

V. Ruiz Barros & J. KingChildren’s Hospital Oakland Research Institute, Oakland, CA 94609

Background. Over one-third of all pregnant women in the United States are obese. Maternal obesity can be detrimental for both mother and child because they can develop metabolic disorders, such as insulin resistance (IR), and gestational and type II diabetes mellitus. After mid gestation, pregnant women routinely develop a modest state of insulin resistance to allow an increased amount of glucose and other nutrients to circulate in maternal blood and to be available for the fetus. When women are obese at conception they are likely to be more insulin resistant than non-obese pregnant women; this puts them at a higher risk to develop gestational diabetes mellitus (GDM). Previous studies in obese adults have shown that there is a strong relationship between central adiposity and the development of IR. Currently, no studies have been done to determine if such relationship holds true among obese, pregnant women. Because current techniques to measure visceral fat thickness (VFT) involve the exposure to radiation, they are inappropriate for pregnant women. However, new ultrasonographic measurements have been developed to determine regional fat distribution changes during pregnancy. Objective. The purpose of this study is to determine VFT, as measured by ultrasound, is related more closely to the measurements of IR than is total body fat (TBF) in obese pregnant women. Additionally, we seek to determine if maternal waist circumference (WC) at mid-pregnancy is a good surrogate measure of VFT. Methods. Glucose tolerant women (n=24) with body fat ≥ 30%, as calculated by body density equations adjusted for pregnant women, were studied at 20 and 34 weeks gestation. VFT was determined by longitudinal ultrasound scanning with a 3.0 MHz probe from 5 cm above the umbilicus to the umbilicus. VFT was defined as the thickness between the anterior wall of the aorta and the internal surface of the rectoabdominal muscle perpendicular to the aorta. Body fat, waist circumference and insulin resistance were measured at 20 and 34 weeks. Waist circumference was measured at the midpoint between the lateral iliac crest and the lowest rib using a measuring tape. Insulin resistance was measured using HOMA-IR, the Matsuda index, the Insulin Area Under the Curve (IAUC) and the Deuterated-Glucose disposal test following a 100g oral glucose challenge test. Results. In a subsample of 13 women studied to date, there was a strong correlation between VFT and increased IR during fasting and after an OGTT at both mid-pregnancy (20 weeks) and late pregnancy (34 weeks). TBF was only related to fasting IR at 20 weeks; there was no relationship between the TBF and IR at late pregnancy. WC was related to IR at 20 weeks, but not at 34 weeks due to expansion of the uterus. Conclusion. As hypothesized, VFT was more strongly correlated with IR than total body fat. Also, waist circumference appeared to serve as a good surrogate of VFT at 20 weeks gestation and, therefore, may be a good simple clinical measure of risk of IR in pregnancy

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Timothy Ryan

Volunteer

University of Notre DameFirst Year

Mentor: Robert O. Ryan, PhD Kasuen Wong

I chose to participate in the CHORI summer research program for students because I believe in the enormous potential science has, and wanted to be a part of it. I had always enjoyed science class at all levels of my education, and am fascinated by nature and the miracle of life. When I learned that my uncle, Robert Ryan, was working in a biomedical research laboratory in Oak-land, I thought maybe that would be something that I would like to do. Coming in and seeing the CHORI labs really sparked my interest in potentially working here. So I asked Robert about any work-related opportunities for undergraduate students during the summer and he told me about the CHORI summer student program. So I applied, was accepted and am here today working in my uncles lab. Medical research has so much potential to do great things in the future, and I am just happy for the great opportunity CHORI has given me to make a contribution to medical research.

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Characterization of the C Terminus of Apolipoprotein A-V

T. Ryan, R.O. Ryan & K. WongChildren’s Hospital Oakland Research Institute, Oakland, CA 94609

Introduction: Apolipoproteins can be found in human plasma and serve a number of purposes. Located on lipoprotein particles, apolipoproteins mostly serve as modulators of lipid metabolic enzymes and proteins, ligands for cell surface receptors and/or acceptors of cellular lipid in the reverse cholesterol transport pathway. Specifically, apolipoprotein A-V has proven to be a potent modulator of plasma triacylglycerol levels. A high level of plasma triacylglycerol is a known risk factor for cardiovascular disease. It has also been shown that the C-terminal end of apo A-V, specifically residues 296-343, readily binds to lipids and may perhaps be the lipid-binding site of apo A-V. Further study of apo A-V structure and characteristics will help reveal how this protein functions to modulate plasma triacylglycerol homeostasis, and perhaps help curb cardiovascular disease in the United States. Objectives: (1) The most important objective of my project is to express and isolate the C-terminal end of apo A-V, specifically residues 296-343. (2) Once I have isolated the desired peptide, the next objective is to characterize the peptide. Hypothesis: My hypothesis is that I can successfully express and isolate the C-terminal end of apo A-V. Methods: Bacterial system for recombinant protein expression; Affinity column purification; HPLC; SDS PAGE. Concluding Remarks: Studying the characteristics of Apo A-V is very important because very little is known about this specific apolipoprotein, and it has been shown to control plasma triacylglycerol levels, which have a positive correlation with cardiovascular disease.

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Georgia Sleeth

Volunteer

Head-Royce High SchoolSophomore

Mentor: Barbara Staggers, MD

My name is Georgia Sleeth and I am going to be a junior at Head-Royce High School next year. I have been interested in medicine ever since seventh grade when I first came in contact medicine and a hospital. When I was twelve years old, I was taken to Children’s Hospital for pneumonia. I was healthy again after two weeks, but ever since my experience in the hospital, I have been fascinated in medicine. I attended the FACES Summer Medical Academy through Children’s Hospital last summer. At FACES, I observed everything from a laparoscopic surgery to a pathology lab. The FACES program truly expanded my interest in medicine and opened my eyes to different fields of medicine.

This summer at CHORI, I have had the opportunity to work with Dr. Barbara Staggers and the residents at the Teen Clinic on Telegraph Avenue. Dr. Staggers introduced me to the difficult task of providing for adolescents. As Dr. Staggers discussed in a speech of hers, adolescents are the healthiest group of people, however; adolescents have the highest death rates. The three leading causes of death among adolescents are: car crashes from under the influence, homicide, and suicide. All of these, as Dr. Staggers pointed out, are preventable. By working with Dr. Staggers this summer, I have developed a new fascination in adolescent medicine and in changing these statistics.

I would like to thank Dr. Staggers for giving me the opportunity to construct my own research project and trusting me to do so. This is my first time doing clinical research and I am rather young. Dr. Staggers and the other residents at the clinic have made me feel very comfortable with organizing my own research and I plan to continue my work with Dr. Staggers for my remaining two years in high school.

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Obesity and Stress Reduction Techni�ues

G. Sleeth & B. StaggersChildren’s Hospital & Research Center at Oakland, Oakland, CA 94609

Introduction: Obesity is a growing epidemic in America. It is very important that we emphasize to our adolescents the importance of eating well and maintaining a healthy weight. It is necessary that we focus on adolescents because 70% of adolescents that are overweight will become overweight or obese as adults (Source 1). As obesity is the second leading cause of preventable deaths after smoking, we must develop a system to help prevent these teens from leading an unhealthy life (Source 3). Objective: The main goal of this project is to see if there is a relationship between stress and obesity. Healthy eating is a source of stress reduction; however people are more often inclined to eat comforting, and fatty food when they are feeling stressed (Source 2). By prescribing various stress reduction methods as well as a balanced diet and exercise plan, I believe that adolescents will notice a decrease in their stress. I believe that this reduction in stress will allow for adolescents to focus on eating well and losing weight in a healthy manner. Methods: I made a survey to give to all patients that were obese or at risk of obesity (a BMI of 28 or above). In the survey, I asked questions relating to their stress, sleep, eating, and exercise habits. Based on the responses, I will prescribe various stress reduction methods, such as breathing and muscle relaxation exercises; as well as start them on an exercise and diet plan. I am planning on monitoring the patients through a period of time and discussing the progress. I will work to institute a program into the teen clinic about healthy eating and exercise with an emphasis on stress reduction techniques. Results: Although I am still in the process of establishing my quality care project, I do have results from the surveys I have distributed to the patients. 60% of the patients that received this survey were experiencing stress in their lives and of that 60% that are stressed, 92% were unhappy with their weight. I believe, based on the survey results, that weight does affect the patients stress level. This is why I believe that a clear exercise and diet plan as well as providing stress reduction exercises for the patients will allow them to focus on losing weight in a healthy way. Anticipated Conclusion: A healthy diet and continuous exercise are stress reducers as well. I believe that when the patients pair stress reduction techniques with eating well and exercising, they will find a reduction in their stress and be able to continue this healthy lifestyle. I am going to be working for the next couple years to develop a program at the Teen Clinic on Telegraph Avenue that focuses on stress reduction techniques with eating and exercise plans.

Bibliography:1. “Childhood Obesity: A Growing Concern” http://library.gnc.com/HealthConcernArticle.aspx?id=224&CategoryID=12&lang=en2. 2. “Stress Reduction Techniques.” http://intelihealth.com/IH/ihtIH/WSIHW000/24602/24639/289016.html?d=dmtContent#Vis3. “Obesity second only to smoking as preventable cause of death.” http://www.ecu.edu/csadmin/news/inthenews/archives/2005/10/101005packetobesity.cfm

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Tara Streich-Tilles

Funded by the Elizabeth NashFoundation

Yale UniversityThird Year

Mentor: Horst Fischer, PhD Beate Illek, PhD

From kneeling at the edge of tide pools to examining insects under a microscope as a young child, I have always enjoyed immersing myself in the study of science. I will graduate from Yale University with a B.S. in Molecular, Cellular and Developmental Biology in 2009, and I will receive my M.P.H. in Global Health from the Yale School of Public Health in 2010. Interning with the maternal and child health nonprofit organization Infante Sano in the Dominican Republic, and working as a Spanish interpreter at the HAVEN Free Clinic in New Haven, CT, have been formative in my desire to study and pursue medicine through a set of lenses crafted in my study of public health. With this enriched and broadened vision I aspire to promote health both in the United States and abroad.

Juxtaposed with my exploration of the clinical elements of medicine are the three summers of research at the Children’s Hospital Oakland Research Institute. My time at CHORI this summer is a step in my ongoing exploration of science that combines my interest in research and medicine. I feel fortunate to be able to build upon the foundation of knowledge and experience of my previous two summers in the Fischer/Illek Lab as I continue to investigate the intricate pathways of hydrogen peroxide production in the lungs. I thank Dr. Fischer and Dr. Illek for sharing their vast knowledge and passion for research, and for supporting me as I pursue my dreams.

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Stimulation and Control of H2O

2 Secretion in Lung Epithelial Cells

T. Streich-Tilles, B. Illek & H. FischerChildren’s Hospital Oakland Research Institute, Oakland, CA 94609

Introduction: The NADPH oxidase Duox-1 is the underlying membrane protein for the generation of H

2O

2 across the apical membrane in lung epithelial cells. This mechanism may be important to

innate host defense; however, excess generation of hydrogen peroxide (H2O

2), which can occur

in the Cystic Fibrosis lung concurrently with infection, may lead to the destruction of lung epithelial cells. Objective: Our studies attempt to determine the effect of pro-inflammatory agents on the regulation of H

2O

2 release into the lung lumen and the modification of this response by Compound

M. We further studied the modulatory effect of siRNA knockdown of Duox-1-mediated H2O

2

production. Methods: 16HBE14o-, CFBE41o- wtCFTR cl.10, and JME cells were cultured in 96-well plates. The effect of the bacterial products Lipopolysaccharide, Flagellin and Pyocyanin, fungal product Zymosan, and pro-inflammatory cytokine Interleukin-1β on H

2O

2 production was

measured using the Amplex Red assay. AMAXA nucleofection of JME cells was used to reduce the expression of Duox-1 prior to the Amplex Red experiments. Results: The dose-dependency of cellular H

2O

2 production response to LPS stimulation was tested in 16HBE14o- cells using LPS

from E. Coli 0111:B4. Incubation with 10uM Compound M for 24 hours prior to addition of LPS resulted in a decrease in H

2O

2 production. Even with LPS stimulation at 50ug/mL, H

2O

2 production

was reduced to 37.7% of control levels and to 49.5% of LPS stimulated H2O

2 production after 3

hours. The cellular response to LPS (E. Coli 0111:B4, Sigma), Flagellin (Salmonella typhimurium, Invivogen), Il-1β (Human, Recombinant, Sigma), Zymosan A (Saccharomyces cerevisiae, Sigma), and Pyocyanin, was tested in 16HBE14o- and CFBE41o-wtCFTR cl.10 cells. In both cell types Zymosan stimulated H

2O

2 production to the greatest extent; supplementation with Zymosan at

500ug/mL resulted in a 1.5-fold increase in H2O

2 production compared to the control after 3 hours.

AMAXA nucleofection with Duox-1 and Nox-4 siRNA was used to determine the pathway through which Zymosan-activated H

2O

2 production occurs. Three Invitrogen StealthTM Select siRNA samples

of both Duox-1 and Nox-4 (HSS122734, HSS122735, HSS122736, HSS121312, HSS121313, and HSS121314) were tested with nucleofection Solution V to determine the knockdown potential of each. Transfection with Duox-1 HSS122736 siRNA resulted in a 22% reduction in H

2O

2 production

compared to the control that warranted further investigation. Supplementation with 500ug/mL Zymosan resulted in a greater increase in H

2O

2 production than 10ng/mL Il-1β; as such, Zymosan

was used to induce an inflammatory response in further experiments. In an attempt to determine a protocol that would result in a better transfection efficiency, two AMAXA nucelofection solutions, Basic Epithelial Solution and Solution V were compared. In addition, the knockdown efficiency of Duox-1 HSS122736 siRNA was compared to that of Duox-1 2725 siRNA. GFP fluorescence readings after 48 hours indicated that the use of Basic Epithelial Solution resulted in a higher transfection rate than Solution V. It was observed that transfection with Duox-1 HSS122736 siRNA resulted in a reduction in H

2O

2 production whereas transfection with Duox-1 2725 siRNA

had no effect or increased H2O

2 production. Even with Duox-1 HSS122736 siRNA knockdown

of Duox-1, Zymosan stimulation resulted in a 6.70-fold increase in H2O

2 production compared to

unstimulated siRNA-knockdown cells after 3 hours. Conclusions: Compound M has proven to be a promising inhibitor of H

2O

2 generation by NADPH oxidases under inflammatory conditions. Our

data indicate that Zymosan treatment of cell cultures increases H2O

2 release from lung epithelial

cells. However, siRNA knockdown of Duox-1 suggests that the Zymosan-induced inflammatory response occurs through a mechanism that does not involve the activity of Duox-1, perhaps implicating Duox-2, another NADPH oxidase found in the lung epithelium. Future nucleofection experiments with JME cells should be conducted using Basic Epithelial Solution and Duox-1 HSS122736 siRNA for effective transfection and Duox-1 knockdown.

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Dagim Taddesse

Funded by the NIH/NHLBIDiversity Student Program

University of California BerkeleyThird Year

Mentor: Edward Lammer, MD David Iovannisci, PhD

My name is Dagim Taddesse senior Molecular and Cell Biology major at the University of California, Berkeley. I feel very fortunate for participating in the CHORI summer research program. First, I did not have research experience before I came to CHORI due to lack of research opportunities during my educational career although I have taken many lab courses. I did not have a chance to do research in either high school or junior college because the academic environment did not provide the resources to support research projects to be carried out. However, the CHORI Summer Research Programs has provided me the tool, technique, and opportunity to have my firsthand research experience. I feel very proud to be in the program and I am very confident that this research experience will be a great asset for my long term career goals.

I have learned more about professional research exclusively from the CHORI Summer Research Faculty and would like to practice it in the future because I know this will impact my long term educational career goals in a positive way. At the University of California, Berkeley, being a molecular and cell biology student, I have been exposed to intensive principles of life science and now at CHORI, I have applied what I have been taught theoretically. CHORI makes sense out of what Berkeley taught me in a definitive way.

In general, the CHORI Summer Research Programs has provided the environment which has exposed me to fascinating research procedures to gaining research experience while learning about the scientific processes, which will be the foundation for my research experience. As a molecular and cell biology student, I am well aware of the benefit of research and its significant effect on society. That is why I decided to spend the summer with this program and I am very glad by doing so.

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Maternal Smoking, Genetic Variation of N-Acetyltransferase-1(NAT-1), and Risk of Gastroschisis

D.Y. Taddesse, D.M. Iovannisci, K.A. Schultz & E.J. LammerChildren’s Hospital Oakland Research Institute, Oakland,CA

Introduction: Gastroschisis is a severe, life-threatening birth defect characterized by a right-sided abdominal wall defect through which intestine and other abdominal organs lie outside of the body. Nearly unique among birth defects, its frequency is increasing in many states and countries; doubling among California over the past two decades, and showing an essentially marked increase among younger mothers. Objectives: We hypothesize that single nucleotide polymorphisms (SNPs) within the infant’s genes encoding detoxification enzymes may alter the fetus’ ability to metabolize and excrete the toxic compounds found in cigarette smoke. High levels of toxins may therefore accumulate in the developing fetus, and complex with bio-molecules such as DNA, RNA and proteins disrupting their normal function, and interfering with the normal fetal developmental program. In this study, the interaction between maternal smoking during pregnancy and the gene encoding the detoxification enzyme N-Acetyltransferase1 (NAT1) has been investigated as a possible influence for increasing the risk of gastroschisis. Methods: Extracting DNA: Genomic DNA was obtained from dry blood spots on newborn screening cards (Guthrie Filter papers) collected from children born in California. DNA was extracted from the blood spots using a modification of the salting-out method. Samples were resuspended in 10μl of TE buffer. DNA Amplification: Due to extensive sequence identity between NAT1, NAT 2, and NATP, NAT1 amplification will be performed in two steps. The first amplification will employ primers flanking the NAT1. The second amplification will employ NAT1 pre-amplicon as template for the hemi-nested amplification. This creates an allele-specific restriction recognition site by using the mis-matched primer because the SNP doesn’t reside in a naturally occurring restriction site. This PCR design generates an amplicon containing an ASE I restriction site when T, but not A, is present at nucleotide 1088. (PCR: The purpose of a PCR [Polymerase Chain Reaction] is to make enough copies of the gene so that it could be analyzed.) Results: A total of 224 samples have been genotyped. As a result, we have 34 or 15.18% variant homozygous (A), 95 or 42.4% wild type homozygous (T), and 95 or 42.4 heterozygous (TA). The data has been sent to California Birth Defect Monitoring Program (CBDMP) and combined with maternal interview data concerning smoking behavior during pregnancy. The combined data will be used to calculate the risk of gastroschisis given the genotype of the infant and the level of exposure to tobacco smoke. Conclusion: We predict that the at-risk genotype is the homozygous altered gene sequence and the at-risk behavior is the mother smoking cigarettes during pregnancy. We predict that alterations in the infant’s genes encoding detoxification metabolic activities will negatively impact the developing fetus’ ability to metabolize and excrete the toxic compounds found in cigarette smoke. These toxins may then accumulate to excessive levels when mother smokes during pregnancy and subsequently complex with the natural bio-molecules within the cell, inhibiting their activities and disrupting the normal developmental program during these very critical times throughout embryogenesis.

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Alex Vidal

Volunteer

University of California Los AngelesFirst Year

Mentor: James Policy, MD

Hello my name is Alexander Vidal and I am enrolled in the CHORI Summer Student Research Program. I graduated from Miramonte High School and I am currently enrolled at the University of California Los Angeles planning to attain an undergraduate degree in Biology and to hopefully attend Medical School in the future. I grew up in Orinda very close to Children’s Hospital Oakland and attended many appointments and interacted with many of the doctors at these facilities. It was one of these experiences at Children’s Hospital Oakland that spurred my interest in becoming a doctor and especially an orthopedist. As a child, I was a very active athlete and I was operated on by Dr. Policy, who is now my mentor, for a baseball elbow injury when I was 17. I have been interested in this field of medicine ever since. This summer I have been able to do research on orthopedics, with a specialty in the exact arthroscopic elbow surgery that was performed on me. My research and observations of multiple surgeries has truly been an invaluable experience giving me great hands on knowledge in this medical field.

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Trauma vs. Osteochondritis Dissecans Arthroscopies In the Pediatric Elbow

A.Vidal & J. Policy

Children’s Hospital & Research Center at Oakland, Oakland, CA 94609

Introduction: Elbow injuries are very prevalent among the youth of the United States today. Elbow trauma injuries can result from car accidents, sports injuries, or broken bones that do not heal correctly. Osteochondritis Dissecans is another rising elbow problem, caused by the overuse of the elbow joint. OCD, as this is problem is commonly referred to, manifests itself among the most active young athletes, especially pitchers and others who perform repeated throwing motions. OCD is caused by the breakdown of the articular cartilage which covers the Humeral, Radial, or Ulnar head and leads to the death of the bone. Both of these elbow problems can lead to pain, stiffness, soreness, loose bodies breaking off, and/or the loss of one’s range of motion in the elbow. Comparing the surgical success rates between these two different injuries could be extremely beneficial for patients and doctors in the future. Objective: The objective of this study is to review 21 patients’ arthroscopic surgeries for OCD and trauma, in order to discover the varying success rates of these two injuries with regards to 15 different variables. We hypothesize that the treatment of OCD patients will have a higher success rate than that of trauma patients with regards to their post operation feelings of pain, soreness, stiffness, and range of motion. Methods: This study was conducted at the Children’s Hospital Oakland’s Outpatient Building in the Orthopedic Clinic. Here we selected 21 patients from the patient database who had had arthroscopic elbow surgeries for either OCD or trauma, from the period of June 2000 to December 2007. From this study group of 21 patients we reviewed their records for the various variables included in this study. Results: The results of this study were that 100% of the patients suffered from pain, stiffness, soreness, and/or loss of range of motion prior to surgery and 100% of patients were able to return to normal activities after their surgeries. Among the 14 OCD patients, 9 of them (64.3%) had no pain, soreness, or stiffness following their surgery, and among the 7 trauma patients, only 3 of them (42.9%) were free of pain, soreness, and stiffness following their surgery. Also, of the 14 OCD patients, 7 of them (50%) were able to get back close to full range of motion, whereas 4 of the trauma patients (57.9%), were able to get back close to full range of motion following their surgery. Conclusion: From the data we can conclude that patients having surgery for OCD have a significantly higher probability for pain, stiffness, and soreness free post surgery symptoms in comparison to the trauma patients. However, we can also conclude that trauma patients having arthroscopic elbow surgery have a higher probability of regaining full or close to full range of motion in comparison to the OCD patients.

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Sonja Washington-White

Funded by the NIH/NHLBIDiversity Student Program

California State University East BayPostbaccalaureate

Mentor: Cassandra Calloway, PhD

My name is Sonja Denise Washington-White. I am a recent graduate of the University of California, Riverside. On June 16th of last year I earned my degree in Psychology finishing in four years. Before my junior year in college I realized that I wanted to change my major to Psychology. I wanted to study the human mind. Though I changed my major I never kept my eyes from being focused that I wanted to be a Pediatrician. Immediately after graduating from the University of California, Riverside I enrolled in school to complete my courses for medical school. As a research student this summer at CHORI I plan to learn about different lab tech-niques and my summer project. This program will allow me to gain research experience in a lab. At the end of my program I will present my project. This research project is very beneficial to my career plans as a doctor for I am able to explore different areas of health careers through research.

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Analysis of Caucasian and US Hispanic Population Samples Using a New Linear Array Probe Panel for Targeting Variation in the Mitochondrial Genome

S. Washington-White & C. CallowayChildren’s Hospital Oakland Research Institute, Oakland, CA 94609

Since the 1980’s analysis of mitochondrial DNA (mtDNA) has become a very useful tool for evolutionary studies and for forensic DNA analysis. Currently only two hypervariable regions (HVI/HVII) of the mitochondrial genome (<5% of the genome) are routinely targeted by forensic DNA laboratories. A new linear array assay which targets 15 variable regions (including the 2 hypervariable regions) distributed throughout the mitochondrial genome has been recently developed. This new assay has the potential to significantly improve the informativeness of mtDNA analysis for forensic DNA applications. In this study, we evaluated this expanded linear array probe assay to determine if the power of discrimination will be significantly increased, specifically among populations with a large number of common HVI/HVII types, such as the US Caucasian and US Hispanic populations.

We present here population data to illustrate the increased discrimination power of this new assay. We examined 2 US population groups: US Caucasians and US Hispanics. These groups were chosen because they have the lowest discrimination power in the HVI/II region. We used 5 and 10- plex PCR assays to amplify the DNA samples. Gel electrophoresis was used for verification of amplification and to determine product yield. We used two immobilized probe panels (5 and 10- plex) to target 61 polymorphic sites of the mitochondrial genome. The 5- plex probe panel consists of 59 probes and targets variation at 14 HVI sites, 11 HVII sites, 8 coding region sites and 2 variable region I sites. The 10-plex probe panel consists of 46 probes and targets 17 coding region sites, 4 variable region I sites and 5 variable region II sites. We used these linear array probe assays to determine the frequency of mitochondria DNA types within specific population groups and the frequency of sequence variants for each of the probe regions. Using this information we calculated the discrimination power for the expanded array and compared it to the HVI/II results to determine if the discrimination power increased within the two population groups studied (US Caucasian and US Hispanic). In addition we identified variants that give a weak or no signal and determined amplification success.

Data from this study will allow us to determine if examining regions outside the commonly targeted HVI/II regions will increase discrimination power within the two groups. Examining regions outside the commonly targeted HVI/II regions will also allow us to identify new sequence variants. Future studies will allow us to do further analysis of other population groups.

100

Julie Willems

Volunteer

St. Louis UniversityThird Year

Mentor: Rose Ellen Morrell, MD

Ever since I was little I can remember having a passion for helping others. As I child, I rescued all sorts of animals from a baby bird that was kicked out of its nest to earthworms that were stranded on the sidewalk after a night of heavy rainfall. As I have grown, my love of caring for others has continued to flourish and has been affected by numerous personal experiences.

The summer before my junior year in high school, I traveled with 20 other students to Namibia. During my month long stay in Africa, I became very aware of the wide disparities and inequalities that exist in the world. I could not understand how people could allow other people, especially the children, to suffer from starvation and perfectly curable diseases. When I returned, I felt very disheartened and confused but I still felt my deep-rooted passion for helping others, which is why I decided to study nursing.

This past year in the nursing program, I completed a 4-month rotation in public health nursing. During my clinical, I worked in some of the most dangerous and impoverished areas of Saint Louis and witnessed first hand how unfair the world can be. I became aware that disparities and inequalities don’t just exist in foreign, third world countries but also in my own neighborhood. I learned the importance of patience and persistence and I once again saw that there is no magic wand that can be waved to cure all of the world’s ailments overnight.

These previous experiences are some of the reasons that I was drawn to the CHORI program. During the program, I witnessed first hand the love and compassion that this hospital and its staff gives to its patients. I also had the opportunity to see and be a part of the numerous community outreach programs. The environment of this hospital is geared towards allowing patients and their families to thrive, something that is critical when it comes to health care. In that same environment, I too have had the opportunity to develop and thrive. This program has deepened my passion for others and I hope to take everything that I have learned and reach out to others by pursuing a career in public health. My experiences here at CHORI have left me greatly indebted, especially to those who mentored me. I would like to thank Dr. Rose Ellen Morrell, Dr. Christina Lo, Dr. Barbara Botelho, Carole Reilly and Lorena Sanchez for all of their love, support and inspiration.

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2� Hour Blood Pressure Monitoring in Pediatric Patients with a BMI above the ��th Percentile and a Normal Clinic Blood Pressure

J. Willems, R.E. Morrell, C.Lo & B. BotelhoChildren’s Hospital & Research Center at Oakland, Oakland CA 94609

Introduction. Obesity is a problem of epidemic proportions. Recent articles report that over 32 percent of American children are overweight. The large increase in the prevalence of obesity has lead to sequela previously diagnosed in adults to be diagnosed in children. Cardiovascular disease is one such sequela. Cardiovascular disease has many risk factors, but one of the most significant and preventable is hypertension. The American Academy of Pediatrics recommends that blood pressure be screened at least once a year after the age of three. A diagnosis of hypertension is made when the blood pressure is found to be above the norms, based on sex, age and height percentile, on three separate readings. However, blood pressure is not static. It is ever changing and influenced by a variety of factors. Therefore, many physicians who suspect hypertension in a patient will use an ambulatory blood pressure monitor to examine the patients 24 hour blood pressure cycle. A recent position paper, from the American Society of Hypertension, states that ambulatory blood pressure monitoring allows for the identification of high risk patients irrespective of their clinic blood pressure. 24 hour monitoring allows a physician to look at the patient’s dynamic blood pressure pattern during sleep and wake cycles and in their normal daily environment. A normal blood pressure cycle will have a dip of more than 10% when the patient is sleeping. The amount of nocturnal dipping is significant because numerous studies have indicated that there is a strong correlation between a decrease in the nocturnal dip and the development of cardiovascular disease. Studies have also shown that there is a strong correlation between obesity and hypertension, which can lead to cardiovascular disease. Hypothesis. The aim of this study is to examine obese children, above the 95th percentile for body mass index, who have a normal clinic blood pressure. The researchers believe that these children are not truly normotensive. They believe that the data from the 24hour monitor will show that the children are hypertensive throughout much of the monitoring period and also show a decreased nocturnal dip. Both of these findings indicate a possible diagnosis of masked hypertension, which places the patient at an increased risk for cardiovascular disease. Methods. The children are recruited at the pediatric primary care clinics. Upon receiving the proper consents, the children are then connected to the device for 24 hours. After the 24hour period the family returns the monitor to the clinic and the data is then entered into the computer program which analyzes the data using an algorithm. The results are then interpreted by the Nephrologist and reported back to the primary care physician and the family. Results. The study is not complete, but preliminary findings suggest that the hypothesis is valid. The early findings illustrate that the blood pressure was above normal for a significant period of time and also showed a decrease in nocturnal dip. Conclusion. These preliminary findings suggest that obese children who are found to be normotensive in clinic may have what is known as masked hypertension. This suggest that obese children need closer blood pressure monitoring, such as the use of a 24hour blood pressure monitor, in order to prevent hypertension and its sequela.

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Chris Wong

Volunteer

Acalanes High SchoolJunior

Mentor: Barbara Staggers, MD

For the last few years, I have had many interests, refusing to specialize in one area at the cost of abandoning others. For this reason, I enjoy exploring different potential career opportunities to begin to understand the different options I have before choosing a career track. I have an interest in mathematics and art, especially drawing and watercolor, and I continue to pursue these interests. At the same time, I continue to read novels and write fiction, two other activities that interest me. I have also had an interest in psychology and some of the other “softer” sciences, like history. I use to have little interest in history, but my former history teacher brought history to life and helped me to become interested in it.

The CHORI research program gives me an opportunity to learn more about psychology and medical career tracks. I did not know if I would like medical careers (mainly because of my fear of blood) but I have found the program and working with patients to be interesting. I am trying to understand how the patients think and how their minds influence their behaviors. It is somewhat of a puzzle, a problem that requires clever thought to solve, which are the kinds of challenges I enjoy. Working with other summer interns, medical students, residents, and Dr. Staggers is both enjoyable and interesting because we all have different perspectives and this make collaboration interesting.

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Improving Contraception and Condom Use in Adolescents

C. Wong & B. StaggersChildren’s Hospital & Research Center at Oakland, Oakland, CA 94609

Background: Adolescents are one of the most high risk groups in terms of sexual behavior and contraceptive usage. Studies have been done in other countries like Australia and certain parts of African. These studies state that teenage behavior and certain societal values make teens more vulnerable to STIs and unwanted pregnancies and less likely to use contraception or condoms. Objectives: My project focuses on the contraceptive habits and condom usage of adolescents in the Bay Area. The hypothesis is that an unwanted pregnancy or an STI will be more influential on condom and contraception usage than sexual education. My objectives are to understand the sexual and contraceptive behaviors of teens, and the motives behind those behaviors.There are only a few studies that pertain to contraception and condom usage in American teens. Methods: For this study, I gave surveys to the sexually active patients in the Teen Clinic that asked them about their sexual habits, contraceptive and condom habits, knowledge about condoms and contraception, their lives, and other factors that may influence their behavior. Results: This data shows that teens are unreliable in their usage of contraception because they will often forget to take the pill or not visit a clinic for a depo injection. Seeing others have STIs or unwanted pregnancies and getting either an STI or pregnancy themselves, is a large influence on teenage behavior. It also makes teens who felt invulnerable to pregnancies or STIs understand that they are as susceptible as everyone else. Goals and Expectations: The end goal is to improve sexual education so that it is more meaningful for teens which will hopefully improve their usage of contraception and condoms. This study may also help to improve the way the clinics handle teens with STIs and unwanted pregnancies. Overall, this project is meant to reduce the rate of STI transmission in teens and to reduce the number of unwanted pregnancies.

Background sources:

“Sex, contraception, and health.” Australian Family Physician. Vol 36. No 8. August 2007.“Adolescents’ views of and preferences for sexual and reproductive health services in Burkina Faso, Ghana, Malawi, and Uganda.” Afr J Reprod Health. 2007.

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Angela Wu

Volunteer

University of California BerkeleyFourth Year

Mentor: Ervin Epstein, MD Jean Tang, MD, PhD

I have always been interested in the sciences, ever since deciding that I wanted to become a paleontologist in first grade because they got to study dinosaurs, which was just about the coolest thing my six year old self could imagine. Though my perception of what constitutes science and its component parts has since matured, my fascination with it has never wavered, a fact reflected in the choices I’ve made. I graduated in May of 2008 with a degree in Molecular and Cell Biology from the University of California Berkeley, having spent two and a half of those years studying butterfly flight kinematics and marine stomatopods, but ultimately decided to pursue a career in medicine. This led me to volunteer at the Children’s Hospital of Oakland and eventually to a position as a research assistant here at CHORI.

My projects at CHORI focus on the study of basal cell carcinomas and the Hedgehog pathway dysregulations that cause them. One of these projects is a study of the effect of Hedgehog antagonists on BCCs in mice, with the hopes of eventually testing these drugs on humans. My second project focuses on the effect of serum levels of vitamin D on BCC risk, where I have gathered data from patients suffering from Basal Cell Nevus Syndrome, a rare clinical condition where these patients develop many BCCs, sometimes numbering in the hundreds and even thousands.

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A Clinical Study of Serum Vitamin D Levels in Basal Cell Nevus Syndrome Patients

A. Wu, J. Tang & E. Epstein Children’s Hospital Oakland Research Institute, Oakland, CA 94609

Introduction: Basal cell carcinoma (BCC) is the most common type of skin cancer, affecting close to 1 million Americans a year. BCC carcinogenesis occurs due to inappropriate activation of the Hedgehog (HH) signaling pathway, commonly due to mutational inactivation of the tumor suppressor, the Patched1 (Ptch1) gene. Ptch1+/- patients have a rare syndrome known as Basal Cell Nevus Syndrome (BCNS) and develop tens to hundreds of BCC tumors at a young age, and could significantly benefit from new ways to prevent BCCs. Recently, our lab has found that vitamin D inhibits BCCs in murine cell lines and prevents BCCs in mice. We have observed that some of our BCNS patients have hundreds of BCCs, while others develop far fewer. We wish to determine the factors that influence vitamin D status in BCNS patients, and whether high vitamin D levels may be correlated to the number of BCCs. Objectives: To investigate whether serum vitamin D levels are associated with BCCs, sunscreen use, and UV exposure in BCNS patients. Methods: For this observational clinical study, I performed a chart review of 26 BCNS patients that were previously enrolled in a clinical trial. I extrapolated data on their BCC tumor number, sunscreen use, UV exposure, and season of blood draw. Serum levels of vitamin D (25 hydroxy-vitamin D) were determined via radioimmunoassay (ARUP Labs). Multivariable regression techniques were used to examine if there is an association of BCC numbers, sunscreen use, UV exposure and serum vitamin D levels in BCNS patients. Results: We found that 50% of the BCNS patients in this cohort had insufficient (<32ng/l) or deficient levels (<20ng/ml) of vitamin D. This is significant in that low vitamin D Ievels are usually found in elderly cohorts while the average age of our cohort was 40 years old. This low level of vitamin D was not explained by sunscreen use or UV exposure. As a control, vitamin D levels were significantly associated with the season of blood draw, being significantly higher in the fall and summer when compared to winter serum vitamin D levels. Vitamin D levels in this limited cohort did not correlate with BCC numbers. Conclusion: The discovery that 50% of the BCNS patients had insufficient or deficient levels of vitamin D was unexpected. These patients may need to be orally supplemented with vitamin D, not only for possible inhibition of the hedgehog pathway and thus BCC growth, but also to prevent vitamin D-deficiency related reasons for bone loss.

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Alex Wulff

Volunteer

University of OregonSecond Year

Mentor: Ronald Krauss, MD Sally Chiu, PhD

My name is Alex Wulff and I am a junior at the University of Oregon in Eugene. I have grown up in a medical household because my father is a Cardiologist. Some of my earliest recollections are tagging along with my father as he performed his rounds at UCLA. I used to love to sit on the windowsill and watch the helicopters bring in patients and land on the roof across from my dad’s office.

I played competitive volleyball throughout high school and did not give a lot of consideration to pursuing a career in medicine at that time. I participated in two Junior Olympics and was nominated for the all Junior Olympic team in 2006 and was recruited to play at the University of Hawaii but felt that would interfere too much with my studies. During high school I also participated in the Boy Scouts and earned the rank of Eagle Scout at age 15.

I attended a Catholic high school that put great emphasis on helping those less fortunate. I participated in the Lasallian youth program which is designed to help the less fortunate members of the community through fundraisers and volunteer social service. Through these experiences I found the joy in helping others, which reignited my desire to devote my life to a career in service.

At the University of Oregon I enrolled as a business major but after taking a few classes in the field realized that I wanted something that was a little more interactive. I realized that I wanted to pursue a medical career after participating in a humanitarian trip to Peru with my father to provide medical aide to the indigenous people in remote villages. I helped to triage patients, dispense medications, provide translation, and even perform minor noninvasive procedures. I saw first-hand the miraculous work that was being done by the medical team of physicians, nurses, and non-medical personnel like myself.

My time at CHORI has been more than I expected. My mentor Sally Chiu is friendly and energetic and treats me like a peer. Through this experience I have learned more about medicine and research than I could have in the classroom.

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An Angiotensin II Receptor I Polymorphism is Associated with Multiple Metabolic Risk Factors

A. Wulff, R. Krauss & S. ChiuChildren’s Hospital Oakland Research Institute, Oakland, CA 94609

Background. We previously identified a single nucleotide polymorphism (SNP) in the angiotensin II receptor 1 (AGTR1) gene (rs2640543) that is significantly associated with protection from adiposity-induced dyslipidemia, a syndrome characterized by reduced HDL, increased triglyceride, and smaller LDL particle size. Since increased blood pressure (BP) is strongly related to this dyslipidemia, and AGTR1 plays a key role in BP regulation, we tested whether rs2640543 was related to BP or BP response to ramipril, an angiotensin converting enzyme (ACE) inhibitor. Method. rs2640543 was previously genotyped in 149 participants in the Hypertension and Ramipril Pharmacogenetics (HARP) study. The SNP was tested for associations with baseline HDL and triglyceride levels and baseline and change values of office systolic (SBP) and diastolic (DBP) blood pressure, daytime ambulatory SBP and DBP and night SBP and DBP by analysis of variance (ANOVA). Haplotypes including rs2640543 and other previously genotyped AGTR1 SNPs were constructed and tested for associations with blood pressure phenotypes. Results. rs2640543 was associated with baseline triglyceride levels (p=0.02), with the AA genotype having the lowest triglyceride levels, as expected from previous data. Carriers of the AA genotype also tended to have higher HDL levels as expected. This SNP was not significantly associated with baseline BP, but was correlated with greater reduction of daytime ambulatory SBP (p= 0.0157) and DBP (p= 0.0005) in response to ramipril. These correlations remained significant after adjustment for age, race, gender, and BMI (p=0.0415 and p=0.0018, respectively). The GG genotype, which was present in 25.7% of subjects, was associated with the greatest reduction in daytime ambulatory blood pressure with ramipril treatment (- 14.56 SBP, -9.12 DBP). Haplotype analysis did not yield additional information regarding the genetic association of AGTR1 with either lipids or BP regulation. Conclusion. These analyses suggest that a genetic polymorphism in AGTR1 is associated with variations in both lipid and lipoprotein metabolism and BP regulation in response to ACE inhibitor treatment. Further studies will be required to confirm this observation and to investigate its underlying mechanism.

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Johnston Ye

Volunteer

Monte Vista High SchoolJunior

Mentor: Robert O. Ryan, PhD Taichi Yamamoto, PhD

Science has always been a passion for me. Unfortunately, my school has always lacked sufficient science-oriented programs for the scientific student. When I was browsing for summer programs one night, I discovered CHORI, and after another hour of research, I saw that the CHORI research program was specifically what I desired. Because I certainly did not expect admittance into this competitive program, I was understandably surprised when I received the email stating that I had been chosen by the Selection Committee.

I came into the CHORI research environment with no previous experience, and it was initially quite intimidating; however, my mentor delineated every step of my project with detail and patience, and for this, I am forever grateful to him. Since the beginning of my work, I have fostered a newfound passion for science – one that has significantly transcended my previous passion. Moreover, I am now appreciative for scientists and their seemingly unending dedication to their work. Watching them daily in the laboratory and noting how carefully they performed every step of the scientific process has given me inspiration to continue my own scientific endeavors. However cliché this sounds, I am confident that every participant in this program can truly say that scientists are pioneers that venture to cure the world of its long list of diseases. On a final note, I would like to personally thank Taichi Yamamoto for being an outstanding mentor and guiding me through every step of this program with patience and enthusiasm.

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Study of the Interaction between the Receptor-Associated Protein and the Low-Density Lipoprotein Receptor

J. Ye, R. Ryan & T. YamamotoChildren’s Hospital Oakland Research Institute, Oakland, CA 94609

Introduction. As shown by the studies of Brown and Goldstein, the low-density lipoprotein receptor plays a key role in plasma cholesterol homeostasis. This fundamental role is vividly articulated by mutations in the receptor gene that can result in familial hypercholesterolemia, a disease of the cardiovascular system that prompts myocardial infarction. Further detailed studies have shown that LDLR is a key player in facilitating internalization of hepatically derived cholesterol-rich lipoproteins. The receptor-associated protein (RAP) – which consists of three helical domains consisting of 100 amino acids each – was initially discovered as a protein that co-purifies with the LDL-receptor related protein. Studies have revealed that RAP can in fact bind with the LDLR. Moreover, it has recently been published that the third domain (RAP-D3) of RAP exhibits the greatest binding affinity to LDLR family. Objective. Recent studies have shown that RAP facilitates the delivery of the LDLR family proteins to the cell surface by preventing premature interaction of the LDLR and ligands; however, interaction between the two proteins is required in order to do so. It remains to be determined the molecular mechanism of the interaction between LDLR and RAP. Methods. Before the commencement of this project, site-directed mutagenesis was already performed to generate tryptophan-null single cysteine RAP-D3 recombinant protein. RAP-D3 was expressed in E. coli BC 21 cells, and the protein was then labeled at the amino acid cysteine with AEDANS. Soluble LDLR domain C1-699 residues were expressed in human cells, HEK293. Both of the proteins were then isolated by affinity columns. Lastly, the interaction between LDLR and RAP-D3 was monitored through the employment of fluorescence resonance energy transfer (FRET). Results. Two recombinant proteins have been highly purified, and they are ready to be performed for FRET-based receptor binding assay. Conclusion. Further experimentation should reveal the molecular mechanism between RAP and LDLR and give insight for development of new strategies for diagnosis, treatment and/or prevention of cardiovascular disease.

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Maria Zizka

Volunteer

University of California BerkeleySecond Year

Mentor: Julie Saba, MD, PhD

This fall will begin my third year UC Berkeley. I am hoping to major in integrative biology and perhaps continue pursuing a career in medicine upon graduation. I am very thankful to have the opportunity to work with Dr. Julie Saba this summer at CHORI studying and learning about sphingosine-1 phosphate (S1P) and role it and other enzymes play in regulating cell survival during animal development, immunity cancer formation and cancer progression.

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The Effects of Resveratrol on Colon Cancer Cell Lines: Up-Regulation of Ceramide and Inhibition of the Wnt Signaling Pathway

M. Zizka, F. Lu & J. SabaChildren’s Hospital Oakland Research Institute. Oakland, CA 94609

Introduction. Resveratrol is a natural antioxidant found in red wine and grapes that provides protective properties against cancer, heart disease and aging. The mechanisms by which resveratrol acts are not yet fully understood. Previous studies of the effects of resveratrol on highly metastatic breast and prostate cancer cells have shown increased levels of the bioactive lipid ceramide, antiproliferation and proapoptotic cell death. Ceramide regulates cell growth by affecting key signal transduction pathways. We and others have shown that ceramide induces cell death of colon cancer cells. This is likely due to the ability of ceramide to prevent translocation of activated b-catenin from the membrane and cytosol to the nucleus, thereby preventing its induction of bcatenin/TCF target genes that promote survival and proliferation. Objective. Based on our observations of the effects of ceramide in colon cancer cells, I hypothesize that resveratrol, by raising cellular levels of ceramide, may inhibit the Wnt signaling pathway and thereby cause cell death by apoptosis. In coordination with the Saba lab’s study of colon cancer, my experiment will investigate the effects of resveratrol on two different colon cancer cell lines. Methods. I will use cell culture and Western blotting technique to test for ß-catenin expression and localization in colon cancer cells treated with varying doses of resveratrol. In addition, caspase assays will be used to measure the rate of apoptosis in the treated cells versus vehicle-treated control cells. Finally, a TOP/FOP flash reporter will be introduced into colon cancer cells by cDNA transfection, allowing determination of the ß-catenin/TCF transcriptional activity in the presence and absence of resveratrol. Results. Images of colon cancer cells treated with resveratrol show morphological changes including nuclear fragmentation and cytoplasmic enlargement, both of which characterize early cell death by apoptosis. These effects were confirmed by caspase-3 assays. Western blot experiments probing for the expression of active ß-catenin indicate decreased levels of intracellular expression in resveratrol treated cells. Conclusion. Information obtained from these experiments suggest a correlation between resveratrol treatment, decreased levels of ß-catenin, and proapoptotic cell death. Further studies are being conducted to confirm these initial observations, determine the sequence of events and establish whether these effects define the mechanism by which resveratrol acts.

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For more information about this program, please contact:

The Summer Student ProgramChildren’s Hospital & Research

Center at Oakland5700 Martin Luther King, Jr. Way

Oakland, CA 94609summerstudentprogram@chori.org

Or, sign on to:

http://www.chori.org/Education/Summer_Internship_Program/announcements.html