The Twenty-Ninth Annual UNIVERSITY OF ARIZONA UNDERGRADUATE BIOLOGY RESEARCH PROGRAM CONFERENCE
January 20, 2018 Environment & Natural Resources 2 Building
TABLE OF CONTENTS
THE 29TH ANNUAL UNIVERSITY OF ARIZONA UNDERGRADUATE BIOLOGY RESEARCH PROGRAM CONFERENCE
Welcome! 3 Conference Agenda 4 Map of Venue: Environment and Natural Resources 2 Building 5 Today’s Activities 6 2017-2018 UBRP Programs, Participants, and Faculty Mentors 8 Acknowledgements 12 A Thank-You to Our Donors 13 Supporting UBRP 14 2017 UBRP Mentor Awards 15 UBRP Ambassadors 16 Pen Pals Program 17 2017 Small Group Discussion Leaders 18 Abstracts of Posters Presented 19
Support for the 29th Annual UBRP Conference has been provided by:
2 WELCOME!
Dear Students, Faculty, Friends, Family Members, and Guests:
Welcome to the Twenty-Ninth Annual Undergraduate Biology Research Program (UBRP) Conference!
The annual UBRP Conference provides a fantastic opportunity to learn from our undergraduates about what they’ve discovered by doing research, their tribulations and triumphs, and new perspectives they have gained from their mentors. At this conference, my first as Director of UBRP, I am excited to see our students’ work!
UBRP encompasses several programs that provide individualized research experiences to a diverse group of students. During the Summer of 2017, these students conducted research full- time, they met weekly to share their research journey in small groups, and they came together as a community in scientific seminars and field trips. While our students come from varied backgrounds, they are all here today with a common purpose: to communicate what they’ve discovered and how it can better the world and our society.
Approximately 120 undergraduates are presenting at today’s conference, and they have conducted research across the globe – in the Czech Republic, in Tucson, at Northern Arizona University, at Barrow Neurological Institute, and at the UA College of Medicine in Phoenix. Their work represents the breadth of biological research, including topics such as the health effects of tattoo ink, how children learn, the impact of urbanization and growing populations upon surface water, boldness in spiders, novel cancer treatments, and using songbirds to study the human voice.
As any of the students presenting today can tell you, conducting scientific research isn’t easy. The posters you see today represent hard work, trial and error, teamwork, patience, sleepless nights, missed meals, and a great deal of tenacity. Today, I welcome you to talk with our students about their adventures in research, and share in celebrating the excitement of discovery and the spirit of scientific collaboration. I hope you enjoy seeing what these students have accomplished!
Enjoy the conference!
Jennifer Cubeta
Jennifer Cubeta, M.Ed. UBRP Director
3 29TH ANNUAL UBRP CONFERENCE AGENDA ENVIRONMENT & NATURAL RESOURCES 2 BUILDING, 1064 E. LOWELL STREET
12:00pm – 1:00pm CHECK IN & OPENING RECEPTION GROUND AND SECOND FLOORS • Check in at the Registration Table • Enjoy refreshments in the Cafe, meet & network with UBRP students, faculty mentors, alumni, and guests • Explore hands-on science activities (Ground Floor) • Preview students’ posters (Second Floor)
1:00pm – 2:00pm KEYNOTE ADDRESS ROOM N120, GROUND FLOOR • Welcome by Jennifer Cubeta, UBRP Director • Introduction of Keynote Speaker by Dr. Philip Malan • Keynote Address: “Shedding (Green) Light on Alternative Pain Management” by Dr. Mohab Ibrahim, UBRP Alumnus, Director of the Comprehensive Pain Management Clinic at Banner University Medical Center South, and Assistant Professor in the Departments of Anesthesiology & Pharmacology at the University of Arizona College of Medicine • “UBRP Up Close” by Tiffany Cho and Kai Aragaki, UBRP Ambassadors President and Secretary, and Dr. John Szivek, UBRP External Advisory Group Chairperson • Acknowledgments of Service to UBRP • Logistics, Jennifer Cubeta, UBRP Director
2:00pm – 4:30pm UBRP POSTER SESSION SECOND FLOOR: ROOMS S215, S223, S225, & S230 • Odd numbered posters present from 2:00pm – 3:15pm • Even numbered posters present from 3:15pm – 4:30pm
4:30pm – 5:00pm CLOSING: AWARDS PRESENTATION & DOOR PRIZES ROOM N120, GROUND FLOOR • Outstanding Graduate Student Mentor Award • Outstanding Faculty Mentor Award • Door Prizes • Student Participation Certificates
4 MAP OF VENUE: ENVIRONMENT AND NATURAL RESOURCES 2 BUILDING
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SECOND FLOOR
5 TODAY’S ACTIVITIES
OPENING RECEPTION ACTIVITIES 12:00pm – 1:00pm
Meet and Greet (ENR2 Café & Courtyard) Arthropod Diversity (ENR2 Courtyard) Grab a bite to eat and connect with UBRP students, See arachnids, giant centipedes, and stinging insects! alumni, faculty, and guests! Hosted by Dr. Justin Schmidt, a 2015 winner of the IgNoble Award and 2016 author of the book The Shark Anatomy and Coral Bleaching Sting of the Wild. (ENR2 Courtyard) Learn the anatomy of sharks and how they play an Arizona Stressbusters (Room 120A, Inside Café) important role in our oceans. Understand the Feeling stressed? Get a free 5-minute backrub from complicated world of corals and their conservation University of Arizona volunteers! issues. Hosted by the Marine Awareness and Conservation Society (MACS). Learn How to Extract DNA! (ENR2 Courtyard) Come learn and perform the steps to extract DNA UBRP 2017 Slide Show (Room N120) from living things! Hosted by the Molecular & See what UBRPers have been up to! Cellular Biology (MCB) Club.
The Brain Bus (Parking Bay, North Side of ENR2) Support UBRP See a human brain and spinal cord, learn what Visit the UBRP Registration Table to contribute to happens when the brain experiences a concussion, UBRP. T-shirts are available for $10; all proceeds will and what to do if you get a concussion. go towards UBRP student support.
KEYNOTE ADDRESS 1:00pm – 2:00pm
SHEDDING (GREEN) LIGHT ON ALTERNATIVE PAIN MANAGEMENT by Dr. Mohab Ibrahim
INTRODUCTION OF KEYNOTE SPEAKER BY DR. PHIL MALAN, PROFESSOR EMERITUS OF ANESTHESIOLOGY AND PHARMACOLOGY
Dr. Mohab Ibrahim is Director of the Comprehensive Pain Management Clinic at Banner University Medical Center South and an Assistant Professor, Clinical Scholar Track, in the Departments of Anesthesiology & Pharmacology at the University of Arizona College of Medicine. Dr. Ibrahim came to the United States from Egypt in 1993. He initially enrolled at Pima Community College and then transferred to the University of Arizona. He took a student worker position in Dr. John Law’s lab, working under the supervision of Dr. Rolf Ziegler studying changes in lipid content of mosquito fat bodies and oocytes during adult life under different conditions.
In UBRP, Dr. Ibrahim chose to work with Dr. Phil Malan, who studied pain and the means of treating it. Ibrahim worked in the Malan lab as a UBRP student from 1997-1998, receiving
6 his BS in biochemistry with a minor in chemistry and mathematics in December of 1998. He earned a Master’s in pharmacology and toxicology from UA and completed a PhD in pharmacology and toxicology at UA in 2004 under the guidance of Dr. Malan. From 2004 to 2008 he was a UA medical student and then a surgical intern at UA. From 2009 to 2012, Dr. Ibrahim was an anesthesiology resident at Brigham and Women’s Hospital, which is affiliated with Harvard University, and he spent a year as a Clinical Pain Fellow at Massachusetts General Hospital. After spending a year in Las Vegas as an Interventional Pain Management Physician at the Pain Center of Nevada, Dr. Ibrahim came back to Tucson to assume his current position.
Recently he and his colleagues, including UBRP mentor Dr. Rajesh Khanna, have been experimenting with how exposure to green LED can mitigate pain in both rats and humans.
THE UBRP POSTER SESSIONS! 2:00pm – 4:30pm
If you’ve never attended a poster session before, keep in mind that our students are here to talk to you about the research they’ve done. You are not expected to be an expert in science; we invite our visitors to simply be curious and to ask questions! Can you walk me through your poster? How did you get involved in research? What excites you about doing research? What is the ‘take home’ message from your poster? Our students will be happy to share their research experiences with you! Along with the list of abstracts located in this booklet, you can use the Topical Guide to UBRP Conference Posters handout to help you identify posters of interest.
To give our own students a chance to see each other’s work, half of our students will be presenting their posters from 2:00pm to 3:15pm and the other half will be presenting from 3:15pm to 4:30pm.
AWARDS PRESENTATION & DOOR PRIZES 4:30pm – 5:00pm
UBRP thanks the following organizations and companies for donating door prizes:
University of Arizona School of Theater Film & Television
7 2017-2018 UBRP PROGRAMS, PARTICIPANTS, AND FACULTY MENTORS
UNDERGRADUATE BIOLOGY RESEARCH PROGRAM (UBRP) The Undergraduate Biology Research Program (UBRP) is an educational program designed to teach students science by involving them in biologically-related research. Students are paid for their time doing research where they develop an understanding of scientific method and receive a realistic view of research. They also acquire the tools necessary to be successful in post-graduate studies should they choose careers related to biology or biomedical research. Funding for UBRP students comes from private donors and the Western Alliance to Expand Student Opportunities (WAESO), and internal funds from the UA Provost, Senior Vice President for Research (ORD Research Fellows), BIO5, and the deans of the Colleges of Medicine, Science, Public Health, Pharmacy, and Agriculture and Life Sciences. We gratefully acknowledge this support!
Student Mentor Andrew Alamban Janis Burt Adam Aragaki Ronald Lynch Emma Armstrong Jean-Marc Fellous Kayenat Aryeh (ORD Research Fellow) Andrew Paek Sri Sai Swetha Atluri (ORD Research Fellow) Ronald Lukas Olivia Austin Jennifer Barton Areen Badwal (ORD Research Fellow) Julie Miller George Barnum Shaowen Bao Rachel Bear (ORD Research Fellow) Daniela Zarnescu Bailey Bellaire Jon Njardarson Shreya Bellampalli (ORD Research Fellow) Rajesh Khanna Ian Burton (ORD Research Fellow) Noel Warfel Danielle Cannon (ORD Research Fellow) John Konhilas Briggs Carhart Wulfila Gronenberg Ethan Carlson David Baltrus Shane Carr Jason Yuan Matthew Chaung Daniela Zarnescu Lindsey Chew (ORD Research Fellow) Rajesh Khanna Tiffany Cho (ORD Research Fellow) Haijiang Cai Kathryn Chung Jamie Edgin Dez Coleman Tally Largent-Milnes Nicole De La Pena (ORD Research Fellow, WASEO) Carol Barnes Alyssa Lyn Fortier Ryan Gutenkunst Steven Fried Michael Brown Margret Fye (ORD Research Fellow) Janis Burt Nathaniel Gallegos (WASEO) Lee Ryan Emily Galloway (ORD Research Fellow) Tally Largent-Milnes Emma Harrell (ORD Research Fellow) Jacob Schwartz Dhanwant Hunjan Jil Tardiff Jason Juang Daniela Zarnescu John Kim Todd Vanderah Christopher Kouris Mark Beilstein Sakthi Kumar Anita Koshy Bohan Li Timothy Secomb
8 Student Mentor Albert Liu Carol Dieckmann Kailyn McFarlane Nicole Marrone Kendra Meer Joanna Masel Caitlin Moffett Heddwen Brooks Jibriel Noun Jon Njardarson Abigail O'Conner Daniela Zarnescu Diana Perez Mary Peterson Savannah Perno Joseph Blankinship Lauren Pisani Linda Restifo Caroline Plecki (ORD Research Fellow) Mark Beilstein Jessica Renger Jessica Andrews-Hanna Jose Rios-Monterrrosa (ORD Research Fellow, WAESO) Victor Hruby Rachel Sadler Wulfila Gronenberg Matthew Scandura Daniela Zarnescu Laura Scherliess (ORD Research Fellow) Gene Alexander Hannah Schmitz Russell Witte Yannick Schreiber John Jewett Ahmad Shahin May Khanna Tala Shahin (ORD Research Fellow) David Armstrong Catherine Sikora (ORD Research Fellow) Terence O'Keeffe Charis Springhower (ORD Research Fellow, WAESO) Jacob Schwartz Alyssa Sullivan (ORD Research Fellow) Rebecca Gomez Alex Summers Stephen Cowen Guangzhe Sun Eric Weterings Arjun Syal Lalitha Madhavan Meagan Tran Yitshak Zohar Neeraj Vij Joyce Schroeder Karen Wang Goggy Davidowitz Greg Wheeler Theodore Trouard Meng-Han Ashley Wu Timothy Bolger Ryan Zenhausern Jerome Lacombe
AMERICAN SOCIETY FOR PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS SUMMER UNDERGRADUATE RESEARCH PROGRAM (ASPET SURF) The ASPET SURF Program, funded by a grant from the American Society for Pharmacology and Experimental Therapeutics, supports five undergraduate students per year who work under the mentorship of ASPET members. The program goal is to introduce undergraduate students to pharmacology research, using authentic, mentored research experiences to heighten student interest in careers in research and related health care disciplines.
Student Mentor Esther Bae Todd Vanderah Michael Gee Ronald Lukas Caleb Kim John Streicher Carolyn Stine John Streicher Trinny Tat Rayna Gonzales
9 THE BECKMAN SCHOLARS PROGRAM The Beckman Scholars Program is designed to help stimulate, encourage and support research activities by exceptionally talented undergraduate students, who ultimately will become leaders in their scientific and professional pursuits. The Beckman Scholarship at the University of Arizona provides funding for students to conduct in-depth research with one of 15 stellar research mentors in UA’s College of Science. Funding for this program is provided by the Arnold and Mabel Beckman Foundation.
Student Mentor Stephen Yao Daniela Zarnescu Ben Zaepfel Daniela Zarnescu
ENVIRONMENTAL HEALTH SCIENCES: TRANSFORMATIVE RESEARCH UNDERGRADUATE EXPERIENCE (EHS-TRUE) EHS-TRUE, funded by the National Institute of Environmental Health Sciences, provides two years of paid training and research experience in an environmental health sciences research group. The program targets students from backgrounds under-represented in the sciences. The goal of EHS-TRUE is to prepare undergraduates from under- represented backgrounds to enter graduate programs in the environmental health sciences. Additional individual student support was provided by WAESO.
Student Mentor Jonathan Blohm (WAESO) Mary O'Rourke Casey Calderon (WAESO) Nathan Cherrington Rebeca Gardner (WAESO) Raymond Runyan Cheyenne Grabiec Marti Lindsey Christian Jennings (WAESO) Jeong-Yeol Yoon Ricardo Lira Jr. (WAESO) Eli Chapman & Walt Klimecki Juliana Ordine (WAESO) Shane Snyder Miguel Pacheco (WAESO) Richard Vaillancourt Ayumi Pottenger Helena Morrison Sergio Salguero Helena Morrison Karen Serrano (WAESO) Julie Neilson Ruby Sierra (WAESO) Scott Boitano
PARTNERSHIP FOR NATIVE AMERICAN CANCER PREVENTION (NACP) The Partnership for Native American Cancer Prevention (NACP) is a collaboration between Northern Arizona University, the University of Arizona’s Cancer Center, and the Native American Research and Training Center. The mission of the NACP is to alleviate the unequal burden of cancer among Native Americans of the Southwest through research, training and community outreach programs in collaboration with the communities they serve. Funding for NACP is provided by the National Cancer Institute; additional individual student support was provided by WAESO.
Student Mentor Meucci Ilunga (WASEO) Christina Laukaitis Gabriel Kellogg (WASEO) Laura Meredith Nancy Pham Rajesh Khanna Thane Rosette (WASEO) Ronald Heimark Leonard Seanez Ronald Heimark Roxanne Vann (WASEO) H-H. Chow
10 PROZKOUMAT! RESEARCH PROGRAM IN THE CZECH REPUBLIC Prozkoumat! (which means explore in the Czech language) provided two undergraduate students with a ten-week summer research experience at the Institute of Parasitology, Czech Academy of Sciences, in Ceske Budejovice, Czech. The students prepared for their research experience by taking a spring semester course that provided an introduction to Czech culture and history.
Student Mentor Randall Eck Zdenek Paris Kimberly Skvarla Dan Sojka
MINORITY ACCESS TO RESEARCH CAREERS (MARC) PROGRAM MARC is a research, mentoring, financial and academic opportunity for undergraduates belonging to a group considered underrepresented in biomedical research and who have interest and potential to pursue a career in this broad field. The program provides scientific training, financial support, mentoring, assistance in preparing for graduate school, and networking opportunities. MARC is funded by the National Institutes of Health.
Student Mentor Alexandre Cavalcante Todd Camenisch Jacob Croft William Montfort Tyler Espinoza Indraneel Ghosh Mariajose Franco Justina McEvoy Nadia Ingabire Anne Cress Jayme Jackson Koenraad Van Doorslaer Heber Lara William Montfort Marianne Madias Jeong-Yeol Yoon Brittany Williams Daniela Zarnescu
11 ACKNOWLEDGEMENTS
UBRP is funded by private donors, internal contributions, and external grants.
Internal support comes from the offices of the University of Arizona Provost, Senior Vice President for Research, and BIO5, and the Colleges of Agriculture and Life Sciences, Medicine, Pharmacy, Public Health, and Science.
External funding for UBRP comes from: • The American Society for Pharmacology and Experimental Therapeutics for ASPET-SURF • The Arnold and Mabel Beckman Foundation for the UA Beckman Scholars Program • The National Cancer Institute (U54 CA 143924) for the Partnership for Native American Cancer Prevention (NACP) • The National Institute of Environmental Health Sciences (R25ES025494) for Environmental Health Sciences - Transformative Research Undergraduate Experience (EHS-TRUE) • The Western Alliance to Expand Student Opportunities (WAESO) National Science Foundation (NSF-LSAMP) Cooperative Agreement No. HRD-1101728
UBRP would simply not be possible without the help of individuals who have given generously of their time. A hearty thank-you to:
• The faculty, post-doc, graduate student, and staff mentors who educate our students. The Annual UBRP Outstanding Mentor Awards are only a small reflection of our gratitude for your time and commitment!
• The UBRP Ambassadors - You have not only organized field trips, social activities, and educational experiences; you have helped to build life-long friendships, networks, and an enduring UBRP community.
• The UBRP Pen Pals – Thank you for touching the lives of young people and for giving them the encouragement to know that their dreams are within reach.
• The 2017 Summer Small Group Leaders – Your guidance, training, and advice got students off on the right foot last summer, and will carry them far! Thank you for volunteering.
• Our volunteers – Thank you to all of our volunteers who have given of their time and talents to UBRP throughout the year! A special thanks to Dr. Stacey Tecot and Dr. Henry Johnson granted UBRP permission to use their beautiful photography.
We are grateful for your support!
12 THANK-YOU TO OUR PRIVATE DONORS!
For 29 years, the Undergraduate Biology Program has partnered passionate undergraduates with UA scientists to create unique and invaluable hands-on research experiences. UBRP participants study everything from Type 2 diabetes effects on the heart, to the ecology of Gila Monsters, from the transmission of HIV between mother and child, to the genetics of potential biofuel crops, and much more.
These opportunities for students are made possible by support from internal contributors, external grants, and our generous donors, especially the 2017 Friends of UBRP!
2017 FRIENDS OF UBRP:
MESQUITE MEMBERS ($2,000+) Drs. John Hildebrand & Gail Burd (in honor of Dr. Michael Wells) w Mr. Robert J. & Mrs. Kim A. Nelson Dr. Sheldon L. Trubatch & Ms. Katharina Phillips
OCOTILLO MEMBERS ($1,000+) Dr. Susheela Carroll w Dr. Miriam C. Ruth w Mr. Norman P. Soloway & Ms. Kay Ransdell Dr. Teri Suzuki and Mr. Oleg Lysyj w Dr. John A. Szivek
CREOSOTE MEMBERS ($500+) Dr. Richard Austin w Prof. Carol Bender w Mr. Robert F. Bender w Dr. Sajiv Boggavarapu w Dr. Margaret Briehl Mr. Brian D. Massey w Dr. Sarah K. Nelson-Taylor w Dr. Elena Plante w Mr. Robert E. Smith Ms. Samantha Szuter w Dr. Allison L. Titcomb w Drs. VK Viswanathan & Gayatri Vedantam
AGAVE MEMBERS ($250+) Dr. Craig A. Aspinwall w Dr. Leonid Bartik w Mr. Richard P. Edelman w Dr. and Mrs. John Enemark Mr. Kirtland & Mrs. Nancy Gardner w Dr. Wulfila Gronenberg w Dr. Ronald P. Hammer w Dr. Paul A. Klekotka Dr. Christina M. Kochel w Dr. Janna Mundt w Dr. Kenneth R. Teter w Drs. John Umbreit and M. J. Demetras Dr. Ilya Vilinsky w Mr. Doug & Mrs. Andrea Wellington
And thank you to donors who have contributed to UBRP in 2017 and 2018!
The American Endowment Foundation Dr. Henry Johnson The American Online Giving Foundation Mr. John S. L. Kim Dr. Anne K. Chung Dr. Michael S. Kuhns Ms. Alison E. Comrie Mrs. Shelley Lieser Mrs. Jennifer L. Cubeta Mr. Cesar A. Medina Eli Lilly and Company – Matching Gifts Mr. Juan Mena-Gonzalez The Fix – Arizona’s Mac ‘N Chz Headquarters Network for Good Dr. Jeffrey Frelinger (in honor of Carol Bender) Ms. Donna Njemanze Mr. David A. Gonzales Panda Restaurant Group (Panda Express) Ms. Shaina H. Hasan Raytheon Company – Matching Gifts Dr. James T. Hazzard Dr. Eugene G. Sander Ms. Xi He Dr. Daryn A. Stover Juice It Up Smoothies (Jamba Juice) Drs. Leslie Tolbert & Paul St. John Mr. Rohith Jayaram Dr. Josette Weibrecht
A special thank-you to our UBRP Advisory Board:
Dr. John Szivek, Chair Dr. Henry Johnson Dr. Sheldon Trubatch Dr. Nathan Ellis Dr. Teri Suzuki Dr. Ken Wertman Dr. John Enemark Ms. Samantha Szuter Dr. Raymond Woosley
13 UBRP NEEDS YOUR SUPPORT!
Contributions from donors are key in equipping UBRP to support college students in gaining the necessary experience to be competitive for jobs, graduate schools, and professional schools. Our goal for 2018 is to raise $30,000 for student support.
We hope to achieve our fundraising goal to continue to offer the I UBRP experience to as many students as possible, and hope you will consider joining us in this endeavor! Donors are welcome to give online at www.ubrp.arizona.edu/donate, or at the registration table during the Conference. UBRP
Those contributing $250 or more to UBRP as a single gift or in accumulation during the 2018 calendar year will become members of our Friends of UBRP Yearly Membership Program!
Friends of UBRP Membership Levels:
SAGUARO MEMBERS (~$450/month) $5,500 supports one UBRP student conducting research for an entire calendar year! Saguaro Members receive a behind-the-scenes lab tour with a UBRP student, have the opportunity to meet with students at the UBRP Annual Conference in January and enjoy reserved seating at the keynote address. They also receive a program highlight from a current student and are acknowledged on UBRP’s website. Donations of $5,000 or more also qualify donors for a 1-year membership in the College of Science’s Galileo Circle.
PALO VERDE MEMBERS (~$300/month) $3,500 supports one UBRP student conducting research on a part-time basis during the academic year! Palo Verde Members receive a behind-the-scenes lab tour with a UBRP student, have the opportunity to meet with students at the UBRP Annual Conference in January and enjoy reserved seating at the keynote address. They also receive a program highlight and are acknowledged on UBRP’s website.
MESQUITE MEMBERS (~$175/month) $2,000 supports one UBRP student conducting research on a full-time basis during the summer! Mesquite Members have the opportunity to meet with students at the UBRP Annual Conference in January and enjoy reserved seating at the keynote address. They also receive a program highlight and are acknowledged on UBRP’s website.
OCOTILLO MEMBERS (~$100/month) $1,000 enables a UBRP student to travel to an attend a scientific meeting to present his or her research! Ocotillo Members enjoy reserved seating at the UBRP Annual Conference keynote address, receive a program highlight and are acknowledged on UBRP’s website.
CREOSOTE MEMBERS (~$50/month) $500 funds one month of full-time student research during the summer! Creosote Members receive a program highlight and are acknowledged on UBRP’s website.
AGAVE MEMBERS (~$20/month) $250 enables a student to attend the annual UBRP ethics retreat! Agave Members are acknowledged on UBRP’s website.
Contributions of any amount are welcome and appreciated!
Please visit www.ubrp.arizona.edu/friends-of-ubrp for more information on Friends of UBRP. The University of Arizona Foundation is a tax-exempt 501(c)3 organization and your gift is tax deductible.
14 2017 UBRP MENTOR AWARDS
Beginning in 2008, on the occasion of the 20th anniversary of UBRP, we celebrated the outstanding mentorship UBRP students enjoy by creating the first Outstanding UBRP Faculty Mentor and the first Outstanding UBRP Graduate Student, Postdoctoral Fellow, or Research Specialist Mentor Awards. Candidates are nominated by current UBRP students and UBRP alumni. A committee composed of students reviews nominees and selects the award recipients.
2017 Outstanding UBRP Faculty Mentor
Dr. Daniela Zarnescu Professor, Molecular & Cellular Biology Nominated by Matthew Chaung
2017 Outstanding UBRP Graduate Student Mentor
Lindsay Guzman Doctoral Student, Chemistry Nominated by Yannick Schreiber
Nominees for the Outstanding UBRP Faculty Mentor Award are:
Dr. Todd Camenisch, Associate Professor, Pharmacology & Toxicology Dr. Rebecca Gomez, Professor, Psychology Dr. John Jewett, Assistant Professor, Chemistry & Biochemistry Dr. Helena Morrison, Assistant Professor, Nursing
The University of Arizona has a culture that supports undergraduate research. This is something to be celebrated!
15 2017-2018 UBRP AMBASSADORS
The UBRP Ambassadors, the reincarnation of the UBRP Undergraduate Student Advisory Group (USAG), are charged with the responsibility of helping to create community among undergraduate researchers by organizing social activities, providing feedback to the program director, and representing UBRP in speaking to on- and off-campus groups. This year’s officers and members of UBRP Ambassadors are:
Tiffany Cho Jason Juang President Treasurer
Neeraj Vij John Kim Vice President Volunteer Chair
Adam (Kai) Aragaki Matthew Chaung Secretary UBRP Pen Pals Coordinator
Members:
Andrew Alamban Areen Badwal Esther Bae George Barnum Shreya Bellampalli Jonathan Blohm Dez Coleman Steven Fried Emily Galloway Caleb Kim Sakthi Kumar Albert Liu Caitlin Moffett Michael Ragone Matthew Scandura Tala Shahin Charis Springhower Carrie Stine Ashley Wu Stephen Yao
16 2017-2018 UBRP PEN PALS
During the 2008-2009 academic year, UBRPer Misha Pangasa, in conjunction with sixth grade teacher Patricia Robles-Medina at Mansfeld Middle School, initiated the Pen Pals Project. UBRPers volunteer to correspond with sixth grade students during the course of the year. Every May and in December, UBRP students host their sixth-grade Pen Pals in a morning of science activities on campus.
The 2017-2018 Pen Pals are:
Andrew Alamban Kai Aragaki Emma Armstrong Olivia Austin Esther Bae Rachel Bear Bailey Bellaire Casey Calderon Swati Chandra Matthew Chaung Tiffany Cho Randall Eck Maggie Fye Emma Harrell Jason Juang John Kim Sakthi Kumar Arturo Morales Miguel Pacheco Diana Perez Savannah Perno Jessica Renger Rachel Sadler Matthew Scandura Laura Scherliess Karen Serrano Charis Springhower Alyssa Sullivan Meagan Tran Dillon Yup Ryan Zenhausern
17 SUMMER 2017 UBRP SMALL GROUP LEADERS
UBRP students meet in small groups every week during the summer to discuss their research experiences with their peers. Faculty, postdocs, graduate students, and research-experienced undergraduates volunteer their time to facilitate these groups and to mentor undergraduate researchers.
We feel incredibly fortunate that these individuals volunteered their time and talents to serving as small group leaders in Summer 2017! We deeply appreciate their contributions to enriching UBRP students’ experiences.
Anand Annadurai Matthew Schmit Postdoctoral Research Associate Graduate Student, Neuroscience
Tiffani Begay Ariana Stickel Training Program Coordinator, NACP Graduate Student, Psychology
Margaret Briehl Jean-Paul Wiegand Professor, Pathology Graduate Student, Neuroscience
Alison Bockoven Keaton Wilson Postdoctoral Research Associate Postdoctoral Research Associate
Lindsey Crown Stephen Yao Graduate Student, Psychology Beckman Scholar
Kelsey Nation Benjamin Zaepfel Graduate Student, Neuroscience Beckman Scholar
Brittany Peterson Robert Zinna Post-Doctoral Research Associate Postdoctoral Research Associate
18 LIST OF ABSTRACT TITLES AND PRESENTERS 29th ANNUAL UNDERGRADUATE BIOLOGY RESEARCH PROGRAM CONFERENCE (in alphabetical order by presenter’s last name)
MEMBRANE RECONSTITUTION OF SIGNAL TRANSDUCTION EVENT OF A T CELL CO-RECEPTOR PAUL ACOSTA, YUNLONG ZHAO, ENFU HUI
T cell mediated immunity is dictated by signals transduced through many different receptors on the membrane surface. The receptors then bind to the respective ligands from the target cell. These receptors trigger the destruction of tumorous and virus infected cells. However, these cells hijack some co-receptors to escape immune attack. The fundamental mechanism of co- receptor signaling is needed for rational design of immunotherapies. Our laboratory developed a membrane reconstitution system to investigate the precise mechanism of immunoreceptor signaling in T cell. This reconstitution system, combined with live cell imaging and cell culture assays, allowed us to identify CD28, a costimulatory receptor, as the major target for the immune checkpoint receptor programmed-cell-death-protein-1 (PD-1), which is a viable cancer immunotherapy target. Here we used the reconstitution system to determine the ability of PD-1 to recruit cytosolic factors, including the phosphatase and SAP, an adaptor protein that was reported to bind to co-receptors in natural killer cells that contain similar structural motif to PD-1. Our data suggest that Shp2, but not SAP, bind to PD-1, suggesting Shp2 as a major effector of PD-1.
MODELING EFFICIENCY OF DIFFERENT TASK ALLOCATION STRATEGIES IN ANTS ABRIELLE AGRON, ANNA DORNHAUS
Ants perform many different types of tasks in the nest, such as brood care, undertaking, and debris removal. Using a simple agent-based model made in NetLogo, we examined the efficiency of two different task allocation strategies for different numbers of ants and “task-objects” which represented the tasks present in a nest. The two task allocation strategies were “foraging for work” in which ants worked on any task object they encountered, and “response thresholds” in which each ant has various “thresholds” for different tasks, and only worked on a task object they encountered if the demand of that object exceeded their personal threshold. Existing literature suggests that the “foraging for work” strategy, which implies uniform workers, is more efficient in colonies with less workers, while the “response thresholds” strategy, which implies specialized workers, is more efficient in colonies with more workers. To measure the efficiency of each colony, the average maximum demand of the task-objects was measured. The difference of “foraging for work” efficiency and “response threshold” efficiency was taken and plotted against ant numbers to establish a relationship between the strategies. A positive relationship would support the hypothesis that worker specialization is more efficient in larger colonies. A negative relationship would support the hypothesis that worker specialization is more efficient in smaller colonies. No relationship would support the null hypothesis, that efficiency of worker specialization is not dependent on colony size. Current data supports the null hypothesis, although data collection is still in progress and there is reason to believe that the collection of more data points, especially with a larger variation in number of “task-objects” could support different hypotheses.
CX37-13K IS GROWTH SUPPRESSIVE DESPITE INABILITY TO FORM GAP JUNCTION CHANNELS ANDREW ALAMBAN, TASHA K. PONTIFEX, JANIS M. BURT
Connexins (Cx) comprise a family of gap junction proteins that are expressed in multiple cell types, including cardiomyocytes and vascular endothelial cells. Structurally, connexins have four transmembrane domains, two extracellular and one intracellular loop, and intracellular amino- and carboxyl-termini (NT & CT, respectively). Six of these connexins oligomerize to
19 form hemichannels and hemichannels from neighboring cells dock to form a gap junction channel. The hemichannels and gap junction channels support intracellular, transmembrane and intercellular signaling. Cx37 and Cx43 are alternately expressed in arterial vascular endothelium; Cx37 expression is downregulated following injury whereas Cx43 expression is upregulated. Cx37 and Cx43 have inverse effects on cell growth: Cx37 suppresses growth while Cx43 supports growth. These differences suggest that connexins could be utilized, therapeutically, to promote vascular repair or suppress tumor growth. Our lab has used an inducible rat insulinoma (iRin) cell model to investigate the mechanisms underlying Cx37’s anti-proliferative effects. Available data indicate that growth suppression requires that Cx37 form functional gap junction channels where the CT domain is able to interact with channel forming domain in a phosphorylation specific manner. To form such channels, the oligomerized hemichannels must be transported to the cell’s plasma membrane where they dock to form gap junction channels with hemichannels in neighboring cells. Literature has shown that a 20 kDa isoform (20k) of Cx43 –consisting of a portion of the fourth transmembrane domain and the entirety of the CT – helps with trafficking of Cx43 hemichannels to the plasma membrane. We hypothesized that the corresponding 13 kDa isoform (13k) of Cx37 – with structure analogous to that of the 20k isoform of Cx43 – will similarly facilitate trafficking of Cx37 hemichannels to the plasma membrane and, possibly, exert a growth suppressive effect independent of, and synergistically with, gap junction formation. To determine whether Cx37-13k exerts a growth suppressive effect, we plated iRin37-13k cells and monitored proliferation over the course of three weeks. We found that cells expressing Cx37-13k were not as growth suppressed as those expressing full-length Cx37, but were more growth suppressed than cells with no Cx37 expression. Both immunofluorescence and immunoblotting data suggest that Cx37- 13k localizes to the plasma membrane. These results suggest that despite not being able to form functional gap junction channels, the 13k isoform has growth suppressive properties. This research was supported by UBRP in part by funds from the College of Medicine, and NIH grants HL122443.
INCREASING KISSPEPTIN-10 APPARENT AFFINITY THROUGH HETERO-MULTIVALENT TETHERING ADAM ARAGAKI, C. WEBER, R. LYNCH
Targeting cells in vivo with high specificity is challenging due to the commonalities shared between cell types. Antibody-based approaches have strength in their ability to bind with exquisite specificity, but fall short in terms of cell access due to their large size. Targeting single receptors that are overexpressed on a certain receptor is generally error prone due to the presence of the receptor on other cell types, as even minute receptor expression can result in undesired side effects upon binding. Our lab has shown that simultaneously targeting multiple receptors, that as a combination are specific to the cell of interest, enhanced cell type specificity. We have utilized a modular approach to combine two ligands via a linker chain, creating a multivalent ligand. By choosing ligands that match the receptor combination, cells can target with relatively high avidity and specificity. This approach also can be used to increase the sensitivity to low affinity ligands by linking them to a higher affinity component to increase their local concentration at the membrane. Since the high affinity ligand binds first, this also provides specificity of activity to cells expressing the two complementary receptors. In this project, a high affinity (20 nM) ligand Glucagon Like Peptide-1 (GLP-1) was linked to a fragment of the neuropeptide Kisspeptin (Kiss-10, 5 uM Kd). We studied the ability of GLP-1/Kiss-10 to exhibit Kiss effects at concentrations where GLP-1 is effective, but monomeric Kiss-10 is not. Funded in part by the American Diabetes Association and by UBRP with funds from BIO5.
INFLUENCE OF SLEEP-AFFECTING DRUGS ON MEMORY CONSOLIDATION IN RATS EMMA ARMSTRONG, JEAN-MARC FELLOUS
Memories are consolidated during sleep, and can be activated for further consolidation by use of sounds associated with those memories. This project focuses on the effect of sleep-altering drugs on the ability of rats to consolidate memories during sleep when those memories are targeted for reactivation using brief sounds. Rats are trained to learn a set of three active feeders and then ingest melatonin, caffeine, or zolpidem before memory consolidation occurs. After a sleep period, they are tested for
20 recall. Longer sleep durations should lead to better recall; however, it is possible that these drugs may affect reconsolidation significantly beyond the effects expected from sleep duration. Results thus far indicate that zolpidem both increases sleep and recall performance, while caffeine reduces sleep and recall performance. Melatonin produces mixed results. None of these drugs show a significant behavioral effect separate from that expected based on the change in sleep length. These results shed light on some of the cognitive consequences of commonly used sleep altering substances such as caffeine, zolpidem, or melatonin. Funding in part from UBRP with funds from the Office of the Provost.
FOXO3A RESPONSE TO TARGETED THERAPIES IN SINGLE CANCER CELLS KAYENAT ARYEH, LISA SHANKS, ANDREW PAEK
One of the biggest advancements in cancer treatment has been the development of targeted therapy. Unlike standard chemotherapy, targeted therapies go after specific alternations in cancer cells, minimizing the side effects in patients. However, many targeted therapies are not maximally effective because not all cancer cells die in response to the treatment. FOXO3A is a transcription factor that controls whether cells live or die in response to several different targeted therapies including EGFR inhibitors. We have tagged FOXO3A in lung and breast cancer cells using CRISPR/Cas9. We are using these tagged lines to follow the FOXO3A dynamics of single cancer cells in response to the EGFR inhibitors to better identify patterns associated with cell death.
Funded in part by the Undergraduate Biology Research Program (UBRP) with funds from the Senior Vice President of Research and Friends of UBRP, and the Paek Lab.
AMYLOID BETA-INDUCED ALTERATIONS IN BASAL FOREBRAIN CHOLINERGIC NEURON (BFCN) MORPHOLOGY AND SURVIVAL ARE REGION SPECIFIC SRI SAI SWETHA ATLURI, ILEANA LORENZINI, JASMINE GILL, RONALD J. LUKAS, PAUL WHITEAKER, ANDREW A. GEORGE
Alzheimer’s disease (AD), a progressive neurodegenerative disorder, is one of the most common causes of mental deterioration in the elderly. Several studies have correlated the cognitive severity of early-onset AD with an early loss of BFCNs. Concurrently, new-onset epileptic seizures occur in patients with AD, but the nature and underlying reasons for these seizures are unclear. Given this uncertainty, this project seeks to define the relationship between amyloid beta (Ab), the main component of amyloid plaques involved in AD, and basal forebrain neuronal hyperexcitability as it relates to the survival and cytoarchitectural complexity of BFCNs. We examined the role of the different isoforms of Ab (oligomeric and fibrillar) in inducing dysmorphic changes to the cholinergic neurons in the three main regions of the basal forebrain: Medial Septum Ventral Diagonal Band (MSVDB), Horizontal Diagonal Band (HDB), and the Nucleus Basalis (NB). Organotypic cultures from ChAT-eGFP transgenic mice were incubated in the different isoforms of Ab for 10 days and then imaged using confocal microscopy. Volumetric and Sholl analysis were used to measure differences in neuronal volume and the complexity of dendritic processes of cholinergic neurons within the MSVDB, HDB, and the NB in the presence or absence of Ab. Our findings indicate a significant decrease in the number of BFCNs in the MSVDB (p<0.001) and the NB (p<0.05) when treated with oligomeric or fibrillar Ab. While there was a significant loss of BFCNs in the HDB when treated with oligomeric Ab (p<0.05), there was no significant loss when treated with fibrillar Ab. After morphometric analysis, we observed a significant decrease (p<0.05) in soma volume for HDB BFCNs when treated with fibrillar Ab. However, there was no significant change in dendritic length or complexity in the presence and absence of Ab. These interactions may be uniquely specific to certain cholinergic circuits within the basal forebrain and suggest novel and potentially productive therapeutic strategies to combat neurodegeneration in a brain region affected early in AD. This work was supported by UBRP with funds from the Senior Vice President for Research.
21 BIRDSONG AS A MODEL FOR AGING VOICE AREEN BADWAL, JOHANNA POERTNER, JULIE MILLER, ROBIN SAMLAN
As humans naturally age, vocal production deteriorates resulting in communication deficits and decreased quality of life. Vocal deficits include pitch, loudness and speech rate changes. These deficits are exacerbated in those with neurodegenerative diseases affecting motor control pathways, such as Parkinson’s disease. Although vocal deficits have been characterized in the human aging population, there is a critical gap in knowledge about the neural mechanisms that contribute to the vocal difficulties. To address this question, we use the zebra finch songbird as an optimal model for studying the neural basis for aging voice. The zebra finch songbird has similarities to human vocal learning and production and like humans, they continually fine-tune their vocalizations throughout their life. Zebra finches have song-dedicated brain nuclei that closely approximates human speech and language areas. In the current study, we analyzed features of birdsong in young and elder adult male zebra finches using Pratt, an acoustic analysis program used to evaluate human voice production. We developed a set of common measures to compare birdsong and human voice data. Song was analyzed for acoustic features such as frequency, intensity, speech rate and articulation rate. We hypothesize that the songs of aging finches will experience changes in acoustic features similar to those of the aging human voice. In the future, we plan to identify gene expression changes in the brain related to age- induced changes in acoustic features. Supported in part from UBRP with funds from the Senior Vice President for Research.
ACTIVATING THE A3 ADENOSINE RECEPTOR TO TREAT HIV-INDUCED NEUROPATHY ESTHER BAE, HONG ZHANG, DANIELA SALVEMINI, TALLY M. LARGENT-MILNES, TODD W. VANDERAH
HIV-induced neuropathy, a common form of chronic pain, affects a large population of those infected with HIV – an estimate of 37 million people around the globe. Opioids, although being the main analgesics for HIV pain, lack efficacy in this patient population. The national opioid epidemic has headlined the issue of overprescribing narcotics and narcotic abuse, which is largely due to these substances activating the brain’s reward pathway. Found on the surface of the HIV envelope, glycoprotein 120 (gp120) mediates glial cell receptor activation and release of proinflammatory cytokines in the central nervous system resulting in neuropathic pain. It was previously reported that patients with chronic neuropathic pain have dysregulation of adenosine signaling – adenosine serving as an important endogenous regulator of inflammation. Here we asked whether targeting the adenosine axis with a selective A3 adenosine receptor agonist, MRS 5698, would reverse HIV-gp120-induced pain. Gp120 was intrathecally administered to rats to induce HIV neuropathy over the course of 3 weeks. Pain behaviors were assessed using von Frey (mechanical allodynia) and Hargreaves’ (thermal hypersensitivity) assays on days 7, 14, and 24 days post gp120 injection. We found that rats developed increased sensitivity to mechanical and thermal stimuli. Gp120 rats were injected into the intraperitoneal cavity (i.p.) with MRS 5698 or vehicle (4.4% DMSO in saline) on day 24; pain thresholds were then measured over a 2-hr time course. We found that MRS 5698-treated rats had higher pain thresholds to mechanical stimuli compared to those of the vehicle group. MRS 5698-treated rats also showed an increased latency to thermal stimuli with a peak at 90 minutes, and returned to baseline over the time course whereas, the vehicle group had no trend. These data suggest that activation of the adenosine A3 receptor is a potential alternative to opioids for the therapeutic management of HIV-induced neuropathy.
Funding, in part, by the American Society for Pharmacology & Experimental Therapeutics Summer Undergraduate Research Fellows (ASPET/SURF grant).
22 FUNCTIONAL INTERACTIONS BETWEEN TDP-43 AND TRANSLATIONAL MACHINERY IN A DROSOPHILA MODEL OF AMYOTROPHIC LATERAL SCLEROSIS RACHEL BEAR, BEN ZAEPFEL, SHIZUKA YAMADA, LINH PHAM, DANIELA ZARNESCU
Amyotrophic Lateral Sclerosis (ALS) is a fatal disease that causes progressive neurodegeneration of motor neurons. TAR DNA Binding Protein (TDP-43) has been implicated in the progression of ALS, as well as at the level of pathology. TDP-43 is an RNA- binding protein that is known to regulate many steps of RNA processing. However, little is known of TDP-43’s role in the dysregulation of translation. Several eukaryotic initiation factors (eIFs) have been identified in TDP-43-positive stress granules. Here we show that TDP-43 targets translational proteins and that changing expression levels of eukaryotic initiation factors (eIFs) to reduce translation is neuroprotective in a Drosophila model of ALS. We show genetic and functional interactions between TDP-43 and several eIFs. For example, when various eIFs are co-altered in the context of human TDP-43 in motor neurons of Drosophila, locomotor deficits are rescued and eye depigmentation is suppressed. Results from quantitative PCR demonstrate that TDP-43 alters levels of eIF mRNA transcripts in motor neurons. Furthermore, results from Western blotting suggest that eIFs affect the level of TDP-43 protein found in whole larvae. We will further explore the interaction between TDP- 43 and these eIFs in patient-derived lymphoblastoid cells. As we identify specific translational mechanisms that are dysregulated by the presence of cytoplasmic TDP-43, new targets will emerge for the development of novel therapies for ALS.
Acknowledgements: Muscular Dystrophy Association 255293, National Institutes of Health RO1 NS091299, Arnold and Mabel Beckman Foundation, Undergraduate Biology Research Program with funding from the Office of the Senior Vice President for Research.
SYNTHETIC ROUTES TO APOMORPHINE BAILEY BELLAIRE, KEVIN SCOTT, CHRIS MARSHALL
Dopamine receptors, a class of G protein-coupled receptors, are implicated in a multitude of neurological processes including cognition, memory, and fine motor control. Abnormal signaling results in neuropsychiatric disorders. Apomorpphine is an FDA- approved non-selective dopamine receptor agonist that activates dopamine receptors to produce a biological signaling response to treat Parkinson's disease. Due to lack of selectivity, it has serious side effects. Limited synthetic routes exist for the generation of apomorphine analogues, making it difficult to increase efficacy or reduce side-effects of the drug. We have employed novel chemical methodologies to generate the apomorphine carbon skeleton from scratch. This new synthetic route affords the possibility to produce analogs to increase the biological specificity of apomorphine. Support provided in part from UBRP with funds from the UA College of Medicine.
QUANTITATIVE RISK ASSESSMENT: ARSENIC EXPOSURE FROM DRINKING WATER JONATHAN BLOHM, MARY KAY O'ROURKE, ROBIN HARRIS
Arsenic, a natural occurring metalloid found in our Earth’s crust and soil has been associated with the development of many pulmonary disorders and cardiovascular diseases. In particular, long-term exposure to levels exceeding 100 mg/L in drinking water of inorganic species As3 and As5 are associated with development of cancers. Arsenic exposure data from drinking water in parts of Arizona and Sonora, Mexico is limited. In this study, we used data from the Binational Arsenic Exposure Survey (BAsES) conducted in 2012 to perform a quantitative risk assessment on the relative cancer risk from arsenic in drinking water. The study conducted surveys in 8 different locations known to contain elevated arsenic levels in groundwater, 4 in Southern Arizona and 4 in Sonora, Mexico. The measurements taken included arsenic concentrations in unfiltered drinking water, arsenic concentrations in urinary output of participants and questionnaires used to assess demographics and average fluid ingestion rates. Exposure assessment models were used to quantify the average daily intake of arsenic at each location. Then
23 probabilistic uncertainty analyses were performed using Monte Carlo simulation software to obtain relative cancer risks at each different geographic location. Preliminary data indicate that at each of the 8 locations in Arizona and Mexico, the risk of cancer based on arsenic exposure exceeded the acceptable risk limit of 106 carcinogens per lifetime. Supported by NIEHS R25 ES025494 and Western Alliance to Expand Student Opportunities (WAESO) Louis Stokes Alliance for Minority Participation (LSAMP) National Science Foundation (NSF) Cooperative Agreement No. HRD-1101728.
PIGMENT OR POISON? A COMPREHENSIVE LOOK AT TATTOO INK IN THE US JONATHAN BLOHM, AYUMI POTTENGER, BECA GARDNER, CASEY CALDERON, CHEYENNE GRABIEC, CHRISTIAN JENNINGS, JULIANA ORDINE, KAREN SERRANO, MIGUEL PACHECO, RICARDO LIRA JR., RUBY SIERRA, SERGIO SALGUERO, CAROL BENDER, WALT KLIMECKI
Today roughly 3 in 10 Americans have tattoos. However, research and regulation on tattoo ink is limited. In the Fall 2017 semester, the students of EHS-TRUE conducted a thorough literature review on the risks and regulations of tattoo ink in the US. We collaborated with both international scientists conducting research on the toxicology of tattoo ink, as well as scientists from the FDA concerned with regulation of inks. Our initial findings suggest an abundance of potential adverse health risks associated with different tattoo inks commonly used today. These risks range from a variety of cancers, to autoimmune deficiencies, to inflammatory reactions all resultant from unregulated mutagenic, genotoxic and carcinogenic substances found in tattoo inks. However, our research is subject to limitations due to sampling biases in current tattoo studies. The lack of regulation of tattoo inks in the US is unacceptable given the widespread popularity of tattoos, and the potential health risks that tattoo inks may pose. Thus, more research on the link between health effects and tattoo ink, leading to better regulation of ink composition, is imperative to mitigating the health risks of getting a tattoo.
BOLDNESS AS A HERITABLE TRAIT IN BLACK WIDOW SPIDERS CHARLES BRADLEY, NICHOLAS DIRIENZO, ANNA DORHAUS
Many non-morphological traits, like behavioral propensities, can be passed from a parent onto their offspring. One such behavior, which is defined as the likeliness of engaging in risky behavior, is referred to as “boldness”. To demonstrate if this fundamental aspect of behavior is heritable, a series of experimental trials were performed on adult western black widow spiders (Latrodectus hesperus) and their offspring. For these spiders, boldness can be exhibited as increased number of prey attacks, heightened foraging habits (constructing prey-capturing web structures), and emergence rates from egg sacs. For the adult spiders, the prey attacks were assessed with the application of a vibratory stimulus to the web (simulating entangled prey). Another evaluation of adult boldness was the number of constructed “gum-footed” web lines, which are almost exclusively used for prey capture (and not defense). For the offspring of the adults used in the trials, the boldness was assessed by the rates of emergence from the egg sac (at week one and at week two). The resulting data was arranged into two comparative sets: “Attack Number vs. Emergence Rate” and “Gum-Footed Lines vs. Emergence Rate”. From these data sets, the results were found to be non-significant. Examination of other factors, such as the boldness traits of the copulatory male spiders or the effects of proximal offspring cannibalism, can be further explored.
24 TWO NOVEL TYPE SIX SECRETION SYSTEMS IN THE ENTOMOPATHOGENIC BACTERIUM XENORHABDUS BOVIENII MAY IMPACT BACTERIAL COMPETITION, MOTILITY AND BIOFILM FORMATION CHRISTINE BRADSHAW, REBECCA MCQUADE, S. PATRICIA STOCK
Xenorhabdus bovienii is a Gram-negative bacterium that exhibits a dual lifestyle, behaving as a mutualist to Steinernema nematode species and as a pathogen to insects. The genome of X. bovienii encodes two type VI secretion systems (T6SS), which allow Gram-negative bacteria to transport effector proteins directly into target cells. In previously studied bacteria, T6SSs play a key role in pathogenesis and in competition with other bacteria, as well as in coordinating group behaviors such as motility. The roles of the T6SSs in X. bovienii are unknown but X. bovienii interacts with other bacteria and eukaryotic cells in its lifecycle. In this study, we generated three X. bovienii mutants: one lacking hcp (a crucial structural gene) from T6SS1, another lacking hcp from T6SS2, and a double mutant lacking hcp from T6SS1 and T6SS2. The mutants were used in bioassays to assess the role of the two T6SSs. Preliminary results show no differences in melanization and mortality in insects infected with the T6SS mutants compared to insects infected with wild-type X. bovienii. In vitro bacterial competition assays indicate that the hcp1 mutant is defective in competition when competed against other X. bovienii strains or the wild-type, whereas the hcp2 mutant is not defective. The hcp1 and hcp2 mutants are similar to the wild-type strain in motility and biofilm formation however, the hcp1/2 mutant is defective in both suggesting a redundant role for the two T6SSs in these behaviors. We speculate the two T6SSs may play distinct roles in bacterial competition, motility, and biofilm formation which may contribute to the success of the mutualistic relationship between X. bovienii and Steinernema nematodes. This research is supported by The University of Arizona’s NIH IRACDA Grant (PERT) and The Center for Insect Science.
ALTERED HEPATIC TRANSPORTER AND ENZYME EXPRESSIONS IN NASH/NAFLD CASEY CALDERON, HUI LI, ERICA TOTH, NATHAN CHERRINGTON
The disposition and metabolism of the environmental toxicants and clinical drugs can be summarized in four processes: absorption, distribution, metabolism, and elimination (ADME). ADME processes are mediated by metabolizing enzymes and transporters throughout the body. Alterations in these enzymes may negatively impact ADME pathways, lead to variable responses to xenobiotics and induce potential toxicities. Liver is the primary organ for ADME processes. Liver diseases, such as Nonalcoholic Steatohepatitis (NASH) can alter the functions of transporters and enzymes. NASH is an advanced form of nonalcoholic fatty liver disease (NAFLD) which is a condition in which fat deposits form in the liver and is thought to be a hepatic manifestation of metabolic syndrome. NASH differs from NAFLD in that it is marked by inflammation of liver tissue as well as irreversible liver cell damage. If left untreated, NASH may lead to cirrhosis and liver cancer. NASH is thought to be induced by a two-hit mechanism; the first “hit” is the accumulation of fat deposits in the liver (NAFLD), while the second “hit” is caused by environmental insults. Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) enzymes are important metabolizing enzymes involved in the metabolism of both endogenous and exogenous compounds. Hepatic transporters such as Multidrug resistance-associated protein 2 (MRP2) and Breast Cancer Resistant Protein (BCRP) are responsible for the disposition of numerous pharmaceutical drugs. We have found via western blots that NASH livers show significant alterations in the expression of these enzymes and transporters compared with healthy livers. These changes may have an effect on the ADME of many environmental compounds. Finally, NASH should be considered as a source of inter-individual variation in the response to environmental exposures. The research is supported by funding from National Institute of Environmental Health Sciences, Grant #1-R25-ES025494. Further acknowledgment is given to Western Alliance to Expand Student Opportunities (WAESO) Louis Stokes Alliance for Minority Participation (LSAMP) National Science Foundation (NSF) Cooperative Agreement No. HRD-11017 for providing additional funding in support of this research.
25 PROTEOMICS UNDERLYING MENOPAUSE SUSCEPTIBILITY TO PATHOLOGICAL CARDIAC REMODELING DANIELLE CANNON, MARISSA LOPEZ-PIER, MATTHEW KOPPINGER, GERRIE FARMAN, HEDDWEN BROOKS, MIENSHENG CHU, JOHN P. KONHLIAS
Cardiovascular disease (CVD) is the leading cause of death in women, contributing to 1 out of every 3 deaths per year. Our lab has shown CVD susceptibility increases between premenopause and postmenopause, yet the underlying mechanisms during the transition are still unknown. We have found that 5’AMP-activated-protein kinase (AMPK) interacts with estrogen and has a signaling pathway that is suppressed in postmenopause compared to premenopause cardiomyocytes. We believe that AMPK plays a cardioprotective role in premenopausal women and has the potential to be used as an effective treatment for CVD. AMPK can enter the nucleus to directly alter histone activity and cardiac gene expression, yet a greater understanding of the specific interactions between AMPK and transcriptional regulators is needed. Our research aims to determine the nuclear activity that influences the transition from premenopause and postmenopause by utilizing an in vivo AMPK activator (APEXBIO;A76692).
To model the natural progression of menopause, intact ovaries of female mice were injected with 4-vinylcyclohexene diepoxide (VCD), a chemical compound that accelerates ovarian degeneration. Estrous cycles were monitored with vaginal cytology to affirm pre-, peri-, and postmenopause in all mouse groups. Angiotensin II was administered at a hypertensive dose to stimulate pathological cardiac remodeling. After AMPK activator was dosed for 2 weeks, left ventricle tissue was collected and western blot techniques were used to assess nuclear dynamics by measuring pAMPK(T172)/AMPK, histone H2B, and transcriptional regulator BRD4 activity.
We found that H2B and BRD4 interact with AMPK in total tissue and isolated histone lysates. H2B is present in premenopausal control mice and postmenopausal mice with pathological cardiac remodeling. BRD4 is present in both premenopausal and postmenopausal mice, yet there is a significant (*p<0.05) increase in BRD4 activity in postmenopausal mice that is augmented by pathological cardiac remodeling. Our results show that AMPK plays an active role in the underlying dynamic nuclear activity that occurs during the transition to menopause and during cardiac remodeling. This research was supported by UBRP with funding from the Senior Vice President for Research, and American Heart Association (16GRNT31390006).
EVALUATING CHEMOSENSORY PROCESSING IN THE SPIDER PERIPHERAL AND CENTRAL NERVOUS SYSTEMS BRIGGS CARHART, SKYE LONG, WULFILA GRONEBERG
Spiders and other arachnids have been primarily studied for their mechanoreception and their visual capabilities to navigate throughout their environments, but not enough is known about their chemosensory or olfactory processing. Chemosensory hairs (‘sensilla’) are found on the tarsi of spider legs and on the pectens of scorpions (arachnids). Spider brains, primarily from local Hogna (wolf spider) species, underwent various staining and tracing protocols to parse out possible processing centers for chemosensory and olfaction in the peripheral and central nervous systems. Across the animal kingdom, olfactory processing centers are distinguished by their organization based on spherical suib-units, called glomeruli. Osmium stained brain slices show putative glomerular structures in the neuropil of the spider. Protocols of live electrophoresis and direct neural track injections were performed to backfill these structures, but the anatomy of the spider made these experiments more complicated than expected and specific steps in the protocol need to be adapted to work in arachnids. Our results suggest that the anatomy of the arachnid olfactory pathways differs from those in insects and that spiders may rely more on chemosensory reception than previously assumed from studies on the optic and mechanosensory tracts.
Funding: UBRP with funds from the Office of the Provost, and NSF Grant 1456221
26 RESISTANCE TO BACTERIAL MEDIATED KILLING COMPLEXES IN P. SYRINGAE ETHAN CARLSON, KEVIN L HOCKETT, DAVID BALTRUS
Pseudomonas syringae is a ubiquitous, bacterial plant pathogen, of which there exists a multitude of pathovars capable of causing disease in a variety of agricultural crops. As part of its lifestyle P. syringae grows on the surface of leaves, as a non- pathogenic epibiont, disease only occurs upon infiltration of the bacterium into the leaf interior. As a result of competition for space and resources on the leaf surface Pseudomonas syringae produces bacteriocins (syringacins), which are proteinaceous toxins that inhibit the growth of closely related strains of bacteria. Previous work done in the lab has allowed us to isolate and characterize different phenotypes of syringacin resistant strains of P. syringae pv. phaseolicola (Pph), and pv. glycinea (Pgy), and recover specimens of both pathovars that show heritable resistance to syringacins between generations. Our work has been focused on looking at the genetic basis of resistance and the effect that resistance plays in the production of different types of bacteriocins within the same bacterial genus. Support provided in part from UBRP with funds from Office of the Provost.
OVERACTIVATION OF CA2+/CALMODULIN-DEPENDENT PROTEIN KINASE IV CONTRIBUTES TO ENHANCING PULMONARY ARTERIAL SMOOTH MUSCLE CELL PROLIFERATION IN PATIENTS WITH IDIOPATHIC PULMONARY ARTERIAL HYPERTENSION SHANE CARR, SHANSHAN SONG, KANG WU, ZIYI WANG, JASON X.-J. YUAN
2+ 2+ A rise in cytosolic free Ca concentration ([Ca ]cyt) in pulmonary arterial smooth muscle cells (PASMCs) causes pulmonary vasoconstriction and increases PASMC proliferation, leading to concentric arterial wall thickening. Ca2+/Calmodulin-Dependent 2+ Protein Kinase IV (CaMK IV) is part of a family of serine/threonine kinases that are activated by an increase in [Ca ]cyt. STIM2, a 2+ 2+ Ca sensor in sarcoplasmic reticulum (SR), has been suggested to mediate increases in the resting [Ca ]cyt in PASMCs from patients with idiopathic pulmonary arterial hypertension (IPAH). PDGF-R is a tyrosine kinase receptor that, when activated by PDGF, can cause cell proliferation. Our hypothesis is that overactivation of CaMK IV upregulates STIM2 and PDGF-R, which stimulate PASMC proliferation in patients with IPAH. In this study, we found that the protein level of CaMK IV is significantly increased in PASMCs from patients with IPAH (1.00±0.588 vs 11.4±1.29, n=3 normal subjects and 5 patients, p=0.001). Knockdown of CaMK IV in IPAH PASMCs with CaMK IV specific siRNA significantly decreased the protein level of STIM2, PDGF-R α and PDGF-R β, and also significantly decreased cell proliferation, which is indicated by the decreased EdU incorporation rate, an indicator of DNA synthesis, and total cell number in IPAH PASMCs. Together, these data indicate that overactivation of CaMK 2+ IV in IPAH PASMCs, due to upregulation of CaMK IV and/or increased resting [Ca ]cyt, contributes to enhancing PASMCs proliferation by upregulating STIM2 and PDGF-R. CaMK IV activation-induced upregulation of STIM2 will further raise the 2+ resting [Ca ]cyt and subsequently activate CaMK IV, forming a positive feedback loop. The overactivation of CaMK IV may be an important mechanism involved in sustained pulmonary vasoconstriction and excessive pulmonary vascular remodeling in patients with IPAH. This work was supported by UBRP with funds from the Office of the Provost, and grants from the National Heart, Lung and Blood Institute of the National Institutes of Health HL135807 and HL125208.
LINE-1 ACTIVITY IN DEVELOPMENTAL CARDIAC EMT AND ITS CONSEQUENCES IN HEART MORPHOGENESIS ALEXANDRE CAVALCANTE, TODD CAMENISCH
Congenital heart defects (CHD) are malformations in the structure of the heart that cause defective functioning of the cardiovascular system. CHD are a leading cause of newborn mortality in the first year of life in the United States. Some of the diseases resulting from CHD include: coronary artery disease, valve disease, high blood pressure, congestive heart failure, arrhythmia, and stroke. A key process in heart morphogenesis is epithelial-to-mesenchymal transition (EMT), a process that
27 produces the cardiac mesenchyme. Cardiac developmental EMT causes increased cellular motility and invasive phenotype, which are essential for proper formation of non-myocyte heart cells, which contribute to structures such as the heart valves, interventricular septum, and coronary vessels. Developmental EMT in the heart is mediated by the transforming growth factor β2 (TGFβ2) signaling pathway through canonical SMAD activity. Long Interspersed Element-1 (LINE-1 or L1) is a retrotransposon that constitutes 17% of the human genome and is active during early stages of development before it is silenced. Through collagen gel invasion assays using ORFeus mice heart explants, we found that, when aberrantly expressed in later stages of development, LINE-1 disrupts cardiac developmental EMT by preventing SMAD activity in the TGFβ2 signaling. The forthcoming consequences are disrupted EMT and loss of cardiac mesenchymal cells. In summary, LINE-1 reactivation in later stages of development is antagonistic to developmental cardiac EMT, predisposing the onset and progression of heart malformations.
Funding: NIH MARC Training Grant: T34-GM-08718
COPPER HOMEOSTASIS MODULATES LOCOMOTOR FUNCTION AND ALS PHENOTYPES IN VIVO MATTHEW CHAUNG, BRIGGS CARHART, SAMANTHA MACKLIN-ISQUIERDO, ROBERT KRAFT, DANIELA C. ZARNESCU
Menkes disease is an infantile-onset lethal disease1. It is characterized by failure to thrive and underdeveloped nervous systems. The disease is caused by a mutation in the N terminus of the ATP7A P-type ATPase transporter, which is responsible for the uptake of copper, an important metal ion required for early embryonic neural development1. Interestingly enough, when this same ATP7A protein is mutated in the C-terminus, patients develop adult-onset distal motor neuropathy rather than Menkes disease. This kind of patient mutation has been reported in the literature1 and does not show clinical signs of altered copper levels. For this reason, this mutation is particularly striking given that it leads to motor neuron degeneration and may be linked to the progression of Amyotrophic Lateral Sclerosis (ALS). To investigate this, we utilized the fruit fly Drosophila as a model organism, which is capable of recapitulating ALS/neuromuscular diseases with symptoms including locomotor dysfunction and shortened lifespan that are also seen in patients2.
Through our investigation, we have found that altering endogenous Drosophila ATP7 function (dmATP7) leads to locomotor phenotypes. Interestingly, when we knockdown or overexpress the dmATP7 copper transporter we observe similar locomotor defects, signifying that the regulation of copper is very precise and motor neuron function is highly sensitive to perturbation. In addition, when we express the ATP7AWT or a patient-derived mutation we obtained from our collaborators, we continue to see locomotor phenotypes. This is quite promising for the development of a novel fruit fly model aimed at identifying potential therapeutic agents. Furthermore, we also report that Tar DNA Binding Protein-43 (TDP-43) a pathological hallmark of ALS exhibits genetic interactions with altered copper homeostasis. We found that knockdown or overexpression of dmATP7 modulates TDP-43 induced locomotor defects.
Funding was provided by generous benefactors, NIH NS NS091299 (to DCZ) and the Undergraduate Biology Research Program (UBRP, to MC and BC, with funds from the Office of the Provost).
HIGH-THROUGHPUT CHEMICAL SCREENING IDENTIFIES SGM-45 AS A SELECTIVE INHIBITOR OF N-TYPE VOLTAGE-GATED (CAV2.2) CHANNELS LINDSEY CHEW, Z. SHUJA, V. GOKHALE, X. YANG, Y. JI, A. MOUTAL, Y. WANG, A. DÓRAME, S. S. BELLAMPALLI, T. W. VANDERAH, M. KHANNA, H. M. COLECRAFT, R. KHANNA
Inhibition of voltage-gated calcium (Cav) channels is a potential therapy for many cardiovascular and neurological diseases. Neuronal Cav1/Cav2 channels are typically composed of α, β and α2δ subunits. The β-subunits of voltage-gated Ca2+channels are cytoplasmic proteins that increase the surface expression of the pore-forming α subunit of Ca2+ channels and regulate the
28 biophysical properties of the channel. They do so via a high-affinity protein-protein interaction pocket with the α-subunit of Cavs. Thus, targeting the Cav α-β interaction should result in a new class of calcium channel antagonists with therapeutic potential in nervous system disorders involving dysregulation of calcium. To date, there are no small molecules that physically and selectively disrupt the α-β protein-protein interaction. Here, structure-based virtual screening of a commercial library of 500,000 small molecules docked to the β-subunit led to the identification of 66 compounds. Compound SGM-45 binds to Cavβ2a, inhibits calcium influx via N-type voltage-gated calcium (Cav2.2) channels reconstituted with different beta subunits (except beta 4) and in rat dorsal root ganglion (DRG) neurons, and is a selective inhibitor of Cav2.2. We also found decreased surface Cav2.2 in DRGs following incubation with SGM-45, implicating block of surface expression as a mechanism of action of SGM-45. Constellation pharmacology revealed actions of SGM-45 on a heterogeneous population of DRGs including those responsive to acetylcholine, mustard oil, ATP, histamine, and capsaicin. Of relevance for pain, we found that SGM-45 decreases calcium influx during periods of high activity by promoting accumulation of channels in an inactivated state. As certain pain conditions have been associated with hyperexcitability of sensory neurons, it is possible that SGM-45 could allow for increased accumulation of inactivated Cav2.2 in these neurons. Finally, SGM-45 reversed mechanical allodynia and thermal hyperalgesia in rats subjected to a plantar incision of the paw, an accepted model of human post-surgical pain. Our study will generate new tools to investigate Cav α-β interactions and underscores the importance of targeting this interaction for development of pain therapeutics.
Funding: University of Arizona UBRP with funds from the Senior Vice President for Research (L.A.C); University of Arizona Neuroscience and Cognitive Science Research Award (L.A.C.); University of Arizona Honors College Spirit of Inquiry Grant (L.A.C); National Scientist Development from the American Heart Association (SDG5280023 to R.K.); Neurofibromatosis New Investigator Award from the Department of Defense Congressionally Directed Military Medical Research and Development Program (NF1000099 to R.K.)
EFFECTS OF SLEEP QUALITY ON DELAYED EPISODIC MEMORY IN PRESCHOOL AGED CHILDREN KATHRYN CHUNG, KATHERINE HUGHES, JAMIE EDGIN
Sleep is an important physiological state that aids consolidation of long-term memories and facilitates learning. One type of long-term memory is episodic memory – the autobiographical memory for events. As the hippocampus continues to develop into adulthood, episodic memory improves. The Deferred Imitation task, a memory task that targets episodic memory, tests nonverbal hippocampal encoding and recall of action sequences which can be adjusted to be age appropriate. We investigated how sleep quality affects the episodic recall performance of typical developing 3-year olds with Deferred Imitation. We used actigraphy to collect sleep measures of activity and light levels to measure sleep quality. The Deferred Imitation task was administered to each participant twice, initially to measure encoding, and then following a two-week delay to test recall. We hypothesized that the sleep quality variables, efficiency and fragmentation, will correlate with encoding and recall in 3-year olds, with a greater effect for long-term recall. It was found that the correlation between sleep efficiency and episodic recall was borderline significant (r(18) = 0.465, p = .052). The correlation between sleep fragmentation and episodic recall was not significant (r(18) = -0.359, p = .144). This implies that while sleep efficiency affects episodic memory recall, the amount of sleep fragmentation does not affect the episodic memory recall of typically developing 3-year olds. This research was supported by the NIH (RO1 HD088409 R1), the LuMind Institute, and UBRP with funds from the Office of the Provost.
EFFECTS OF CHRONIC AND ACUTE MORPHINE TREATMENT ON NUCLEAR FACTOR NF-KB DEZ COLEMAN, AUSTEN THOMPSON, WILLIAM STAATZ, TALLY LARGENT-MILNES, TODD VANDERAH
Breast cancer is the second leading cause of death among women. Tumor metastases in the bone can induces pain (cancer- induced bone pain; CIBP) in 75-90% of late-stage patients. Opioids are prescribed to manage this pain, but they may enhance bone loss and tumor growth to further increase pain. Preliminary data suggests that morphine treatment increases
29 phosphorylation of NF-κB subunit p-65 via the traditional pathway which mediates upregulation of inflammatory cytokines. Our research focuses on discovering the relative engagement of the described, nuclear factor NF-kB pathways within a murine breast cancer cell line in response to acute (1 hour) and chronic (24 hour) morphine treatments on cancer cell intracellular signaling. In vitro studies used the murine mammary adenocarcinoma cell line (66.1) treated with morphine sulfate for 1 hour and 24 hours. Our previous study suggests morphine increase phosphorylated and nuclear translocation of NF-κB p-65. Now, using western blot, we observed morphine dose-dependent increase in p-100 cleavage to p-52 through the nontraditional pathway. These data suggest morphine causes an increase in activation of NF-κB pathways, which are known to regulate transcription of pro-inflammatory genes. Supported by UBRP with funding from the University of Arizona Office of the Provost.
TYROSINE HYDROXYLASE AND CALCIUM BINDING PROTEIN EXPRESSION IN THE NORADRENERGIC SYSTEM OF AGED PRIMATES NICOLE DE LA PENA, DANIEL GRAY, WONN PYON, CAROL A. BARNES
The norepinephrine neuromodulatory system is a key component in regulating various aspects of memory and cognition. With aging, changes in the noradrenergic system have been related to changes in cognition, implicating this system as a key target in understanding the aging brain. One possible source of this dysfunction could be changes in the calcium dynamics of aged neurons, as it has been repeatedly shown that calcium-dependent processes are altered in aging neurons. Such increases in Ca2+ concentration can have a number of consequences on intracellular function, including the induction of plasticity mechanisms, such as long-term potentiation and long-term depression, which could contribute to age-related memory decline. Calcium binding proteins (CaBPs) regulate intracellular calcium concentration by acting as calcium buffers and sensors. Parvalbumin, calretinin, and calbindin are the most common CaBPs in the nervous system, and, in general, these proteins are expressed by non-overlapping populations of neurons. With age, the expression of these proteins has been shown to change in density in specific regions across the brain, suggesting they may contribute to the calcium dysregulation observed in senescence. Whether the expression of CaBPs in the nervous system of aged primates correlates with lifespan changes in cognition remains poorly understood. The first goal of the project is to determine whether the expression and extent of co- localization of tyrosine hydroxylase (TH), a precursor to both dopamine and norepinephrine, and the three CaBPs changes in the locus coeruleus with age. The second goal of the project is to correlate the neurochemical data with behavioral data of young and aged rhesus macaque monkeys to observe if changes in CaBP expression within TH+ neurons in the locus coeruleus correlates with age-related behavioral change. To this end, brains were extracted from behaviorally characterized rhesus macaque monkeys and sectioned serially. Tissue slices containing the locus coeruleus will be double labelled for TH and the targeted CaBPs using immunofluorescent labelling techniques. Because of the auto-fluorescence of the age-accumulating pigment granules known as lipofuscin, we first needed to address how to control lipofuscin-induced auto-fluorescence within our tissue so that the fluorescent signals for TH and CaBPs could be detected, even when lipofuscin invaded the emission range of those fluorophores. An image acquisition protocol was developed by using spectral imaging and linear unmixing to eliminate the native auto-fluorescence. This method was shown to outperform traditional dye-based methods of removing native fluorescence with Sudan Black. With this protocol in place, we can now begin to collect neurochemical data from the locus coeruleus, which can elucidate whether changes in CaBP expression within the noradrenergic system predicts behavioral changes in senescence.
Funded by: UBRP with funds from the Senior Vice President for Research, Western Alliance to Expand Student Opportunities (WAESO) Louis Stokes Alliance for Minority Participation (LSAMP) National Science Foundation (NSF) Cooperative Agreement No. HRD-11017, AG003376, RR
30 ANT DEFENSE AND FORAGING TIMES ANNIE DIXON, SARA HU, ANNA DORNHAUS
The competition among rock ants, Temnothorax rugatulus, is driven by the need to acquire more nesting space. Here we investigate how ants defend themselves against rival colonies in the immediate area and how long foragers stay out of the nest if they encounter an enemy ant. Previous work looked at what would drive colonies to competition, and we studied how ants behave when in competition with another group. In this study we placed two separate ant colonies into one arena and studied how they reacted to each other.
DARPA NO: D162-005-0227, TOPIC NUMBER: SB162-005
RNA MODIFICATION IN THE TREMBLAYA TRIPARTITE ENDOSYMBIOSIS RANDALL ECK, ZDENEK PARIS, EVA HEGEDÜSOVÁ, SNEHA KULKARNI, FILLIP HUSNIK
Tremblaya princeps has the smallest known bacterial genome lacking many genes of metabolism necessary for survival. Just as unique, Tremblaya princeps is a partner in a three-way endosymbiosis. Multiple Tremblaya princeps live within each cell of the Planococcus citri mealybug abdomen, furthermore, multiple Moranella endobia cells, a bacteria with a genome four times larger than Tremblaya princeps, live within each Tremblaya princeps bacteria. It is hypothesized that Tremblaya princeps compensates for its degenerated genome by importing proteins from both the Planococcus citri mealybug, in exchange for exporting amino acids it cannot obtain from its diet, and Moranella endobia. It has been previously shown, Tremblaya princeps lacks DNA sequences that encode for amino-acetylated tRNA synthases, which attach amino acids to the tRNA molecules that transport them to become part of proteins. By a series of northern blots, it is demonstrated that Tremblaya princeps do transcribe their own tRNA molecules, providing evidence that they import amino-acetylated tRNA synthases from Moranella endobia (whose genome does not lack these sequences) as opposed to directly importing amino-acetylated tRNAs from Moranella endobia. To provide further evidence to this hypothesis, it should be shown through acid-gel electrophoresis that these tRNAs of Tremblaya princeps are amino-acetylated. The Rlm1 enzyme adds a methyl group to a cytosine at the 1962 position in 23S rRNA in E. Coli which improves tRNA recognition and peptidyl transfer. The genome of the Planococcus citri mealybug contains sequences for the Rlm1 enzyme that was transferred to its genome from a previous endosymbiont. It has been previously shown to be transcribed and localized in the abdomen. Bisulfite sequencing of Tremblaya princeps 23S rRNA shows a 5-methylcytosine modification at the corresponding location indicating Tremblaya princeps imports this enzyme from the mealybug in a metabolic cooperation of their endosymbiosis. By further exploring this relationship, insights can be gained into organelle evolution and cellular cooperation.
ENGINEERING ALLOSTERIC REGULATION IN SRC FAMILY KINASES VIA BH3 DOMAIN INSERTION TYLER ESPINOZA, MATTHEW BIENICK, INDRANEEL GHOSH
Kinases play a critical role in all aspects of cell signaling. A variety of diseases, including cancer, have been linked to aberrant kinase activity. However, with more than 500 kinases coded for in the human genome, it can be a challenge to study the activity of a specific kinase. Many kinases share strong structural similarities, which can lead to problems with off-target binding when using pharmacological inhibitors. Developing methods to modulate the activity of a specific kinase could help in understanding the roles that kinases play in cell biology. To alter the activity of a specific kinase we took a domain insertion approach. We inserted the BH3 domain of Bad into two Src family kinases, Lyn and Yes, to allow for possible allosteric regulation. Bad is an intrinsically disordered protein, but its BH3 domain forms an alpha-helix upon binding the protein Bcl-XL. We proposed that the inserted BH3 domain of the engineered kinases would become structured and form an alpha-helix upon binding Bcl-XL,
31 disrupting kinase activity. We further hypothesized that ABT-737 or A-115463, small molecules that disrupt Bcl-XL /Bad binding, would serve as antidotes and restore the catalytic activity of the kinase. We have shown the success of this system within mammalian cells. Cells expressing a kinase containing the inserted BH3 domain exhibited increased phosphorylation, whereas cells expressing both the BH3 domain containing kinase and Bcl-XL displayed decreased phosphorylation. Subsequent treatment of inhibited cells with ABT-737 or A-115463 restored phosphorylation. Together these results indicate a possible method to allosterically regulate the activity of a specific kinase.
Funding provided by: NSF CHE-1506091, NIH 1RO1GM115595-01 and T34 GM08718, Luceome Biotechnologies
GALLERIA MELLONELLA AS A COMPLEMENTARY MODEL ORGANISM TO STUDY THE ENTEROPATHOGENIC E. COLI TYPE 3 SECRETION SYSTEM ISABEL FORLASTRO, REBECCA MCQUADE, V. K. VISWANATHAN, S. PATRICIA STOCK
Enteropathogenic Escherichia coli (EPEC) is a common diarrheal pathogen that affects approximately 0.8 million children per year in developing countries. EPEC uses a complex bacterial structure called the Type III Secretion System (T3SS) to inject bacterial effector proteins directly into intestinal epithelial cells, manipulating the cells’ behavior and causing disease. Rabbits are a commonly used model to study EPEC. However, rabbits have several limitations as hosts including expense, specialized handling, ethical concerns and federal regulations regarding the use of laboratory vertebrates. Insects are potential complementary models for studying bacterial pathogenesis in vivo. Benefits of using insect hosts include low-cost, ability for mass infections, and analogous immune strategies to vertebrates. We hypothesize that larvae of the greater wax moth Galleria mellonella (GM) can serve as an effective model in studying the EPEC T3SS. In order to determine if EPEC can infect GM we injected EPEC into the body cavity (hemocoel) of the insects. We observed that live wild-type EPEC kills GM and that heat-killed EPEC does not kill, indicating that EPEC actively infects GM. To explore the mechanism of killing, we also considered EPEC mutant strains that lack different components of the T3SS. These mutants were defective in killing GM, indicating that EPEC kills these insects in a T3SS-dependent manner. To identify tissue localization of EPEC, we injected YFP-labeled EPEC into GM and observed fluorescence along the insect gut. We are currently adapting this model to test potential T3SS inhibitors and to study individual T3SS effectors. We are also attempting to model oral infections in insects to more closely represent human infection.
ENSURING THE ROBUSTNESS OF DOMINANCE AND SELECTION INFERENCE ALYSSA LYN FORTIER, TRAVIS STRUCK, RYAN GUTENKUNST
Dominance describes the degree of recessivity or additivity of a certain allele. When paired with information about selection, the dominance coefficient helps us understand a population’s history. However, most population genetic models fix the dominance coefficient at 0.5, meaning completely additive, though this is not biologically justified. We evaluated ways to infer the dominance coefficient along with selection parameters, then determined the limitations of our methodology. We hypothesized that small errors in the underlying demographic model could produce hugely inaccurate dominance coefficients. Using simulated data under realistic demographic assumptions, we optimized simpler demographic models as well as models of selection and dominance to determine under what conditions we could accurately infer the correct parameters. We determined that dominance inference is most consistent for populations with small divergence times. Applying the methodology to dog and wolf populations, we show that the traditional assumption of 0.5 may not be reasonable for many population genetic models. This research was sponsored by UBRP with funds from the Senior Vice President for Research, Private Donors, and NSF grant #DEB-1146074.
32 DEREGULATION OF LONG NONCODING RNAS IS VITAL FOR RHABDOMYOSARCOMA TUMORIGENESIS MARIAJOSE FRANCO, MIREYA L. HERRERA-HERRERA, MADHAVI BATHINA, XIANG CHEN, JUSTINA D. MCEVOY
Rhabdomyosarcoma (RMS), in developing skeletal muscle, is the most common soft tissue sarcoma in children. The molecular events that lead to RMS tumorigenesis remain unknown, making therapeutic development challenging. The two major histological RMS subtypes are ERMS, which displays several somatic mutations, and ARMS, characterized by a translocation that produces the fused PAX3/7-FOXO1 gene. RMS malignancies overall have lower mutation rates than adult solid tumors. Based on this prior work, we propose that non-genetic mechanisms potentially contribute to RMS formation. Preliminary analyses of RMS patient-derived xenografts led to discovering a group of novel long non-coding RNAs (lncRNAs) that are unique to RMS. Some of the lncRNAs are expressed in both subtypes and some in only one. This finding prompted further study because deregulation of lncRNA activity is often associated with the promotion of cancer hallmarks such as cell survival. Preliminary knockdown of one of these lncRNAs, lnc19_31, led to rapid cell death in RMS cell lines and increased expression of pro- apoptotic genes such as TP53. This knockdown produced a complete loss of PAX3-FOXO1 expression, indicating lnc19_31 may be a major driver of RMS oncogenesis. In addition, the expression of the adjacent transcription factor ZNF536 decreased, thus suggesting potential cis-regulatory activity for lnc19_31. ZNF536 knockdown also induced cell death. Together, these data led us to hypothesize that lnc19_31 is essential for cell survival through expression of ZNF536. These findings could translate into therapies targeting lnc19_31. This research was supported by the University of Arizona Cancer Center and Molecular and Cellular Biology Department as well as the MARC Training Grant (T34 GM08718).
INFLUX OF WATER MEDIATES G-PROTEIN-COUPLED RECEPTOR ACTIVATION STEVEN FRIED, SUCHITHRANGA M.D.C. PERERA, ANNA R. EITEL, NIPUNA WEERASINGHE, UDEEP CHAWLA, MICHAEL C. PITMAN, ANDREY V. STRUTS, MICHAEL F. BROWN
Rhodopsin is the photoreceptor of the rod cells that is responsible for scotopic vision in dim light. It is also a canonical member of the Class A (“rhodopsin-like”) family of the G-Protein-Coupled Receptor (GPCR) superfamily, which together govern a wide variety of physiological signaling processes. Utilizing rhodopsin as a model GPCR, we explored the role of water in the structural activation mechanism of GPCRs through the use of osmotic stress techniques. Our motivation for this inquiry stems from nanosecond molecular dynamics simulations of rhodopsin following photoactivation, which reveal a bulk influx of water into the protein core. In order to experimentally validate these simulated results, we subjected the protein within its native lipid membranes to environments of varying degrees of osmotic pressure, using different molecular-weight polyethylene glycols as osmolytes to create the osmotic effect. By briefly exposing the protein within these environments to a high-intensity green light, while recording time-dependent UV-visible spectra, we were able to measure the fraction of protein that had transitioned to the fully active metarhodopsin-II (MII) conformation, the receptor state capable of activating G-proteins. Repeating these trials over a range of pH values, we were able to generate pKA values for each osmotic environment that mark the equilibria between fully-active MII and an inactive conformation called metarhodopsin-I (MI). We discovered that high-molecular weight osmolytes favored the more closed, inactive MI conformation of the protein, likely by withdrawal of water from the inner core. Small osmolytes, on the other hand, penetrated into the protein core and caused an opposite effect, stabilizing the active MII conformation to an even greater extent than occurred in the control environments without added osmolytes. Based on these data, a new model is proposed for the functional role of water in the GPCR signal transduction, enabling a wet-dry cycling mechanism that amplifies the activation of G-proteins. These results necessitate a new understanding of GPCR activation, in which the influx of water plays a critical role in establishing the active receptor conformation. This project was supported by the NIH Grant EY026041 and by UBRP with funds from the University of Arizona Office of the Provost.
33 THE IMPACTS OF FAMILY HISTORY OF ALZHEIMER’S DISEASE AND EDUCATION ON WHITE MATTER INTEGRITY NATHANIEL GALLEGOS, ARIANA STICKEL, LEE RYAN
Alzheimer’s disease (AD) is a multifactorial neurodegenerative disease. Some AD risk factors are age, gender, apolipoprotein e4 status, and having a family history (FH) of AD. FH of AD has not been well studied. Other factors, like education, may delay the onset of AD. Our study investigated the effects FH of AD on white matter microstructure in those with no signs of dementia. Diffusion magnetic resonance imaging scans were collected on 28 subjects with a FH of AD and 29 subjects without a FH of AD. Groups were matched on age, education, gender, and apolipoprotein e4 status. Diffusion images were collected in 25 directions. Fractional anisotropy (FA) measurements were then computed in the uncinate fasciculus and the inferior longitudinal fasciculus (ILF) bilaterally, two regions susceptible to damage in the early development of AD. FSL was used to preprocess the images. DTI-tk was used to create a custom template, extract regions of interest, and obtain FA values in such regions per person. General linear models controlling for age, education, and gender found no differences in FA between the FH groups.
In a secondary analysis, we examined the interaction between education and FH on white matter integrity. Subjects were split into two education groups, high (>14 years) education and moderate to low (≤14 years) education. When controlling for age and gender, those with a FH of AD and a low education, had significantly lower FA in the uncinate compared to those with FH and a high education and those with no FH. There were no differences in FA between the latter three groups to each other. We found the same interactive pattern in the ILF. These results suggest that education is protective of white matter in the presence of AD risk. Future analysis will investigate whether the interaction between FH of AD and education is protective across the entire brain or only in specific tracts (e.g., the uncinate and ILF). The protective effects of education should be further investigated in a longitudinal study of those with and without a family history of AD at various stages of dementia progression.
Funding provided in part from UBRP with funds from the Office of the Provost and Western Alliance to Expand Student Opportunities (WAESO) Louis Stokes Alliance for Minority Participation (LSAMP) National Science Foundation (NSF) Cooperative Agreement No. HRD-11017.
INVESTIGATING KCL-INDUCED EXPRESSION CHANGES OF SODIUM-PROTON EXCHANGER NHE1 AT THE BLOOD BRAIN BARRIER EMILY GALLOWAY, KARISSA E. COTTIER, JOHN KIM, THOMAS P. DAVIS, TODD W. VANDERAH, TALLY M. LARGENT-MILNES
An estimated 38 million people in the United States suffer from migraines. The characteristic, recurrent episodes of headache involved in migraine can be intensely painful and last up to 72 hours if untreated. About 25% of migraineurs have aura- associated effects, such as dark spots in the visual field, before the onset of headache. An electrical phenomenon called cortical spreading depression (CSD) has been linked to migraine with aura. CSD is a propagating wave of depolarization followed by suppression of neuronal activity with corresponding changes in ionic balance. Studies have shown that the firing rate of pain- sensing neurons, called nociceptors, increases in the meninges after initiation of CSD. Neither the mechanisms underlying CSD initiation and propagation nor those leading to activation of craniofacial pain centers in the brain are known. Dysregulation of pH occurs during CSD and drops in pH can activate trigeminal pain fibers in the face. We hypothesize that changes in expression of sodium-proton exchanger NHE1, an antiporter implicated in the maintenance of pH homeostasis, may play a role in migraine pathophysiology. After injection of cortical KCl to induce CSD events, periorbital allodynia increases and remains elevated over a 240-minute time-course in vivo. Female rats have lower pain thresholds and more facial allodynia, and concurrently expression lower levels of NHE1 in whole brain samples analyzed by Western blotting. These results concur with the higher rate of aura and females and the concurrent higher incidence of CSD. We sought to recapitulate our findings using an in vitro system using cells that comprise the blood brain barrier/neurovascular unit. GPNT rat endothelial cells were treated with increasing concentrations of KCl and negative relationship was discovered between NHE1 expression and KCl concentration; similar results were found when either astrocytes or microglia cell lines were used. These studies suggest that ionic changes in the brain, modeled through KCl, may regulate NHE1 expression, thus making the brain more susceptible to pH changes and possibly
34 contributing to CSD or migraine initiation. Future studies will investigate signaling cascades mediating this regulation of NHE1 expression.
Research supported by by NIH grant R013025390, with support provided in part by UBRP with funds from the Senior Vice President for Research.
METASTASIS OF OLFACTOMEDIN-1 TREATED GLIOBLASTOMA STEM CELLS REBECA GARDNER, RAYMOND RUNYAN
Epithelial to mesenchymal transition (EMT) is an essential developmental process that evidence suggests may also be promoting cancer metastasis in Glioblastoma stem cells. Glioblastoma is known to be one of the most aggressive and lethal brain tumors found in adults due to its increased ability to invade. Olfactomedin-1 (OLFM1) and its presumed receptor, Latrophillin 2 (LPHN2), seem to play an important role in the regulation of cell invasion during the EMT process. An upregulation of OLFM1 has been shown in various cancers such as Glioblastoma. Two variants of OLFM1, 1 and 3, have been previously tested and found that Variant 1 increases invasiveness. The purpose of this experiment was to test a subtype of Glioblastoma (GB62) and its ability to metastasize through a transwell invasion assay treated with variants 1 and 3, potentially discovering a target to inhibit metastasis and increase survival of Glioblastoma patients. This research was funded by the National Institute of Environmental Health Sciences, Grant #1-R25-ES025494 and Western Alliance to Expand Student Opportunities (WAESO) Louis Stokes Alliance for Minority Participation (LSAMP) National Science Foundation (NSF) Cooperative Agreement No. HRD-11017.
DESIGN AND FUNCTIONAL CHARACTERIZATION OF a6* AND a7* NACHRS MICHAEL GEE, E.N. MOCA, B. EATON, L.M. LUCERO, R.J. LUKAS, P. WHITEAKER, A.A. GEORGE
Nicotinic acetylcholine receptors (nAChRs) are composed of homologous subunits and assemble to form a wide range of receptor subtypes that differ in their physiological role in the central and peripheral nervous systems. Cholinergic synaptic degradation in the hippocampus and cell death in the basal forebrain are two early onset characteristics of Alzheimer’s disease (AD), a progressive neurodegenerative disorder associated with a decline in memory and cognition. The disorder is also characterized by abnormally high levels of amyloid-β peptide (Aβ1-42). Although Aβ1-42 has been reported to functionally inhibit the homopentameric α7-nAChRs in hippocampal neurons, heteropentameric α7β2*-nAChRs have been found to experience greater sensitivity to functional inhibition by Aβ1-42. In comparison, α6β2*-nAChRs (expressed on dopaminergic projections from the VTA to the dorsal striatum) have been associated with nicotine-evoked dopamine (DA) release in mesocorticolimbic DA reward system, and play an important role in the mediation of nicotine reward and addiction. To understand the effects of subunit stoichiometry on the structure-function relationship of α6β2*-nAChR subtypes, we engineered α6β2*-nAChR concatemers with subunits differentially “linked” at the translational level and performed two- electrode voltage clamp electrophysiology on Xenopus oocytes to measure α6β2*-mediated macroscopic function. In addition, single-channel electrophysiology was performed on mammalian SH-EP1 cells transiently transfected with α7* or α7β2* concatemers to examine differences in channel kinetics between constructs. Funding is provided by the ASPET/SURF grant to the University of Arizona.
35 SA’ÁH NAAGHÁÍ BIK’EH HÓZHÓÓN: A NAVAJO INSPIRED RESEARCH FRAMEWORK CHEYENNE GRABIEC, MARTI LINDSEY
When conducting research in tribal communities, such as the Navajo Nation, it is important that the researcher understands Navajo culture and implements it into the research. This work seeks to take the Bi-Directional Partnership Paradigm that is used by the Southwest Environmental Health Sciences Center (SWEHSC) to illustrate how SWEHSC collaborates with partners on research projects and translates it into the four-step planning and learning process from the Navajo philosophy of Sa’áh Naagháí Bik’eh Hózhóón (SNBH). The Navajo philosophy of SNBH is an educational philosophy that guides Navajo life in harmony within the world and universe and uses a four-step planning and learning process of: thinking, planning, living, and reflecting. To translate SNBH in terms of the Bi-Directional Partnership Paradigm we took each step of the planning and learning process and described them in terms of the research process. Thinking represents collaboration to identify an issue to be researched; planning represents the steps to be taken in addressing the issue; living represents the implementation of the plans; and reflecting represents the evaluation of the steps taken. Both the Bi-Directional Partnership Paradigm and the four- step planning and learning process of SNBH are circular to represent the continuous process of research. Overall, this research framework was designed to be implemented in research on the Navajo Nation. The future direction is to implement this framework in upcoming environmental health literacy research on the Navajo Nation.
Funded by the National Institute of Environmental Health Sciences, Grant #1-R25-E and the National Institute of Environmental Health Sciences Grant #P30ES006694.
INVESTIGATING THE NATIVE ROLE OF RNA-BINDING PROTEIN FUSED IN SARCOMA (FUS) IN TRANSCRIPTION AND DEVELOPMENT OF AMYOTROPHIC LATERAL SCLEROSIS (ALS) EMMA HARRELL, JACOB SCHWARTZ
Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease that causes a drastic breakdown of motor neurons, resulting in eventual death by respiratory failure. Fused in Sarcoma (FUS) is a nuclear RNA-binding protein that has been found to associate with the carboxy terminal domain (CTD) of human RNA polymerase II (RNA Pol II) and other transcription machinery to impact the process of transcription. In its mutated form, FUS has been implicated in ~5% of familial ALS cases, as well as in 1% of sporadic ALS cases. Although the mutated form of FUS has been implicated in the development of ALS, little is known about its native function. Specifically, evidence shows that FUS interacts with the CTD of RNA Pol II and other transcription machinery within the nuclei of cells; this suggests that FUS serves to influence the process of transcription. How these interactions specifically affect the efficiency of transcription in cells, however, is still relatively unknown. To determine how FUS affects the process of transcription in cells, a HeLa cell in vitro model of transcription was adopted to explore the wildtype function of FUS in the role of human transcription. An in vitro transcription assay in which produced RNA is DNase treated, reverse transcribed, and analyzed through quantitative polymerase chain reaction (qPCR) was used to determine transcriptional efficiency; the conditions of the in vitro reaction are modified in order to test how transcriptional efficiency of several genes, including green fluorescent protein (GFP), are affected by the presence of FUS. Current experimental results suggest that the addition of FUS to the transcription assay increases transcriptional efficiency of the target gene. These results provide insight into the role of FUS in the process of human transcription and help to illustrate the native function of FUS within the nuclei of cells.
This work is supported by startup funds from the University of Arizona College of Science, Federal Work Study Funds, and UBRP with funds from the Senior Vice President for Research and Private Donors.
36 IN VITRO PURIFICATION OF CARDIOMYOPATHY-ASSOCIATED MUTATIONS IN HUMAN β - MYOSIN HEAVY CHAIN DHANWANT HUNJAN, SARAH LEHMAN, JIL TARDIFF
Hypertrophic cardiomyopathy (HCM) and Left Ventricular Non-compaction (LVNC) are two distinct forms of genetic cardiomyopathies that can arise from point mutations within the cardiac sarcomere. HCM is typically characterized by thick ventricular walls and stiffening of the ventricles, while LVNC is characterized by irregular trabeculation and sponge-like appearance of the ventricular wall; both lead to impaired heart function. Interestingly, similar point mutations in the enzymatic thick filament protein β-Myosin Heavy Chain (β-MyHC), have been implicated in both forms of cardiomyopathies. One such example is the HCM-associated I457T and LVNC-associated I467T, both of which are found in close proximity to the ATP-binding pocket of the motor domain. We hypothesize the replacement of a hydrophobic isoleucine with a non-polar threonine in a known hydrophobic ATP-binding pocket will induce a structural alteration that subsequently affects ATPase activity of the motor domain. Of note, limitations in the ability to express and purify a functional motor domain has historically restricted molecular understanding of β-MyHC mutations. However, recent advances in the expression and purification of functional human β-MyHC have allowed for the expression of the functional motor domain of this protein. This time- and labor-intensive process, currently only performed in two other labs, requires significant optimization of the purification process necessary to obtain the functional proteins. This project primarily focuses on implementing this highly complex technique including site- directed mutagenesis, viral production, and protein expression and purification into the research environment at the University of Arizona. To date, I457T and I467T mutants were made following site-directed mutagenesis via recombinant PCR of the MyH7 gene. Mutants were cloned into a plasmid containing a viral genome for amplification of cDNA in vitro. Subsequent infection of C2C12 myoblasts with virus containing MyH7 gene allowed for production of chaperone-mediated functional myosin protein. Column chromatography was used to purify the myosin protein. We hope to utilize various in vitro assays to study the structural and functional alterations within myosin caused by the point mutations. Ultimately, we aim to link genotype to phenotype and design and implement mutation-specific therapies for early clinical intervention in patients afflicted by these mutations. Support provided in part by UBRP with funds from the Office of the Provost.
PRIMER DEVELOPMENT AND TESTING FOR POLYMERASE CHAIN REACTION ON DNA TEMPLATES OF HOUSE MOUSE AND RELATED SUB-SPECIES MEUCCI ILUNGA, CHRISTINA LAUKAITIS, ROBERT KARN
Polymerase Chain Reaction (PCR) is a synthetic DNA duplication technique that is used to amplify DNA in order to generate millions of copies of a particular segment of genetic code. The product of a PCR can be used to identify the ordering of base pairs in a DNA molecule through a process known as sequencing. The overall goal of this project is to amplify, sequence, and compare Androgen binding protein (Abp) gene regions of related house mouse species and sub-species. This would provide information about the function of ABP on a biochemical level as well as general information about gene duplication. One of the major preliminary stages of this project is the development and testing of PCR primers. Because primers are the cornerstone of the PCR process, the sub-focus of this project is to create a working set of primers able to amplify all the Abp paralogs of the genome mouse (C57BL/6) as well as other related mouse species.
M.I. supported by the National Cancer Institute, Grant #2U54CA143924, through the Partnership for Native American Cancer Prevention (NACP) and Western Alliance to Expand Student Opportunities (WAESO) Louis Stokes Alliance for Minority Participation (LSAMP) National Science Foundation (NSF) Cooperative Agreement No. HRD-11017.
Keywords: PCR, primers, DNA template
37 CHARACTERIZATION OF A NOVEL INTEGRIN β4 VARIANT IN HUMAN PROSTATE CANCER NADIA INGABIRE, JULIE E. MCGRATH, ANNE E. CRESS
While localized prostate cancer is curable, the survival rate of patients with prostate cancer is lower as cancer cells become metastatic. Here, we study adhesion of PC3N cells expressing a unique integrin β4 isoform, β4E to laminin 332. Laminin binding integrins (LBI) are important in the development and progression of cancer. The LBI α6β4 has both cancer promoting and suppressing roles and is known to form strong adhesions mediated by stable hemidesmosome formation. All splice variants of β4 conserve the transmembrane and extracellular domains but variants A-D retain the same cytoplasmic domain as β4. Interestingly, Variant E (β4E) consists of a unique cytoplasmic domain with 114 amino acids. The novel cytoplasmic domain of β4E results in the loss of major protein motifs that may alter cell anchorage mechanisms such as adhesion. Since integrin activation can be dependent upon the cytoplasmic domain, it was important to determine whether cells containing the isoform β4E had reduced adhesion with laminin 332 as compared to cells expressing β4C isoform. In our study, adhesion and crystal violet assays were used to measure absorbance of adherent β4-null PC3-N cells that were transfected with β4C- RFP and β4E- GFP plasmid constructs. The results showed no significant difference in cell adhesion to laminin between the cell groups. These data suggest that β4E isoform is equally capable of supporting adhesion to laminin as compared to β4C isoform. Future experiments will be to test the significance of the unique cytoplasmic domain for supporting integrin signaling and cancer invasion.
This work was supported by the following NIH grants: MARC Training Grant T34 GM08718, RO1 CA 159406, P30 CA 23074, T32 CA 09213 and PO1 HL 126609.
DETECTING PAPILLOMAVIRUS RECOMBINATION IN VIVO JAYME JACKSON, JANA JANDOVA, KELLY KING, KOENRAAD VAN DOORSLAER
Human papillomaviruses (HPVs) are the most common sexually transmitted disease (STD) and can cause cervical and head and neck cancers. The prophylactic vaccine is based on virus-like particles that reconstitute the viral epitopes. This approach raises highly specific antibodies. The current vaccine only protects against a minority of the genital HPVs, thus leaving the vaccinated population susceptible to (oncogenic) HPV types. Recombinant genomes may fail the current prophylactic vaccine. However, whether papillomavirus genomes recombine, and whether the offspring genomes are sufficiently fit to become established in the population remains unclear. The initial work focuses on developing a robust recombination assay in primary human cells. The assay introduces two defective mutant viral genomes into a cell. Recombination between both defective parent genomes restores the ability of the daughter genome to replicate. We successfully generated the required mutant genomes. First, part of the viral genome was replaced by a gene expression cassette allowing for antibiotic selection. To stop viral replication of these selectable genomes, we introduced several additional mutations. The first construct contained a translation termination linker (TTL) interfering with the production of the viral helicase, thus blocking replication in trans. In a second construct, we mutated viral cis binding sites for the replication protein. The mutant genomes were then transfected into primary human foreskin keratinocytes. The RNA and DNA were extracted 96 hours post transfection. Analysis of cDNA through qPCR indicated that the mutant genomes were unable to transcribe E6* and E1^E4 and E1^E4 is required for a successful viral lifecycle. This suggests that these genomes would not be able to survive, as intended. Current work includes testing whether replication can occur and if recombination can rescue the replication defect. This research was supported by NIH MARC Training Grant T34 GM08718 and an UA RDS seed grant to Dr. Koenraad Van Doorslaer.
CHARACTERIZATION OF HEAVY METAL IONS BY MIE SCATTERING AND CLASSIFICATION BY LINEAR DISCRIMINATE ANALYSIS CHRISTIAN JENNINGS, KATHERINE KLUG, JEONG-YEOL YOON
38 Through the use of Mie scattering data and linear discriminate analysis, an approach to characterize and classify heavy metal ions in water is proposed. This method tested nine heavy metal ions and two control cations (1-1000 ppb) in mixtures with carboxylated polystyrene particles (0.40 μm, 0.75 μm, and 0.91 μm diameters). Scattering was measured for each mixture at two angles optimized to distinguish particle aggregation with heavy metal ions. The collective scattering intensities for each heavy metal ion under all conditions were then evaluated through linear discriminant analysis-based statistical classification. Specifically, linear discriminant analysis was conducted under Mahalanobis distance conditions. The models produced by linear discriminant analysis were successful at classifying most of the heavy metal ions under the experimental conditions, but were most effective at identifying Ni(II). This method demonstrates potential for a portable and low-cost heavy metal ion sensor given further data collection and processing to train the linear discriminate analysis models. The research was supported by the National Institute of Environmental Health Sciences through award No. 1-R25-ES025494, the Western Alliance to Expand Student Opportunities (WAESO) Louis Stokes Alliance for Minority Participation (LSAMP) National Science Foundation (NSF) Cooperative Agreement No. HRD-1101728, and U.S. National Science Foundation Graduate Research Fellowship under Grant No. DGE-1143953.
EFFECT OF IMMUNE RESPONSE ON QUEEN ACTIVITY IN BUMBLEBEES DEREK JEZULIN, EVAN KELEMEN, ANNA DORNHAUS
Within a biological system, multicellular organism or a social insect colony, conflict between selection on the individual and on the system as a whole can arise. One potential outcome of this conflict may be phenotypic diversity among the individuals within a system. In bumblebees, these two levels of selection may create the large variation in body sizes that exist within a colony. Queens prefer smaller workers as they are less likely to selfishly reproduce. Workers prefer larger workers because their reproductive success is not solely dependent on the individual, and their larger sisters will be more likely to reproduce. This conflict may create the diversity we see in bumble bee body sizes. To test this, we tried to manipulate the quality of the queen using Lipopolysaccharides (LPS), an identifying molecule on the exterior of gram-negative bacteria, to increase the conflict within the colony. We hypothesize that LPS will evoke an immune response from the queen, which will then cause a decrease in her activity, increase in her activity, or have no effect on her activity. Video analysis of queen bees thus far has lead us to conclude that injection of lipopolysaccharides does not have an effect on queen activity levels.
COMMUNICATION STRATEGY EFFICIENCY OF SOCIAL INSECT FORAGERS DOESN’T DEPEND ON SPATIAL RESOURCE CLUSTERING WES JOHNSON, ANNA DORNHAUS
The emergent behaviors of social insect colonies allow them to adapt to their environments, and these behaviors depend fundamentally on the way individuals communicate. Here we explore how the spatial clustering of resources modifies the communication strategy efficiency of social insect foragers; we use computer simulations to model social insect foragers communicating in environments of varying spatial resource distributions. Foragers of a colony will communicate via repulsive pheromone trials that repel nestmates, attractive pheromone trials that form paths to resources, or a communicable version of the win-stay lose-shift search method where a forager recruits nearby nestmates to search the area a resource has been found in. Nearly 1200 simulations of each communication strategy are run over 10 different degrees of spatial clustering. Preliminary results seem to suggest that communication efficiency does not depend on resource clustering.
39 INVESTIGATING THE EFFECTS OF SMALL MOLECULES ON ALS PHENOTYPES IN A DROSOPHILA MODEL JASON JUANG, ARIELLE TRAN, DANIELA C. ZARNESCU
Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disorder that results in loss of motor function and death three to five years after diagnosis. At the moment, Radicava is the newest and second only FDA approved treatment for ALS that can slow decline and extend lifespan by a few months. There is still a big need for discovering new compounds that can prolong lifespan further and improve motor function.
Several genes including C9ORF72, SOD1, FUS, and TDP-43 contribute to ALS pathology; however, cytoplasmic inclusions related to TDP-43 are identified in more than 95% of cases. To encompass these cases, we work with a Drosophila animal model expressing wild type or mutant TDP-43 that exhibits motor function and lifespan phenotypes. This model is utilized in screening candidate compounds to determine toxicity and potential ability to rescue phenotypes. As of late, we have been focusing on three compounds that may have neuroprotective properties: tauroursodeoxychlic acid (TUDCA), 4-phenylbutyric acid (PBA), and AQU-118. A preliminary screen was conducted through larval turning, a behavioral assay used by our lab to measure changes in motor function. By timing how long treated larvae take to return ventral side down after being turned onto their backs, it is possible to evaluate the efficacy of compounds compared to ALS larvae.
Our results show that after treatment, ALS larvae exhibit a rescue in larval turning times, implying these compounds may be promising candidates. We are currently running additional experiments to measure the effects on TDP-43 levels through western blotting and the effects on lifespan. The results will provide future strategies on how to proceed with future experiments in order to gain a better understanding of the mechanism of action for these compounds and their therapeutic potential in patients.
Experiments funded by the Undergraduate Biology Research Program with funds from the University of Arizona Office of the Provost (to JJ), NIH NS091299 and Aquilus (to DCZ).
IMPLICATIONS OF EDAPHIC DRIVERS ON SOIL MICROBIAL COMMUNITIES GABRIEL KELLOGG, LAURA MEREDITH
Microbial community’s impact soils on numerous levels such as soil health, yet finding key edaphic drivers in relation to these different microbial communities is challenging. The challenge arises in trying to find edaphic drivers that impact microbial communities within soils since they are complex, and soils vary with chemical components, structure and location. In our research we attempt to define how key soil properties vary across soils and the impact they have on microbial communities. Literature has suggested that some of these key edaphic drivers include soil pH, organic carbon (quality and quantity), soil O2 and redox status, soil moisture availability and nitrogen and phosphorus availability (Fierer, 2017). Using statistical analysis such as pairwise correlation analysis, Principle Coordination Ordination analysis (PCO), and Non-Metric Multidimension Scaling (NMDS) we determine whether our key edaphic variables are potential drivers of microbial community composition. Our research provides us with an advantage in that we have various samples across multiple soil types, this will allow us to aim at identifying key edaphic drivers that contribute to microbial communities across all soil types. Determining key edaphic drivers of microbial communities in our set of soils will help expand that understanding to other soil types to better understand drivers of soil health and the development of microbial communities.
G.K. supported by the National Cancer Institute, Grant #2U54CA143924, through the Partnership for Native American Cancer Prevention (NACP) and Western Alliance to Expand Student Opportunities (WAESO) Louis Stokes Alliance for Minority Participation (LSAMP) National Science Foundation (NSF) Cooperative Agreement No. HRD-11017.
Reference: Fierer, Noah. “Embracing the unknown: disentangling the complexities of the soil microbiome.” Nature Reviews Microbiology 15, 579–590, August 2017.
40 PHOSPHATIDYLETHANOLAMINE-BINDING PROTEIN PROMOTES OPIOID ANTI-NOCICEPTION IN THE BRAIN AND SPINAL CORD BY REDUCING βARRESTIN2 RECRUITMENT TO THE MU OPIOID RECEPTOR CALEB KIM, JUSTIN LAVIGNE, KATIE EDWARDS, JOHN M. STREICHER
Opioids have been widely accepted as an effective treatment for chronic pain at a cost of serious side effects, including tolerance and addiction. Side effects are caused in part by desensitization and downregulation of mu opioid receptors (MOR) through recruitment of βarrestin2. Phosphatidylethanolamine-Binding Protein (PEBP) is known to regulate either Raf Kinase or G Protein Receptor Kinase-2 (GRK2) through phosphorylation by Protein Kinase C (PKC), and GRK2 is a major player in the recruitment of βarrestin2. However, PEBP has not been investigated for its role in MOR signaling and opioid response. We first tested this using an in vitro model of βarrestin2 recruitment in MOR expressing U2OS cells. We found that by inhibiting PEBP through siRNA knockdown or the small molecule inhibitor locostatin, we could increase the recruitment of βarrestin2 to the MOR in 2 separate assays. Conversely, we found that activating PKC via phorbol myristate acetate (PMA) led to PEBP phosphorylation/activation, and a reduction in βarrestin2 recruitment to the MOR, which could be blocked by PEBP knockdown. We then tested the role of PEBP in vivo by injection of locostatin into the brain or spinal cord, or CRISPR editing of PEBP in the spinal cord. We found that blocking PEBP in vivo led to a strong reduction in morphine-induced tail flick anti- nociception in both the brain and spinal cord. Together, these results support the hypothesis that PEBP acts to sequester GRK2 and block βarrestin2 recruitment downstream of the MOR both in vivo and in vitro. These results also suggest that enhanced PKC/PEBP activation by opioid drugs could further reduce opioid side effects and enhance analgesic efficacy.
Acknowledgments: ASPET/SURF grant for student support; institutional funds from the University of Arizona.
BLOOD-BRAIN BARRIER: PILOT INVESTIGATIONS ON REGIONAL DISTRIBUTION OF SODIUM- HYDROGEN EXCHANGER AND TIGHT JUNCTION PROTEIN EXPRESSION AFTER CORTICAL KCL INJECTION JOHN KIM, KARISSA COTTIER, EMILY GALLOWAY, TODD VANDERAH, THOMAS DAVIS, TALLY LARGENT-MILNES
Progressive research in pharmacology and neurology has recently centered around the molecular constituents of the blood- brain barrier (BBB) whose restrictive properties are required information for development of effective treatments for neurological disorders such as migraine headaches. Recent studies show implication of the membrane solute carrier Na+/H+ exchanger (NHE) in migraine physiology. Clinical studies show a tripled rate of migraine onsets in women over men in America and an approximately doubled rate worldwide, indicating sex hormones as a regulator of NHE expression and function. To determine the integrity of the BBB in migraine with aura models, expression of the tight junction proteins known to hold the BBB intact were probed for. We carried our experiment via molecular and behavioral techniques utilizing rat models grouped by gender and treatment. Our investigations targeted the sodium/hydrogen exchanger 1 (NHE1) isoform of NHE and the tight junction proteins Occludin, Claudin-5 and Zonula occludens-1. Our three areas of interest were the brain and brainstem in the CNS and the trigeminal nerve in the PNS.
Funded by UBRP with funds from the Office of the Provost and a grant from the Arizona Area Health Education Centers (AHEC) Program. The content is solely the responsibility of the authors and does not necessarily represent the official views of Arizona AHEC.
41 GRP8 PROJECT CHRISTOPHER KOURIS, MARK BEILSTEIN, KYLE PALOS
The current world population is increasing at a rapid rate which will require increased production to ensure global food security. One approach to increasing food supply is to expand the acreage of land under cultivation. However, much of the soil not currently cultivated is unsuitable because it is deficient in critical nutrients. Lack of the essential nutrient phosphate is one of the many deficiencies and the propelling point of our research. Our goal is to evaluate genes underlying the ability of plants to tolerate low phosphate conditions, with the hope of developing more efficient crops capable of growing in low nutrient soils. Our focus is the plant family Brassicaceae, which includes many important crop species like cabbage, broccoli, and kale, as well as the genetic model Arabidopsis thaliana (Arabidopsis). Camelina sativa is an emerging biofuel crop in Brassicaceae, and serves as a powerful comparison point to determine how findings from Arabidopsis apply to more economically important species. Previous research has shown that the RNA binding protein GLYCINE RICH PROTEIN 8 (GRP8) in Arabidopsis plays a role in the plant’s response to soil phosphate levels. Specifically, over-expression of GRP8 increases root hair density and results in better growth under phosphate starvation. We replicated these same phosphate starvation conditions for C. stativa. We utilized overexpression as well as CRISPR genome editing to determine how the three copies of GRP8 present in the genome of C. sativa effect root properties and overall growth under phosphate starvation. We are currently examining the phenotypes of GRP8 overexpression and knockout lines in C. sativa. Our initial findings show changes in root hair density, length, and primary root growth, but further study is required to make significant conclusions. In the future, we will explore the root and shoot phenotypes of GRP8 overexpression and knockout lines in C. sativa. Funding provided in part by UBRP with funds from the Office of the Provost (to C.K.).
ENGINEERING CRE RECOMBINASE SECRETING TOXOPLASMA EFFECTOR MUTANTS TO STUDY TOXOPLASMA EFFECTOR-NEURON MANIPULATIONS SAKTHI KUMAR, JOSH KOCHANOWSKY, ANITA A. KOSHY
Toxoplasma gondii is an intracellular parasite that infects the central nervous system (CNS) in up to a third of the population. Although it causes a life-long, asymptomatic infection, in immunocompromised individuals the infection can be fatal. Recent studies have shown that Toxoplasma secretes effector proteins into the host cell. These effector proteins can alter the host cell’s immune response. In addition, these effector proteins can be polymorphic between different strains of Toxoplasma leading to strain specific manipulations of host cells. One of these polymorphic effector proteins is dense granule protein 15 (GRA15). The GRA15 of certain strains upregulates the NFκB pathway in fibroblasts and macrophages. To date, no studies have addressed how GRA15 might affect CNS cells or the CNS immune microenvironment, especially in the context of in vivo infection. To determine the role of GRA15 in systemic and persistent infection, I used a CRISPR/CAS9-system to generate a deletion mutant that also secretes Cre recombinase (ΔGRA15:Cre). I confirmed that GRA15 was knocked out using diagnostic PCRs to verify that the selected ΔGRA15:Cre parasites were negative for GRA15 and positive for Cre recombinase. I have determined the efficiency of these transgenic strains to cause Cre-mediated recombination using Cre reporter cells that only express GFP after Cre-mediated recombination. Now that I have confirmed the in vitro efficacy of this strain to cause Cre- mediated recombination I will use the ΔGRA15:Cre strain to infect Cre reporter mice that express GFP only after Cre-mediated recombination. In these mice, I can permanently track CNS cells that have interacted with these transgenic parasites. By comparing Cre reporter mice infected with the ΔGRA15:Cre strain or the parental Cre strain, I will be able to determine the role of GRA15 on Toxoplasma’s ability to disseminate to the CNS, establish a chronic infection, and provoke a CNS immune response. I also can isolate the GFP+ neurons from mice infected with the ΔGRA15:Cre strain or the parental Cre strain and use transcriptional analysis to determine how GRA15 alters neuron transcription. Lastly, I would like to thank the Office of the Provost for funding to make this research possible.
42 CD47 AND NITRIC OXIDE SIGNALING IN NORMAL AND CANCEROUS CELLS HEBER LARA, SU CHUNG, WILLIAM R. MONTFORT
Cluster of Differentiation 47 (CD47), a transmembrane protein, emerged recently as a promising target for cancer immunotherapy. Upregulation of CD47 by tumor cells correlates with poor survival in multiple cancer types. Interaction of CD47 with the signal-regulatory protein alpha (SIRP-α) on macrophages and dendritic cells relays a “don’t eat me” signal that blocks phagocytosis and subsequently inhibits cross-presentation, allowing tumor cells to evade immune surveillance.
Soluble guanylyl cyclase (sGC), a key mediator in nitric oxide (NO) signaling, regulates the tumor microenvironment (TME). In endothelial and vascular smooth muscle cells, NO binding to sGC stimulates catalysis and the formation of cyclic-guanosine monophosphate (cGMP). cGMP initiates a molecular cascade that can lead to vasodilation along with other physiological effects. Changes in cytosolic calcium ion concentration influences sGC activity and its inhibition can be attributed to CD47 bound to thrombospondin-1 (TSP-1), an extracellular glycoprotein produced by immune cells. Inactivation of sGC by CD47/TSP- 1 occurs in non-cancerous cells; however, this interaction in cancerous cells remains to be elucidated.
We hypothesize that TSP-1 inhibits sGC activity in normal cells, but not cancerous cells, in the TME through binding to CD47. We further hypothesized that enhanced NO signaling through sGC leads to increased tumor progression.
We are examining the CD47/TSP-1/sGC signaling axis within cancerous (MDA-MB-468) and normal human breast cells (MCF- 10A) Using immunoblots, immunostaining, cGMP assays, QPCR, and protein assays.
Our results indicate that NO activates sGC enzymatic activity in both MDA-MB-468 cells and MCF-10A cells, but to a greater degree in the cancerous cells. Additionally, CD47 in MCF-10A cells runs at a higher molecular weight than expected on SDS- PAGE gels perhaps as a result of post-translation modification or differences in protein binding partners. How CD47 in tumor and normal cells influences NO signaling and tumor progression will be key for understanding the outcomes of anti-CD47 clinical trials. Our studies are beginning to uncover a mechanism in this newly discovered signaling system.
This work was made possible in part by: NIH MARC Training Grant T34 GM08718, NIH GM117357, and the Montfort Research Group, Chemistry and Biochemistry Department at the University of Arizona.
ALGORITHM FOR ESTIMATION OF BLOOD FLOW RATES IN LARGE MICROVASCULAR NETWORKS BOHAN LI, TIMOTHY W. SECOMB
The microcirculation is a network of very small blood vessels that supply oxygen and other solutes to tissues. Theoretical analyses of solute transport provide insight into the effects of vascular network structure on tissue functionality. Such analyses often require knowledge of vessel blood flow rates, which may be difficult to obtain experimentally depending on the size and complexity of the network. The goal of this this project is to improve the computational speed and efficiency of the blood flow estimation algorithm presented in Fry [Microcirculation 2012; 19:530]. The algorithm estimates blood flow rates for a vascular network in the absence of complete information about the boundary inflow and outflow segments by minimizing the deviation of shear stresses and pressures of vessel segments from target values, constrained by conservation of flow. The method requires multiple solutions of large systems of linear equations, in which use of direct methods requires long computation times for large networks. A specialized linear system solver using the Preconditioned Biconjugate Gradient Stabilized Method was developed. This improved algorithm has proven effective in estimating flows in rat mesentery networks, and can be run for a network of 3572 vessel segments in minutes. This project was funded by NIH Grant U01HL133362 and the UBRP with funds from the Office of the Provost.
43
INHIBITION OF THE UBIQUITIN-PROTEASOME SYSTEM IS NOT SUFFICIENT TO REVERSE ARSENITE-ENHANCED INFLUENZA VIRUS INFECTION IN LUNG EPITHELIAL CELL LINES RICARDO LIRA JR., EVA AMOUZOUGAN, WALTER KLIMECKI
Arsenic is a naturally occurring element present in the environment from both natural and human sources. Exposure to arsenic in drinking water has been associated with infectious and non-infectious diseases. Epidemiological data suggests that infants exposed to arsenic have an increased risk of respiratory infections in their first year of life. We are interested in whether arsenic exposure can cause human respiratory cells to become more susceptible to influenza infection. Previous studies from our lab revealed that chronic exposure to sodium arsenite enhanced influenza A virus infection in lung epithelial cell lines. Recent reports suggest that influenza virus depends on several cell-signaling pathways, including protein degradation pathways, for infection and replication. Based on these observations, we hypothesize that treatment with cellular target inhibitors such as MG132, and bafilomycin-A1 could impair arsenite-enhanced influenza A virus infection in vitro. The goal of this project is to determine whether these inhibitors can suppress infection in arsenic exposed cells. Using cell viability assay, we showed that treatment with 1µM of MG132 did not reverse arsenite-enhanced influenza virus infection in A549 cells. This study could help uncover new mechanisms by which arsenic exposure enhances influenza viral infection. This project is funded by National Institute of Environmental Health Sciences, Grant #1-R25-ES025494, and Western Alliance to Expand Student Opportunities (WAESO) Louis Stokes Alliance for Minority Participation (LSAMP) National Science Foundation (NSF) Cooperative Agreement No. HRD-11017.
INVESTIGATING THE PHYSICAL INTERACTION OF TWO PROTEINS IN THE LIPOIC ACID BIOSYNTHETIC PATHWAY ALBERT LIU
Lipoic acid synthesis in bacteria closely resembles the eukaryotic pathway in mitochondria; both use octanoic acid as a precursor to lipoic acid. However, in yeast, it has been observed that a glycine cleavage enzyme subunit, Gcv3, is an intermediate in the pathway and physically interacts with yeast lipoyl-protein transferase, Lip2; the two proteins co-purify from a strain with a carboxyl-terminal tag on Gcv3. In the first step of the pathway, Lip2 transfers the octanoic acid precursor from the acyl carrier protein Acp1 to the Lys102 residue of Gcv3. Why Lip2 and Gcv3 co-purify together remains unknown, since most enzymes are not physically associated with their substrate stably over time. It is hypothesized that the Lip2-Gcv3 complex exists to sequester enough Gcv3 for the lipoic acid biosynthetic pathway, since it is needed also in the glycine cleavage (GC) pathway.
To test whether Lip2 binds to Gcv3 independently of Gcv3 acylation, Lys102 loss-of-function, respiratory deficient mutant strain extracts were used for the co-purification protocol. It was found that association of Lip2 with Gcv3 is independent of the acylation status at Lys102. As a second test of the importance of Gcv3 acylation in the Lip2 association, the mitochondrial fatty acid biosynthetic pathway was blocked by deleting the gene for one of the enzymes in the pathway, Etr1. Surprisingly, the Gcv3-Lip2 complex could not be purified from this extract, though both proteins were present, and Gcv3 was purified quantitatively via the carboxyl tag. To test whether induction of the GC pathway changes how much Gcv3 is associated with Lip2, yeast were grown on minimal medium containing glycine. Less Gcv3 was associated with Lip2, and a high-mass, approximately 400 kilodalton Gcv3-containing complex appeared in the extract that was not apparent in extracts of yeast grown on rich medium. This large complex is presumed to be the GC complex, which has never been purified previously. Currently, the composition of this high-mass complex is being investigated, with a specific focus on the ratio of Gcv3 dedicated to the lipoic acid versus glycine cleavage pathways under various growth conditions. Techniques utilized in the project include mitochondrial extraction, protein purification, Western blotting, Blue Native gel analysis, gel silver staining, etc.
44 This work is funded by UBRP with funds from the UA College of Medicine and private donors, and the Department of Molecular and Cellular Biology.
TESTING INDIVIDUAL-BASED MODELS FOR DIVISION OF LABOR IN SOCIAL INSECTS VIA NUMERIC SIMULATIONS COLIN LYNCH, ANNA DORNHAUS, ROBERT WILSON
The response threshold hypothesis posits that ants divide labor by varying individual ant's reactivity to signals for work in the environment. The point at which an ant responds to this task-associated stimulus is the response threshold. Individuals with low response thresholds become specialists. Essentially, the hypothesis is that the variation in the starting point of a task can account for variation in task allocation. Under this paradigm the task-associated stimulus is defined as a signal whose intensity increases as a function of ants not performing the task. However, we have found that it is also possible that a work signal can decrease over time, The former type of signal is classified as a task initiation stimulus and the latter is a task completion stimulus. In addition, it is also possible that the variance of an endpoint of a task, i.e. the satisfaction threshold, can account for variation in task allocation. These possibilities generate at least four total models that can describe the proximate cause of division of labor. Both response thresholds and satisfaction thresholds can operate on a task initiation stimulus or a task completion stimulus. We have simulated Markov Chain models across a wide parameter space to see which was the most effective at reducing work in the virtual environment.
PAPER ANGIOGENESIS-ON-A-CHIP MARIANNE MADIAS, KATTIKA KAARJ, SOOHEE CHO, PATARAJARIN AKARAPIPAD
Angiogenesis, the formation of new blood vessels, plays a critical role in many biological processes and is a mechanism that remains in need of further study. In vivo methods of studying angiogenesis are expensive and time-consuming, and traditional two-dimensional in vitro assays lack physiological relevancy. The use of organ-on-a-chip (OOC) platforms enables improved in vitro study of three dimensional morphogenetic processes relevant to angiogenesis. Polydimethylsiloxane (PDMS) based OOC’s have been used as the conventional platform for such studies, however paper based systems are a promising new platform for cell-based assay. Paper is advantageous as a platform because it is inexpensive, accessible, and most importantly flexible, presenting the opportunity for a dynamic OOC, as opposed to the rigid and unchangeable form of PDMS based OOC’s. Here we demonstrate a paper based cell culture OOC, fabricated by wax printing hydrophobic barriers enclosing hydrophilic channels on nitrocellulose paper. Rat vascular endothelial cells (RVECs) were used to study cell sprouting within collagen coated hydrophilic channels, using vascular endothelial growth factor (VEGF) and the signaling lipid sphingosine-1-phosphate (S1P) to induce sprout formation on the nitrocellulose OOC. The effect of shear stress was also studied by inducing pulsatile flow on the chip using servo motors run by an Arduino microcontroller. We demonstrate angiogenic sprouting on a paper based OOC, showing that paper-based platforms can provide a simple, inexpensive, and accessible method of studying angiogenesis in a physiologically relevant in vitro environment.
Funding: NIH T34Training Grant 2T34GM008718-19
45 DO BEES GENERALIZE CAUTIOUS SAMPLING BEHAVIOR LEARNED ON UNREWARDING FLOWERS, AND IF SO, HOW? ANDREA MASON, DAVID KIKUCHI, ANNA DORNHAUS
Bees sometimes encounter unrewarding flowers while foraging and when they have these negative experiences while foraging, it has the potential to cause them to forage more cautiously. Negative experiences can make bees probe flowers more cautiously to avoid costly mistakes. This experiment addresses the question: Do bees generalize cautious sampling behavior learned on unrewarding flowers, and if so, how? In this experiment, we used artificial flowers to test how bees generalize cautious probing behavior after exposure to unrewarding flowers. We hypothesized that bees would either: generalize cautious sampling by probing all flowers they have not had positive experiences with, cautiously sampling only flowers they have had negative experiences with, or sampling all flowers cautiously. For this experiment, bees were trained to associate blue and green flowers negatively with water and were trained to associate cyan and gray flowers positively using 2M sucrose. After training, bees were given the test board with the two flowers used in the training trials (blue/green and cyan/gray) and two flowers with intermediate combinations (blue/cyan and green/gray). Their visits to the test board were filmed and later analyzed using the video software VLC to record visit time to the nearest frame. Each bee’s first six visits were recorded and the times were averaged for each flower color combination. A two-way ANOVA was used to test whether visit time to each color combination depended on flower type or individual bee. The results of the test showed a non-significant result for flower color combination (ANOVA, p=0.8028, DF=3). The result for individual bees was asignificant (ANOVA, p=0.0432, DF=2). The results showed bees visited the negatively reinforced blue and green flowers the longest amount of time while visiting the postiviely reinforced cyan and gray flowers and intermediate flowers for around the same amount of time; this result does not reflect any of the hypotheses. The significant result for individual bee suggests that the amount of time bees spent sampling the various color combinations was a relfection of each individual bee, rather than the flower color.
ELECTROPHYSIOLOGIC CORRELATES OF SPATIAL RELEASE FROM MASKING KAILYN MCFARLANE, BARBARA CONE, NICOLE MARRONE, ALI POURJAVID
Background: Speech is difficult to understand when it is mixed with background noise that comes from the same direction as the talker. If the speech signal background noise can be separated from one another there is an improvement in speech detection and discrimination ability known as spatial release from masking (SRM). Previous research using perceptual test methods has demonstrated that listeners with hearing loss have variable benefit from SRM. The aim of this study was to document the benefits of spatial release using the cortical auditory evoked potential (CAEP) in response to speech tokens as noise level and location were varied.
Methods: Adults with normal hearing (n=30) were tested in a sound field under three conditions: signal in quiet, signal and noise co-located at 0° azimuth, and signal and noise spatially separated by 90° and at three signal-to-noise ratios (SNRs): +10, 0, and -5. CAEPs were recorded in response to consonant-vowel speech tokens in each condition. SRM was measured by comparing the latencies and amplitudes of the P1, N1, and P2 peaks of the CAEPs of the co-located conditions to the corresponding CAEP components of the spatially separated conditions. Psychophysical tests of speech perceptio, were completed in the same conditions of varying background noise level and location.
Results: Latencies and amplitudes of the CAEP P1-N1-P2 response components showed a systematic shift as a function of noise location, stimulus, level, and SNR. Co-located conditions across all stimuli, levels, and SNRs had longer latencies and smaller amplitudes than the spatially separated condition with the same SNR.
Conclusions: The systematic shifts in CAEP response parameters as a function of noise location demonstrated an electrophysiologic analog of perceptual spatial release from masking. The latency and amplitude differences found in the spatially-separated conditions suggested greater benefit than was appreciated in the speech perception tests. Release from masking appears to be a result from central auditory nervous system binaural mechanisms combined with ear canal acoustics. These results on normal hearing adults provide a basis for investigation of the differences in SRM that adults with hearing loss
46 experience. K. McFarlane was supported by the University of Arizona Office of the Provost through the Undergraduate Biology Research Program and by the James S. and Dyan Pignatelli/UniSource Clinical Chair in Audiologic Rehabilitation for Adults.
BODY SIZE VARIATION IN COMMON EASTERN BUMBLE BEES: DO MALES HAVE THE SAME AMOUNT OF BODY SIZE VARIATION AS FEMALE WORKERS? ALAINA MICHALES, EVAN KELEMEN, ANNA DORNHAUS
In the bumble bee B. impatiens, body size is highly variable among individual workers. Typical of social insects, workers perform different tasks in the colony such as foraging and caring for brood. However, morphological differences in body size do not necessarily dictate specialization of a task. Instead, a range of sizes participate in a variety of tasks during their lifetime. On the other hand, male bees spend much less time in the colony after emerging. Before leaving the nest to find potential mates, males participate briefly in brood care (Cameron 1985). However, there is no other evidence that males contribute to the colony after leaving the nest. This leads to the question of whether or not variation in worker body size serves a function or benefit to the colony as a whole. The purpose of this research is to examine the potential for selection to be driving highly variable body sizes among bumble bee workers by comparing the differences in variation between males and workers.
ANTAGONISM OF ESTROGEN RECEPTOR ALPHA IN ANGIOTENSIN II-INDUCED HYPERTENSION IN FEMALES CAITLIN MOFFETT, JOSH UHLORN, NATHANIEL HUSBAND, JILL ROMERO-ALESHIRE, AMY KELLY, HEDDWEN L. BROOKS
High blood pressure affects one in three adults in the United States. Sustained high blood pressure, or hypertension, is the leading risk factor for cardiovascular disease and kidney failure. Sex and age are major characteristics in hypertension prevalence. Although young men are more susceptible to hypertension than age-matched women, the opposite is true in older age groups. After entering menopause, estrogen levels drop and women lose protection and have a higher incidence of hypertension. Our aim is to determine how estrogen signaling protects younger women from cardiovascular diseases. Because hypertension is accompanied by a low-grade inflammatory state, in addition to blood pressure, we study the immune system, specifically the role of T cells in the development of hypertension. In this study we treated young female mice with angiotensin II (Ang II) (infused 800 ng/kg/min, for 14d) and with methylpiperidinopyrazole (MPP), a selective estrogen receptor alpha (ERα) antagonist. We had three groups, control, Ang II infused, and Ang II/MPP. On Day 7 of Ang II infusion MPP treatment caused a significant increase in systolic blood pressure (SBP) as compared to control mice (p = 0.0166). However, on Day 14, the Ang II group alone was the only group with significantly elevated blood pressure as compared to control (p = 0.0199). Our previous RNAseq studies identified a cohort of genes that demonstrated a differential expression in female splenic T cells (CD4+) following Ang II infusion (vs con female T cells). In this study, analysis of CD4+ gene expression via qPCR indicated significant downregulation of heat shock protein Hspa1b in Ang II-infused mice (vs con p < 0.001) and Ang II/MPP mice (vs con p < 0.001). Upregulation of the calcium binding protein S100a8 occurred in Ang II-infused mice (vs con p = 0.048) and Ang II/MPP mice (vs con p = 0.003). Inflammatory genes such as vascular cell adhesion molecule VCAM1 (p = 0.020) and chemokine receptor CCR3 (p = 0.034) were upregulated in Ang II/MPP compared to Ang II. These data indicate that while ERα antagonism does not induce severe Ang II-induced hypertension in cycling female mice, we were able to confirm CD4+ expression changes from RNAseq analysis of splenic T cells.
Funding: UBRP with funds from the Office of the Provost, NIH R01HL131834 (HLB), Sarver Heart Center Endowment for Women’s Health Research (HLB)
47 IDENTIFICATION OF GENETIC VARIANTS THAT INFLUENCE HUMAN BODY WEIGHT WITH ALTERED POPULATION BASED FREQUENCY DUE TO ADAPTATION TO ENVIRONMENT TASHA NEZ, PANKAJ KUMAR, DARIN MAHKEE, LESLIE BAIER
Background: Historically, the Pima lived a traditional lifestyle producing corn, squash and beans. Traditional Pima had lower body weight and a lower diabetes prevalence. Today, Pimas live a modern lifestyle with a western diet. Modern Pima also have a high obesity prevalence and one of the highest reported prevalences of Type 2 Diabetes worldwide. In this study, we looked for differences in frequency of genetic variation across different geographical populations that correlate with environmental variables such a latitude and longitude, and further associate with body mass index in the Pima.
Methods: Our group has previously identified genetic variants (i.e. SNPs) that associate with BMI in Pima. Among these variants showing a nominal p value for BMI (p ≤ 0.001), 190 SNPs were present in the dbCLINE database. Among these 190 variants, 24 associated with latitude across 61 global populations. Twelve SNPs had a higher frequency of the derived allele in Pima people versus all other ethnic groups.
Results: We observed 12 SNPs out of 24 that had higher frequencies of the derived allele in the Pima compared to global derived allele frequency. We hypothesized that alleles which are more common in Pimas as compared to all other ethnic groups, which also correlate with environment (i.e. latitude differences) may have an adaptive role in Pimas. Genes in regions surrounding these SNPs were examined for their possible role in obesity. We focused our attention on rs11700219, which is 100 kb downstream of the APMAP (adipocyte plasma membrane associated protein) gene. This SNP was found to be associated with BMI in the Pima (p=0.000847, n=6789) where the derived allele G predicts higher BMI. APMAP has been shown to play a role in adipogenesis and adipocyte differentiation where loss-of-function of APMAP leads to expansion of visceral adipose tissue.
Conclusion: We identified a SNP rs11700219 near the APMAP gene, where the derived allele is much more common in Pima people, is associated with latitude, and also associated with a higher BMI. Based on the important functional role of APMAP in adipogenesis, our data suggests an adaptive role for this SNP in the Pima people.
THE MU-DELTA OPIOID RECEPTOR HETERODIMER PROMOTES ACUTE AND CHRONIC MORPHINE INDUCED DEPENDENCE/WITHDRAWAL IN MICE PAUL NGUYEN, ATTILA KERESZYTES, KEITH OLSON, VICTOR HRUBY, JOHN STREICHER
Opioid drugs like morphine are the gold standard for treating acute and chronic pain, but induce detrimental side effects such as tolerance and dependence. It has been suggested that the mu-delta opioid receptor heterodimer (MDOR) transduces some of these side effects and that heterodimer targeted drugs could be a solution to weaken these side effects. We have thus created an MDOR selective antagonist called D24M. We evaluated D24M in vitro, and found a ~100 fold selectivity for the MDOR over the monomers. Similarly, we performed hot water tail-flick experiments in mice in the presence of various doses of D24M against CYM51010 and Deltorphin-2 (MDOR selective agonist), and DAMGO (MOR monomer selective agonist), and found that D24M potently (A50=2-7.8 nmol) blocked MDOR activity with no activity against the MOR monomer up to 10 nmol. To test the ability of D24M to lessen acute or chronic morphine-induced dependence and withdrawal, we established acute (4 hr) and chronic (4 day) morphine dependence models, with 1 nmol D24M or vehicle injected 5 minutes prior to naloxone precipitation of withdrawal. We found that D24M strongly reduced jumping behavior in dependent mice in both the acutely and chronically models; further confirming that the MDOR promotes withdrawal behavior, and that D24M could possibly be a promising drug candidate for opioid dependence and withdrawal. These discoveries will further determine the role of the MDOR in vivo, and provide a novel tool that could greatly impact opioid heterodimer research.
Acknowledgements: Institutional support from the University of Arizona
48 DOWNSTREAM TRENDS OF IN VITRO BIOASSAY RESPONSES IN A WASTEWATER EFFLUENT- DOMINATED STREAM JULIANA ORDINE, KEVIN D. DANIELS, SHANE A. SNYDER
As a result of urbanization, growing populations, and drought conditions, surface waters are becoming increasingly influenced by wastewater effluents. These complex mixtures can be composed of trace organic compounds and potentially bioactive constituents, which are not fully attenuated by conventional wastewater treatment systems. The aim of this study is to characterize the effluent of two wastewater reclamation facilities (WRF) and the Lower Santa Cruz River, Pima County, Arizona, in which wastewater is discharged. Analytical methods applied include bulk organics parameters (i.e. EEM/TOC) and targeted analysis of contaminants of emerging concern. In addition to the chemical analysis, a variety of bioassays were applied to evaluate the bioactivity of the river. Altogether, the results showed the highest concentrations at the wastewater outfalls, with a decrease in response downstream of the river. Possible causes of these downstream concentration reduction include photo degradation and sediment sorption and deposition. Thus, as wastewaters continue to have greater impacts on surface waters, there is a dire need to monitor water sources and the effluents being discharged in the environment. This research was founded by the National Institute of Environmental Health Sciences, Grant#1-R25-ES025494, and Western Alliance to Expand Student Opportunities (WAESO) Louis Stokes Alliance for Minority Participation (LSAMP) National Science Foundation (NSF) Cooperative Agreement No. HRD-11017.
COMPETITION BETWEEN ANT COLONIES VARY IN STRATEGY VICTOR PAAT, SARA HU, ANNIE DIXON, ANNA DORNHAUS
Ants are noted as social insects. This means that they form colonies, are lead by one or multiple queens, and partake in certain tasks to maintain survivability which include brood-care and scavenging. However, little is known about how collectively they go about solving problems. How ants actually overcome those problems is yet another division of questioning. Specifically, this experiment is set to investigate how ants overcome a practical problem, other ants.
The ants used in this experiment, Temnothorax rugatulus (Commonly known as the rock ant), are known to inhabit forests or deserts, their populations concentrated on the western side of North America. Unique to them, they house themselves in the crevices of rocks or dead trees as opposed to digging tunnels. Ants tend to be territorial, so in terms of competition, Temnothorax rugatulus compete for nest space, total area, and resources.
The Ant Competition Experiment aims to answer questions such as search patterns ants take, interaction change at the inter and intra-colony level, and others, all while under the influence of an enemy colony. While many questions arise regarding ant competition, the focus here is to analyze what strategies, if any, each colony applies to "win" against the other. To test this, two colonies of about the same size are allowed to interact with one another in a closed arena while we analyze their behavior.
DOES FAMILIARITY INCREASE THE PERCEIVED SHARPNESS OF AN OBJECT? DIANA PEREZ, SARAH M. COOK, MARY A. PETERSON
Does memory affect the appearance of an object such that, for a given level of blur, the borders of familiar objects are perceived as sharper than the borders of novel objects? We typically fixate and attend to objects in the visual field; hence, objects are perceived in focus. Memories represent the norm of previously seen objects; for familiar objects, these are likely to be objects with sharp, focused, borders. Perception arises from the integration of the current stimulus and memory representations. Consequently, familiar objects with blurry borders might be perceived as sharper than matched novel objects.
49 We measured participants’ Point of Subjective Equality (PSE) -- the level of blur at which two objects are perceived as equally sharp. On each trial, participants viewed two black-silhouettes on a gray background: one was a silhouette of a familiar object – a table lamp; the other was a matched novel object created by spatially rearranging the parts of the table lamp. One of the objects was the Test, varying in blur from low to high levels across trials; the other was the Standard, which was held at a constant medium blur value. Familiar and novel objects served as Standard and Test equally often; the left/right location of Test and Standard objects varied across trials. Participants’ task was to determine if the two objects were the same or different levels of blur. In a previous experiment, the familiar object was perceived as sharper than its novel counterpart, p < .001. In that experiment, the name of the familiar object preceded the pair of stimuli on ~17% of trials; no effect of this word prime was observed. In Experiment 1, we replicated the effects without a word prime, p < .05, supporting the hypothesis that memory representations sharpen the perception of the borders of familiar objects. Experiment 2 tests the generalizability of these results by using two new sets of stimuli: an anchor and a standing woman, each presented alongside their part-rearranged, novel matches. We expect to replicate the results of Experiment 1, supporting the hypothesis that object memories can alter the appearance of an object.
Funding provided in part from UBRP with funds from private donors.
THE EFFECTS OF WET-DRY CYCLES ON GREENHOUSE GAS EMISSIONS FROM SOUTHERN ARIZONA COMPOST SOURCES SAVANNAH PERNO, JOSEPH BLANKINSHIP
Soil organic carbon (SOC) sequestration has emerged as one of the best options for mitigating and adapting to global climate change. Soil contains more carbon than plants and the atmosphere combined, and there may be potential to store even more SOC with improved land management. For ecosystems in arid climates, SOC has the added benefit of increasing soil water retention, which could help alleviate plant water stress in the future as rainfall patterns continue to become more irregular. Because plant production is limited in arid ecosystems, compost from cities and industry is a possible large scale source of organic carbon for soils, which has co-benefits for diverting waste from landfills and therefore promoting sustainability. Compost application has been shown to enhance SOC stabilization in wetter climates, but in arid regions like southern Arizona, compost application has not been examined. As a first step in determining the suitability of local compost sources for SOC stabilization, our objective was to determine—under irregular moisture conditions and hot temperatures—which sources of compost near Tucson emit the least amount of greenhouse gases. Otherwise, adding compost might actually accelerate global warming. We measured a total of 7 gases including carbon dioxide (CO2), methane (CH4), and nitrous oxide (N2O) from four local composts that were generated from manure, food waste, pecan shells, and landscaping woody waste. The composts were applied to two local soils: a young sandy soil and and old clay-rich soil We found that all types of compost emitted slightly larger amounts of CO2 after the initial wetting event, and then rates decreased to near baseline levels. The other measured gases varied by compost type. In addition, the older soil emitted less CO2 than the young soil before wetting regardless of compost type. However, after the initial wetting, CO2 emission in the old soil outpaced the young soil. These findings suggest that soil physical properties, compost ingredients, and climatic conditions impact the carbon sequestration potential of soil organic amendments. Support provided in part through UBRP with funds from BIO5 and private donors.
ANTI-NOCICEPTIVE PROPERTIES AND FUNCTIONAL CHARACTERIZATION OF BETULINIC ACID, AN EXTRACT FROM THE DESERT LAVENDER HYPTIS EMORYI NANCY PHAM, YINGSHI JI, SHIZHEN LUO, SONG CAI, AUBIN MOUTAL, MAY KHANNA, LESLIE GUNATILAKA, RAJESH KHANNA
Over 1.5 billion people suffer from chronic pain worldwide and at immense cost to society. Current treatments are not effective for all patients and often have adverse side effects including a high potential for abuse. These challenges provide motive to
50 develop a novel, non-addictive pain therapy. Natural products have emerged as probes for the pain pathway. Isolated from the Hyptis emoryi, also known as the desert lavender, NPC-LG-001 was used as a counter irritant for pain by the California Indians in the early 1900s. Here, we evaluated the molecular and behavioral basis of NPC-LG-001 using a combination of molecular biology, functional calcium imaging, natural product biochemistry, and rodent pain models. Depolarization-evoked calcium influx was blocked in a concentration-dependent manner by NPC-LG-001. Using constellation pharmacology, a technique that images live cells to find cell-specific combinations (constellations) of key signaling proteins to define cell populations, we investigated which dorsal root ganglia neuron (i.e. nociceptor) subpopulations were affected by NPC-LG-001. Compared to control neurons, NPC-LG-001-treated DRG neurons exhibited a greater competence to acetylcholine, ATP, and menthol, identifying possible pathways targeting acetylcholine receptors or transient receptor potential cation (TRPA1) channels (AITC responders), ATP, or those transient receptor potential cation, subfamily M, member 8 (TRPM8) channels (menthol responders). The HIV viral glycoprotein gp120 enhances calcium influx through voltage-dependent and glutamate-gated ion channels to induce its neurotoxicity; blocking calcium entry restores calcium homeostasis and alleviates neuronal damage. Intrathecal application of NPC-LG-001 reversed mechanical allodynia induced by gp120. Thus, our data support the use of NPC- LG-001 as a potential therapeutic. Future studies will determine its safety and relative efficacy via an oral route.
N.P. supported by the National Cancer Institute, Grant #2U54CA143924, through the Partnership for Native American Cancer Prevention (NACP).
THE ROLE OF NEURON NUMBER IN THE SMALL-BRAIN PHENOTYPE OF CASK Δ18/ Δ18 DROSOPHILA MUTANTS LAUREN PISANI, JUDITH A. TELLO, LINDA L. RESTIFO
Drosophila melanogaster is used as a model system in genetic studies of intellectual disability (ID). Insects and humans share many genes in which mutations cause ID. The protein CASK is essential for normal brain development. Various mutations in the human X-linked CASK gene lead to microcephaly (due to small brain size) and/or ID. Drosophila CASK Δ18/Δ18 mutants have smaller head and brain sizes when compared to their specific genetic controls, CASK Ex33/Ex33. Primary cell cultures of developing central nervous system (CNS) tissue also revealed that the mutant neurons grow smaller neurite arbors compared to the controls. Smaller neuron size is thought to be a contributing factor to the CASK Δ18/Δ18 small-brain phenotype. Holistic observations from previous cultures suggest there is no difference in the number of neurons obtained from mutant and control CNS. We are investigating neuron numbers within the mutant Drosophila CNS to test the hypothesis that they are normal. If the number of neurons is the same in the mutant and control CNS, then reduced neuron number, from insufficient neurogenesis or excessive apoptosis, cannot be the main cause of the small brain size of CASK Δ18/Δ18 mutants. Parallel cultures of CASK mutant and control neurons dissociated from the whole CNS of wandering larvae were prepared, and the neurons were immunofluorescently stained. The neurons were imaged by fluorescence microscopy at 400X. Neuronal cell body number was counted and compared between samples of mutant and control cultures. The findings will contribute to the understanding of how CASK contributes to brain size in human as well as insect development.
DEVELOPMENTAL DELAYS ASSOCIATED WITH PLANT LONG NON-CODING RNA TRANSCRIPTION DURING SALT STRESS CAROLINE PLECKI, MARK BEILSTEIN, KYLE PALOS
In soils across the world, increasing salinity is creating detrimental environments for essential crops, resulting in loss of arable land. This presents a serious obstacle in the effort to feed the growing human population. To help overcome this obstacle, we are determining how gene expression is regulated in response to high salt stress, with the goal of generating crops capable of growing in semi-saline conditions. We focus on the plant family Brassicaceae, which contains important crop species such as
51 broccoli and cabbage, and the plant genetic model Arabidopsis thaliana. Our studies of gene expression regulation are centered on regions of the genome that are transcribed into long non-coding RNAs (lncRNA). lncRNAs are long stretches of RNA that do not code for a protein, but rather are involved in critical cellular processes such as regulating transcription of protein coding genes. Using a bioinformatics approach, we compared gene expression in Brassicaceae species subjected to salt stress to identify conserved lncRNAs that are consistently upregulated. One of the top candidate lncRNAs we identified is At1NC031460. To characterize the products transcribed from this locus, we determined the length of the lncRNA transcript produced at different stages of development. To explore the function of these transcripts, we evaluated developmental phenotypes to uncover a consistent growth delay in T-DNA knockout lines At1NC031460, a unique characteristic among disrupted lncRNAs. In addition, At1NC031460 showed highly conserved promotor elements upstream of the transcriptional start site and snoRNA motifs within the transcribed region. We are currently studying the effects of editing each of these regions of the genome on the ability of plants to tolerate salt stress. This research was funded by National Science Foundation by the PGRP grant #1444490, and by UBRP with funds from the Senior Vice President for Research, Dr. Kim Espy.
SUB-ANESTHETIC KETAMINE INCREASES MICROGLIA RAMIFIED MORPHOLOGY IN A PRE- CLINICAL MODEL OF LEVODOPA-INDUCED DYSKINESIA AYUMI POTTENGER, HELENA W. MORRISON, MITCHELL J. BARTLETT, TORSTEN FALK
Parkinson’s disease (PD) is a neurodegenerative disease caused by the death of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and is characterized by motor dysfunction. PD has no cure, but symptoms can be treated with levodopa (L-DOPA). Continuous L-DOPA use can cause levodopa-induced dyskinesia (LID). Sub-anesthetic ketamine has been shown to reduce LID, as measured by abnormal involuntary movements (AIMs). The literature suggests that ketamine treatment leads to changes in dendritic spines via the tropomyosin receptor kinase B (TrkB) pathway. Microglia may play a role by phagocytosing neuronal spines. We hypothesized that ketamine would prevent AIMs and increase microglia ramified morphology, an indicator of a microglia response. In order to test this hypothesis, we studied the behavioral and histological outcomes in LID rats with and without ketamine treatment. Rats were injected with a neurotoxin which targets dopaminergic neurons. Rats were then primed for 2 weeks with daily injections of L-DOPA. Twice, rats were treated with ketamine, ketamine + ANA-12 (KA), or vehicle. ANA-12 is an antagonist to TrkB. AIMs were scored every 3-4 days to assess changes in LID. On day 14, ketamine-treated rats showed a reduction in AIMs as compared to controls. This effect of ketamine on AIMs was lost in the KA group; KA animals showed no significant reduction in AIMs compared to controls. A sub-analysis of AIMS scores in ketamine-treated animals revealed two subgroups: ketamine responders (K), and ketamine non-responders (KNR), which showed no difference in AIMs, as compared to controls. Coronal slices were processed for immunohistochemical staining using ionized calcium-binding adapter molecule and tyrosine hydroxylase to visualize microglia and dopaminergic neurons, respectively. The SNpc on the ipsilateral and contralateral side were imaged using confocal microscopy to obtain photomicrographs for image analysis. In the K group, a decrease in the number of microglia and an increase in cell ramification was observed. These data indicate that in the vehicle, KA, and KNR groups, microglia in the SNpc have a lingering response to the injury, which was increased beyond contralateral conditions after ketamine treatment in the K group.
Funding: National Institute of Environmental Health Sciences, Grant #1-R25-ES025494
NEUROGENESIS IN THE AGING BRAIN: A MALE VERSUS FEMALE COMPARATIVE STUDY ALEXANDER REED, M.J. CORENBLUM, L. MADHAVAN
It is known that new neurons are generated throughout life, particularly in specialized niches located in the subventricular zone of the forebrain and the subgranular zone of the hippocampus, in the adult mammalian brain. However, this process of neurogenesis also undergoes a significant decline with advancing age. Previously, using male rats, our laboratory has shown
52 that the age-related decrease in neurogenesis is not linear in nature, but in fact involves a specific temporal pattern of decline marked by a critical period during middle-age (13-15 months). At this time point there is a striking reduction in the survival and neurogenic capacity of neural stem cells (NSCs) (Corenblum et al., Aging Cell, 2017; Ray et al., Cell Transplantation, Under review). We have also shown that a correlative loss in the expression of the redox-sensitive transcription factor, Nrf2, is a key mechanism mediating this phenomenon. However, whether and how neurogenesis patterns and Nrf2 function differ in the female brain, are not well understood. Here, we compare hippocampal neurogenesis across the age-spectrum in male and female rats. Specifically, we immunohistochemically analyzed the proliferative and neurogenic potential of the NSCs using Minichromosome Maintenance Complex Component 2 (Mcm2) and Doublecortin (Dcx) antibodies, respectively. These results indicate that Mcm2 and Dcx expression exhibit an earlier decline (9 months instead of 15) in the female rats compared to age- matched males. Currently, we are further studying this phenomenon by using other NSC markers such as Musashi and Nestin/GFAP, and also characterizing the NSCs expression of Nrf2 in the female rat brain. These studies will support an understanding of hippocampal NSC aging in the female brain, and how it may relate to the increased susceptibility of women to age-related diseases such as Alzheimer’s disease (AD).
GOAL SETTING AND ACHIEVEMENT IN SOCIALLY ANXIOUS INDIVIDUALS JESSICA RENGER, JESSICA ANDREWS-HANNA
Anxiety is one of the most widespread and debilitating forms of mental illness. Social anxiety in particular is thought to affect nearly 12% of people at some point in their lives (Kessler, 2005). It is thought that a contributing factor to social anxiety may be an inability to set and reach goals effectively. To examine this hypothesis, socially anxious individuals were asked to state the three most prominent goals they would like to move towards over the next few weeks. A scoring procedure was developed to categorize the stated goals along the dimensions of specificity, personal relevance and focus to further examine the types of goals set by those with social anxiety. Specificity was determined by the degree of detail provided. Goals were then categorized as intrinsic or extrinsic based on their degree of personal relevance. Intrinsic goals were deemed as highly personally relevant and stemmed from a need to achieve growth or develop a sense of autonomy. Conversely, extrinsic goals were deemed as less personally relevant and aimed at obtaining positive judgments from others (Ryan & Deci, 2000). Intrinsic and Extrinsic goals were then divided according to their focus. Goals that were categorized as approach focused aimed to move towards a desirable outcome. Conversely, avoidance focused goals were aimed at circumventing a potentially undesirable outcome (Elliot, 1999). It was found that socially anxious individuals moved towards their goals significantly less. It was also observed that those with less specific goals reported lower levels of goal attainment. It is thought that these factors may cause the symptoms of social anxiety to persist over time. It is hoped that armed with this knowledge, mentors, life coaches and therapists can better instruct socially anxious individuals about how to most successfully set and reach their goals.
Funding provided in part by UBRP with funds from the Office of the Provost.
SIZE VARIATION WITHIN BUMBLEBEE COLONIES DOES NOT INFLUENCE COLONY EFFICIENCY AND ROBUSTNESS AUBREY REYNOLDS, EVAN KELEMEN, ANNA DORNHAUS
Within complex biological systems exists morphological variation. This is particularly true of systems which exhibit division of labor behavior such as Bombus impatiens, however the specific reason for this morphological variation is unknown. The purpose of this study was to determine how worker size variation within a colony of bumblebees influences efficiency and robustness of the colony. The worker size distribution of paired colonies was manipulated so that one had a standard distribution of worker sizes while the other had only mid-sized bees. Different pairs received either a constant food source or a variable food source to determine how worker size distribution affects both efficiency and robustness. With the data collected
53 so far, we found the percent difference in worker biomass produced has no statistical significance (Wilcoxon test, p=0.5 for constant treatment, p=0.875 for variable treatment). This suggests that size variation in bumblebee colonies is not adaptive and does not give colonies higher efficiency or robustness. Funding from NSF grant IOS-1455983
INVESTIGATING THE STRUCTURAL CHARACTERISTICS OF MC4R SELECTIVE PEPTIDES JOSE RIOS-MONTERRROSA, SAGHAR M. HAGHIGHI, MAJ KRUMBERGER, ALEX PAIGE, YANG ZHOU, MINYING CAI, VICTOR J. HRUBY
The melanocortin receptors are a family of five G-protein coupled receptors, MC1R-MC5R. These five receptors have been associated with a wide variety of processes like skin pigmentation, MC1R, and maintaining energy homeostasis, MC3R. Recently, it was discovered that inhibition of the POMC-MC4R neural pathway impairs cognitive function in Alzheimer’s mice models. Furthermore, activation of MC4R in these mice results in increased cognitive function. As a result, MC4R may be a potential drug target to treat Alzheimer’s disease. One of the main obstacles that presents itself is that MC1R, MC3R, MC4R, and MC5R are activated by one of three melanocortin stimulating hormones (MSH); α-MSH, β-MSH, and γ-MSH. Because of this, designing selective, potent agonists has been difficult. To better understand the structural characteristics that lead to MC4R selectivity, nine novel peptides were created, AIM 1-AIM 9. These peptides were made by modifying the sequence of MT- II, a potent, synthetic agonist of all receptors except MC2R. The modifications made were chosen because of their ability to increase MC4R selectivity or decrease selectivity at other receptors. To test their selectivity, a cAMP activity assay was performed on each receptor, and the EC50 values were compared to MT-II. AIM 9 (Ac-Arg-cyclo[Asp-Pip-D-Phe-(4-Cl)-βArg- βTrp-Lys]-NH2) showed high selectivity for MC4R with an EC50 of 4nM; meanwhile, it had an EC50 at MC1R, MC3R, and MC5R of 240 nm, >1000nm, and 300 nm respectively. This research was funded in part by Western Alliance to Expand Student Opportunities (WAESO) Louis Stokes Alliance for Minority Participation (LSAMP) National Science Foundation (NSF) Cooperative Agreement No. HRD-11017, and by UBRP with funds from the Senior Vice President for Research and Private Donors.
TARGETING CMYC AND ITS LNCRNA BINDING PARTNER PVT1 TO EXAMINE THEIR CO- OPERATIVE FUNCTION IN CONTROL OF TRANSLATION THANE ROSETTE, KELSY GUEST, RONALD HEIMARK
The purpose of this study is to explore the possible pathways that control cMyc turnover and its role in regulation Cap- dependent translation. cMyc can be stabilized from protein degradation by association with long noncoding RNAs (PCGEM1 and PVT1) which sustains its function. Enhancing cMyc turnover is a potential therapeutic strategy to inhibit cMyc oncogenic function in regulation of translation. cMyc target genes that regulate translation include eIF4E and P-4EBP1which are the rate limiting step in cap-dependent translation. PVT1full length transcripts are localized to the nucleus, but recently it was reported that exon 2 of PVT1 is a circular lncRNA (cPVT1) and this resides in the cytoplasm where it can function as a microRNA sponge. miR-455 binds to cPVT1 and is sequestered from its target gene eIF4E. Our approach is to examine the outcomes in treating DU145 prostate cancer cells with validated siRNA molecules to cMYC, PVT1 and cPVT1 and modified Antisense Oligonucleotides (ASOs) to PVT1 which show ~80% knockdown. In an experiment, we observed the effects of treating prostate cancer cells with two different siRNAs that were implemented to inhibit cMYC and compared it to a control siRNA to GL2. Three days after transfection the cells were then analyzed by western blotting for the protein level of cMYC, eIF4E, and P-4EBP1,and signaling proteins in the PI3 kinase pathway. Quantitation of protein bands was carried out by densitometry. These results showed that cMYC levels were reduced by siRNAs, causing downstream affects through the mTOR pathway to also reduce eIF4E. The levels of4EBP1 and P-4EBP1 are currently being determined. We also looked at the effects of treating prostate cancer cells using two different ASOs which were used to inhibit PVT1, an activator and stabilizer of cMYC. These results showed that there was significant knockdown in cMYC, particularly in applying ASO2. This cMYC reduction further caused the levels of eIF4E and P- 4EBP1 to also be reduced. For experiment three, we treated the prostate cancer cells with an siRNA molecule to observe how inhibiting cPVT1 would affect translation in the cell by looking at the concentration of eIF4E, the rate-limiting component in
54 eukaryotic translation. By using siRNA to block cPVT1, miR-455 is free to inhibit eIF4E from translation. The anticipated results of this experiment will prove if the treatments affects translation of essential proteins that influence the cells’ ability to grow and divide. The findings may be useful in developing treatments for those affected by this disease.
T.R. supported by the National Cancer Institute, Grant #2U54CA143924, through the Partnership for Native American Cancer Prevention (NACP) and Western Alliance to Expand Student Opportunities (WAESO) Louis Stokes Alliance for Minority Participation (LSAMP) National Science Foundation (NSF) Cooperative Agreement No. HRD-11017.
INDIVIDUAL INNATE PREFERENCES AND VISUAL LEARNING IN ANTS RACHEL SADLER, R. KEATING GODFREY, WULFILA GRONENBERG
Insects use learning and memory capabilities to navigate and interpret the world around them. Color visual learning has been studied extensively in social insects such as bees, but less is known about the abilities of ants to form new color associations, especially on an individual level. By allowing individual ants to explore a bifurcated maze, half illuminated with ultraviolet light and half with green light, we can determine whether ants have innate color preferences. Based on each ant’s behavioral results, the specimens are conditioned to reverse their innate preferences via a quinine (punishment) vs. sucrose (reward) paradigm. The ants’ behavior prior to and after the conditioning is compared to assess whether or not each ant formed new color associations. Ants are evaluated based on changes to both first choice when entering the bifurcation and distribution of time spent in either half of the maze. My results show that most individual ants have innate color preferences, which vary even amongst ants selected simultaneously from the same colony. Ant capability of learning new color associations is variable amongst individuals; potential brain differences underlying this variability will be investigated via cytochrome oxidase staining of cryosectioned brain tissue of the tested ants. This histochemistry will allow for an exploration of possible metabolic differences in the brains of learners vs. non-learners, thus allowing investigation of potential links between brains and behavior. This project is generously funded via support from UBRP by the Office of the Provost and the NSCS Undergraduate Research Award.
QUANTIFYING DIVERSE MICROGLIA MORPHOLOGIES IN THE EPILEPTIC BRAIN SERGIO SALGUERO, MICHAEL HAMMER, HELENA MORRISON
When healthy, microglia have highly-ramified morphologies necessary to maintain a variety of neuronal and glial physiologic functions. During injury or neuronal dysfunction, microglia have an immediate morphologic response. We have shown that level of ramification and complexity of microglial shapes change to accommodate a shift in microglial function (from surveillance to phagocytosis) within ischemic stroke. As such, microglia morphologies reflect their cell function as well as the health-status of the cells in their micro-domains. We postulate that microglia morphology could be used as a biomarker of brain physiology/pathophysiology. Unlike stroke, epileptic seizures result from neuronal hyperexcitability, alter brain function and may injure the brain without causing visible tissue death. Early infantile epileptic encephalopathy—a rare but devastating type of epilepsy—was modeled in this study using SCN8a mutated mice where the loss of function in neuronal sodium channels leads to neuronal hyperexcitability and intractable seizures. The objective of this study was to determine the usefulness of computer-aided methods to quantify microglia morphologies in the epileptic brain. We hypothesize that 1) microglia morphology in the CA1, CA3 and entorhinal regions are significantly different among SCN8A wild-type, heterozygous and mutant mice, 2) sex differences exist in microglia morphology with and without genetic mutation and 3) that increased microglia morphology will correlate to increased seizure activity. TALEN SCN8atm1768DMm mice were observed for epileptic seizures, euthanized at 20 days and brain tissue was collected. Microglia ramified morphology and complexity were quantified from photomicrographs imaged from brain regions after immunohistochemistry. ImageJ plugins were used to quantify cell ramification (AnalyzeSkeleton(2D/3D)), complexity and shape (FracLac). Preliminary data show that microglia morphology and size are different among D/D, D/+ and +/+ mice at post-natal day 20 in the striatum and motor cortex (p < 0.05). We present
55 simple and highly-sensitive computer-aided methodologies useful in quantifying microglia morphologies in the healthy and injured brain. We expect these analysis methods to be valuable tools to not only link microglia form and function, but also in the use of microglia morphology as a biomarker of injury. This research was funded by NIH grant R25-E5025494 and Accelerate for Success, University of Arizona.
REDUCING ERROR IN ULTRASOUND ELASTICITY IMAGING VIA 3D SIMULATION OF HUMAN TENDON HANNAH SCHMITZ, A. NUNCIO ZUNIGA, L. CLARK, C. FASTJE, D. LATT, R. WITTE
Tendinopathy is degeneration of tendon brought on by overuse or other factors, such as age and obesity. It often results in pain and is causative of reduced physical performance. It is cause for concern that the initial treatment recommendation is to prescribe physical therapy regardless of the extent of damage. Posterior tibial tendon dysfunction (PTTD) is one such tendinopathy for which the first line of treatment is physical therapy and bracing. The problem in providing a better informed treatment decision both for PTTD and several other types of tendinopathy is the lack of a reliable methodology for non- invasively and objectively determining the severity of degradation of the tendon. Ultrasound Elasticity Imaging (UEI) is a non- invasive imaging technique which may prove useful in identifying differences in the extent of damage of tendinopathies. UEI utilizes ultrasound imaging taken while a subject performs an inversion task against a force transducer which captures the inversion force for a duration of six seconds. Ultrasound speckle tracking is used to calculate strain on the tendon during the loading cycle, which is used to calculate the elastic modulus of the tendon, which may be altered with progression of disease as has been demonstrated for several other types of tendinopathy. We are currently using UEI in a study with patients affected by PTTD although it is challenging to assess accuracy of this technique in vivo. This study was designed to quantify sources of error and optimize the protocol by applying the speckle tracking processing and elasticity calculations on a simulated biomechanical model of the tendon using a 3D ultrasound simulation program. For this study, we examined the relationship between correlation and displacement as well as correlation and strain with displacement and strain varying according to magnitude irrespective of time to determine if out-of-plane motion was proportional to changes in displacement or strain. Other factors influencing the correlation that were examined were degree of speckle density and strain rate. Funding for this project was provided by UBRP with funds from the Office of the Provost and private donors, and NIH grant #R21AR065732.
SYNTHESIS AND CHARACTERIZATION OF A NOVEL TRIAZABUTADIENE WITH A CAFFEINE SCAFFOLD YANNICK SCHREIBER, LINDSAY E. GUZMAN, JOHN C. JEWETT
Aryl diazonium ions are attractive chemical biology reagents that have been used to label tyrosine residues of proteins. Triazabutadienes are understudied chemical compounds that degrade to release aryl diazonium ions under mildly acidic conditions, and allow chemical biologists to harness the reactivity afforded by aryl diazonium ion chemistry. Current triazabutadiene scaffolds exhibit limited solubility in aqueous environments, presenting obvious challenges to the translation of this chemistry to a cellular environment. A triazabutadiene containing an alkylated caffeine scaffold was hypothesized to exhibit greater solubility in aqueous environments than the previous scaffolds. A high-pressure methylation reaction was developed to efficiently synthesize an alkylated caffeine salt, which served as a scaffold for the subsequently synthesized caffeine triazabutadiene. 1H NMR spectroscopy studies were performed to evaluate the rate of aryl diazonium release in solution. Contrary to our hypothesis, this compound exhibited minimal solubility in aqueous environments. However, NMR data suggest that the reactivity conferred by the alkylated caffeine scaffold is intermediate to the reactivities offered by current scaffolds. The unique reactivity offered by the caffeine triazabutadiene expedites our understanding of how scaffold composition influences the rate of aryl diazonium ion liberation, and provides an additional avenue for the delivery of aryl diazonium ions. Future research involves modulating the selectivity of aryl diazonium release by appending a protecting group to the
56 triazabutadiene or performing iterative synthesis to investigate the reactivity of triazabutadiene scaffolds derived from other natural organic products.
This research was supported in part by UBRP with funds from the University of Arizona Provost's Office.
THE ROLE OF SIRPA INITIATED CD47 SIGNALING IN BREAST CANCER ANTHONY SCHWENKER, SU CHUNG, SARAH YOUNG, WILLIAM MONTFORT
Cluster of differentiation 47 (CD47) is a transmembrane protein highly expressed in many cancer cell types. It is an important immune checkpoint molecule that interacts with signal regulatory protein alpha (SIRPα) on the surface of macrophages and dendritic cells. This interaction initiates a signaling cascade emanating from SIRPα that inhibits phagocytosis which then prevents cross presentation and T cell activation. This SIRPα signaling has been well studied, however the function of SIRPα initiated CD47 signaling has yet to be determined. Although CD47 has a short cytoplasmic tail, it has been shown to have many binding partners and to trigger various signaling pathways. We hypothesize that the binding of SIRPα triggers CD47 signaling pathways involved in tumorigenesis.
To study CD47 signaling we will use MDA-MB-468 human breast cancer cells and human normal MCF10a cells as our cell models. These cell lines have been confirmed to have CD47 on the membrane through immunostaining. We have also purified the N terminal immunoglobulin variable (Igv) domain of SIRPα, which is known to interact with the extracellular IgV domain of CD47. We will treat our breast cell lines with the SIRPα Igv protein and Co-immunostaining will be performed to confirm binding between CD47 and this domain. To determine if SIRPα initiated CD47 signaling plays a role in cancer, SIRPα IgV treated cells will be analyzed for changes in tumor cell phenotypes including migration, invasion and proliferation. If there are any phenotypic changes, we will investigate the signaling mechanism by which CD47 is altering cancer phenotypes. RNA sequencing assays will be performed to define possible genes of interest.
Currently anti-CD47 humanized antibodies are in phase 1 clinical trials for cancer treatment. Thus our work is relevant as it will further elucidate the signaling mechanism and function of CD47 in tumor cells. Such findings will be important in improving therapeutic strategies targeting CD47. This research was funded by NIH GM117357.
REVEGETATION OF MINE WASTE ROCK SLOPES: INFLUENCE OF PH AND BIOAVAILABLE PHOSPHORUS KAREN SERRANO, RAINA M. MAIER, JULIA W. NEILSON, LYDIA L. JENNINGS
Waste rock piles consist of rock containing target minerals in concentrations too low for economic recovery, and are a major waste stream left over from the mining process. Regulations often require revegetation of these piles prior to mine closure; most of which are not conducive to plant growth due to poor water holding capacity, lack of available nutrients, or deviations from an optimum pH for plant growth. These factors are especially limiting to vegetation in semiarid environments where water is not readily available.
This study evaluated parameters influencing reclamation success using four consecutive years of data from two hydroseeded waste-rock slopes compared to an unseeded slope at the Carlota Copper Mine in Miami, Arizona. Undisturbed off-site areas were used as a standard for a natural environment. Parameters used to evaluate success of plant establishment included substrate pH, electrical conductivity, plant cover, and plant species composition. Substrate alkalinity has been shown to limit the bioavailability of phosphorus, an essential plant growth nutrient. Thus, bioavailable phosphorus was measured on the slopes to determine if substrate pH affects phosphorus bioavailability.
57 The results indicate that the pH decreased over the four years for both the hydroseeded and unseeded slopes, whereas no significant difference was observed in the off-site areas. A strong negative correlation between the pH of the slopes and plant cover was observed. However, there were no significant differences in the decrease in pH over the four years between the hydroseeded and non-seeded slopes. This indicates that weathering alone may be driving the decrease. Analysis of electrical conductivity showed that all values were below the salinity threshold. The influence of substrate pH on bioavailable phosphorus was also reported for all slopes.
Acknowledgements: This research was supported by the National Institute of Environmental Health Sciences, Grant #1-R25- ES025494, the Western Alliance to Expand Student Opportunities (WAESO) Louis Stokes Alliance for Minority Participation (LSAMP) National Science Foundation (NSF) Cooperative Agreement No. HRD-1101728, and the University of Arizona Center for Environmentally Sustainable Mining Academic Industry Research Cooperative funded by KGHM Carlota Copper, Rio Tinto Resolution Copper, and Asarco Mission Mine.
SCREENING OF CRYSTALS FOR CO-STRUCTURES OF TDP-43 RRM DOMAINS BOUND TO RNA AHMAD SHAHIN, LIBERTY FRANCOIS-MOUTAL, MAY KHANNA
Amyotrophic lateral sclerosis (ALS) is a progressive neurological disease that leads to muscle atrophy followed by death. Mutations in transactive response (TAR) DNA Binding Protein (TDP-43), have been linked to the development of ALS. While it is known that TD-P43 can bind to DNA and RNA, to date, there is no high-resolution crystal structure of TDP-43 in the presence of RNA. My research in Dr. May Khanna’s laboratory is aimed at crystallizing TDP-43 in the presence of multiple RNAs known to interact with TDP-43. Detailed analysis of the interaction between TDP-43 and RNA will not only be critical to understanding the molecular pathogenesis of ALS but will also guide our therapeutic development aimed at targeting TDP-43.
Ahmad Shahin, a student in Dr. Khanna’s laboratory in the University of Arizona, is working to understand the optimal conditions of crystallization for TDP-43 in the presence of the relevant RNA binding partners. In order to obtain a crystal structure of TDP-43, he has been on working on optimizing protein purifications and crystal screenings in the absence and presence of RNA. This research was supported by UBRP with funding from the University of Arizona College of Medicine.
ALLOGRAFT ADIPOSE GRAFTING APPEARS TO REDUCE PLANTAR PRESSURE IN PATIENTS IN DIABETIC FOOT ULCER REMISSION TALA SHAHIN, KAIRAVI V. VAISHNAV, JORDAN P. GARTH, ETHAN LARSON, MARCY WATCHMAN, VIGNESH SUBBIAN, DAVID G. ARMSTRONG
Background/Aim: The aim of this study was to determine the initial effectiveness of plantar fat grafting in patients with recently healed diabetic foot ulcers to assess potential plantar pressure reduction, with the ultimate goal of reducing the risk of reulceration.
Methods: Five feet in four patients in diabetic foot remission (ADA Foot Risk Category 3, 100% male, 55.9 ± 1.8yr) received plantar fat grafting via abdominal liposuction to their postulcerative plantar wound. Using wearable pressure sensors (TekScan, Boston, USA), real-time plantar pressure was measured serially. Patients were instructed to walk at a self-selected speed for twenty five meters without assistance other than their medically prescribed footwear. Changes in force and pressure were assessed preoperatively and at 2 months, postoperatively.
58 Results: Using a paired analysis, patients received a mean 45.4 ± 8.0% reduction in peak pressure at the site of previous ulceration following fat grafting (p = 0.002). A postoperative 58.0 ± 28.3% reduction in force time impulse of the site of ulceration, was also noted (p=0.01).
Conclusions: Initial results of this project suggest a potential net protective effect in people undergoing plantar fat grafting/lipofilling at areas at high risk for reulceration as evidenced by plantar pressure reduction. We look forward to further works that may confirm or refute these findings.
This research was funded by UBRP with support from the Senior Vice President for Research.
TOXICOLOGICAL EFFECTS OF ARSENIC-LADEN DUST PARTICLES RUBY SIERRA, CANDY M. RIVAS, JAHAIRA C. VERA, SCOTT BOITANO
Exposure of the toxic metalloid arsenic can be ingested through drinking water or inhaled from arsenic-laden dust particles. Arsenic is frequently found in mine tailings and thus poses a greater risk for people living adjacent to mines. Arsenic ingestion from contaminated drinking water has been associated with increased cancer risk, lung, cardiovascular and gastrointestinal diseases. However, the effects of inhalation of arsenic-laden dust are not as well characterized. Recent computational models predict increases in airborne, respirable dust due to drier climates and increased wind, making mine-adjacent populations more susceptible to arsenic exposure. In our study, part of a larger group from the University of Arizona Superfund, we are investigating the toxicological effects of inhalation of arsenic-laden dust through cell models. We use an immortalized human bronchial epithelial cells (16HBE14o-) as our model system. These cells originate from the epithelial lining of the human bronchi, and thus, represent the initial point of contact for inhaled dusts. To evaluate airway epithelial cell toxicity, we started with an advanced high-capacity toxicity screening device, the xCELLigence real time cell analyzer (RTCA). RTCA allowed for direct comparison of toxicity between dust gathered from mine tailings and from control areas. 16HBE14o- cells were exposed for 24-48 hours to mine tailing and control dusts and continually evaluated for cytotoxicity and compromise in cell-signaling (cell-signaling toxicity). We found dust from the mine site exhibited cytotoxicity at lower concentrations than that observed from the control dust. We have previously shown that arsenic can alter tight junction protein expression in airway epithelial cells and hypothesized that arsenic in mine tailing dust may be a cause of the increased cytotoxicity. Thus, we are now exploring the effects of mine-tailing and control dusts airway epithelial tight junction proteins and transepithelial resistance (TER) using immunocytochemistry and biophysical (EndOhm chamber) measurements, respectively. Tight junction proteins allow for epithelial cells to form a barrier in the airway lumen and thus prevent insults that enter the airway from reaching underlying tissues. To accomplish these experiments, 16HBE14o- cells are grown on permeable support filters and allowed to establish an TER. At this time, cells are exposed to arsenic, arsenic-laden dust or control dust and monitored for changes in TER. Findings from these experiments will be used to further in vitro and in vivo experiments that aim to develop interventions that could offset any detrimental effects of mine tailing dust. This research was funded by the EHS-True program under UBRP. EHS- True is funded by a grant from the National Institutes of Health R25-ES025494. This research was supported in part by the Western Alliance to Expand Student Opportunities (WAESO) Louis Stokes Alliance for Minority Participation (LSAMP) National Science Foundation (NSF) Cooperative Agreement No. HRD- 1101728.
EFFECT OF BEHAVIORAL VARIATION ON LIFE HISTORY STRATEGIES IN BLACK WIDOW SPIDERS CONNOR SMITH, NICHOLAS DIRIENZO, ANNA DORNHAUS
Does behavioral variation influence life history strategies in black widow spiders? Life history can be defined as the choices an organism makes through its lifespan; these include decisions regarding physiology, reproductive strategy, and aspects related to growth and size. Another phenomenon observed in the world around us is individual behavioral variation. These differences
59 in behavior, also known as personality, will be measured by assessing aggression and web structure. One question is if there are between aggressive behavior and web structure, and an individual's life history strategy measured by reproductive output and reproductive emergence rate. Individuals following a “fast" life history are thought to favor foraging, and more total offspring, and less energetic investment in individual offspring. While individuals following a “slow" life history will produce fewer offspring, build more lines that favor the safety of the spider. Both reproductive output and emergence rate of offspring can be observed by analyzing photos egg sacs. Aggression is defined as individual response threshold to a simulated prey-stimulus of mature female black widow spiders. Web structure will be evaluated by comparing the amount of lines in a web specialized for foraging versus lines that support the safety of the spider. These analyses allow us to compare the behavior and web structure of the specimen which laid that particular egg sack to further comprehend its life history strategy adaptations. After conducting statistical analysis of these relationships, it was determined that no significant relationship was found between personality and life history strategies.
CLASSIFICATION OF HIGHER ORDER ASSEMBLIES OF FUS CHARIS SPRINGHOWER, RACHEL VICTOR, DANIEL WIELAND, JACOB SCHWARTZ
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that kills motor neurons which are responsible for voluntary muscular functions such as chewing, talking, and breathing. Symptoms of ALS don’t typically show until an average age of 55 and most people survive only 3-5 years after diagnosis. There is currently no cure for the disease. Mutations in the RNA-binding protein Fused in Sarcoma (FUS) are responsible for 5% of inherited ALS and 1% of spontaneously acquired ALS. The low complexity (LC) domain of FUS is an SYQG-rich region which is largely disordered, so FUS can exist in many different conformational states and FUS also can form higher order assemblies. These assemblies are hypothesized to be the basis for FUS regulation of transcription. Normally, FUS is located in the nucleus of the cell and it polymerizes into assemblies in the presence of RNA. These assemblies also bind RNA Pol II in cells at actively expressed genes. With regards to ALS, it is important to study the formation and behavior of these assemblies, because when FUS is mutated, it can become stuck in the assembly forms. This can cause toxicity and inappropriate gene regulation. We can learn more about individual FUS proteins and can control the transition of the individual proteins into assemblies in vitro. To do this, I set up dynamic light scattering (DLS) assays. I first expressed and purified recombinant FUS protein in E. coli. I then controlled the transition by varying temperature and buffer conditions to accelerate the polymerization and by adding crowding agents to mimic the cellular environment. Using DLS, I found that the hydrodynamic radius of an individual FUS protein is about 4.2 nm and I am also detecting and characterizing several larger sized assemblies. This gives us more information on the structure of FUS and allows for better characterization of the assemblies, allowing us to gain more insight into both normal and diseased FUS activity. I have received funding from UBRP (from the Senior Vice President for Research and Private Donors), the National Institute of Health NS082376, and Western Alliance to Expand Student Opportunities (WAESO) Louis Stokes Alliance for Minority Participation (LSAMP) National Science Foundation (NSF) Cooperative Agreement No. HRD-11017.
HEAT SHOCK PROTEIN 90 PROMOTES MORPHINE ANTI-NOCICEPTION IN THE SPINAL CORD, BUT NOT IN THE BRAIN, IN A MURINE CANCER INDUCED BONE PAIN MODEL CAROLYN STINE, WEI LEI, JOHN M. STREICHER
Heat shock protein 90 (Hsp90) is a ubiquitous and highly expressed regulator for various signaling pathways. Hsp90 has been well studied as a potential cancer therapeutic target, and Hsp90 inhibitors such as 17-AAG have been tested as anti-cancer therapeutics in preclinical studies and clinical trials. However, there have been no reported studies as to whether or not treatment with an Hsp90 inhibitor could affect pain or opioid therapy in cancer patients. Our laboratory previously found that Hsp90 promotes opioid-induced anti-nociception in the brain and represses it in the spinal cord in tail flick, paw incision, and HIV neuropathic pain models, suggesting potential effects in cancer pain treatment. This study was designed to investigate the regulatory activity of Hsp90 on opioid induced anti-nociception in cancer pain using a model of cancer induced bone pain
60 (CIBP). To achieve this goal, female Balb/c mice were injected with 66.1 murine breast cancer cells in the intramedullary space of the right femur. Pain behaviors, including mechanical allodynia, spontaneous flinching, and guarding, were assessed prior to, 7 days, and 13 days after the surgery. Thirteen days after the injection with cancer cells, mice were given intracerebroventricular (icv) or intrathecal (it) injections with either 0.5 nmol 17-AAG or 1% DMSO vehicle, followed by a 24 hour recovery. After 24 hours, all of the mice were subctutaneously injected with 10 mg/kg morphine and pain behaviors were assessed for two hours. We found that it administration of 17-AAG greatly reduced morphine anti-nociception in mechanical allodynia (~64% reduction) without changing spontaneous pain. Meanwhile, treatment with 17-AAG via icv showed no effect on von Frey, flinching, or guarding. These results indicate that Hsp90 in the spinal cord, but not in the brain, modulates opioid- induced anti-nociception in cancer pain. These results also suggest that Hsp90 inhibitors given for cancer therapy could make opioid treatment less effective in cancer patients.
Acknowledgments: ASPET/SURF grant for student support; institutional funds from the University of Arizona.
INDIVIDUAL DIFFERENCES IN CHILDREN'S TENDENCY TO ABSTRACT RULES ALYSSA SULLIVAN, REBECCA GÓMEZ
Adults divide into two types of learners, those who tend to remember specific examples (example learners) and those who tend to abstract rules from the learning examples (rule learners). McDaniel and colleagues trained learners to guess an output for a given input, with output governed by a hidden function. Learners were then tested on learned and new examples. Accurate performance on the new examples demonstrated the ability to use the hidden function to extrapolate beyond the learned examples. Example learners did not extrapolate outputs that matched the shape of the function. Rule learners extrapolated the hidden function. Previous work demonstrated that children can classify objects by learning a single-dimensional rule by age five. McDaniel et al. showed that working memory capacity correlates significantly with learner type. The aim of this project is to test whether the rule learner versus example learner distinction manifests early in development and whether learner type relates to working memory capacity. Children play a computer game where, during learning, they see 6 blocks of 8 randomly- ordered creatures. The child's task is to guess the creature's home (mountains or desert). Four of the 8 creatures display a single-dimension visual feature indicating that the desert is their home (e.g. they are small, or they have a specific shape). The other 4 have a feature consistent with the mountains (e.g., they are large, or they have a different shape). Each creature has a set of non-critical features that example learners might remember. If children display 75% accuracy in any two consecutive blocks of learning trials, they advance to a test of 8 extrapolation trials. The extrapolation trials are organized so that example learners and rule learners will place the creatures in opposite places. Children also complete a measure for working memory capacity. Preliminary results suggest that children do indeed divide into example learners and rule learners. This project has significance for understanding the learning strategies used by young children and refining the understanding of the role of working memory in rule abstraction in young children. Supported in part by UBRP with funds from the Senior Vice President for Research and the Honors Legacy Grant.
ALTERNATIVE TECHNIQUES FOR MANUFACTURE OF CARBON FIBER MICRO-ELECTRODES ALEX SUMMERS, DANIEL HILL, STEPHEN L. COWEN
Accurate and real-time detection of neurotransmitters is important when investigating neurophysiological activity. This can be achieved through fast-scan cyclic voltammetry, a measurement technique that involves passing a voltage through a carbon fiber micro-electrode. This voltage induces an oxidation-reduction reaction in juxtaposed neurotransmitter molecules, such as dopamine. The current produced by this redox reaction can be measured to determine the concentration of neurotransmitter. A challenge of this technique is the difficult and time-consuming process involved in manufacturing reliable electrodes given their small diameter (7 μm). The small diameter makes it difficult to make a strong and reliable connection to a metal connector. In this study, we systematically evaluated three techniques for manufacturing carbon probes in order to determine
61 which technique produced the most reliable recordings. We observed that a technique using carbon colloidal graphite epoxy to make contact with the carbon fiber dramatically reduced manufacturing time relative to two other approaches without any reduction in sensitivity as measured with a flow cell. Thus it can be concluded that the carbon colloidal graphite epoxy has the potential to serve as an alternative to metallic epoxies traditionally used for carbon fiber microelectrode manufacture. Future studies will investigate the longevity of electrodes produced using this approach. This project was supported by UBRP with funds from the UA College of Medicine.
A NOVEL MOUSE MODEL TO EXAMINE NRF2--SYNUCLEIN INTERACTIONS IN THE CONTEXT OF PARKINSON’S DISEASE ARJUN SYAL, A. ANNADURAI, W. DU, D.D. ZHANG, L. MADHAVAN
Parkinson’s disease (PD), a chronic age-related neurological disorder, affects over 10 million people worldwide and over 1 million people in the United States alone. There is compelling evidence from recent studies that the protein Alpha-Synuclein (a- Synuclein) plays an important role in the pathology of PD. In particular, it has been found that the increased expression or aggregation of abnormal forms of a-Synuclein contribute to oxidative and mitochondrial stress causing cellular toxicity and neuronal dysfunction in PD. These findings support the development of PD therapies that reduce a-Synuclein gene expression or block its aggregation. In this context, we are studying the potential of nuclear erythroid factor 2 (or Nrf2), a major stress resistance transcription factor, to protect from a-Synuclein toxicity and cell death in PD. Specifically, we are generating transgenic mice overexpressing a-Synuclein that also either completely lack (a-Synuclein+/Nrf2-/-) or express Nrf2 (a- Synuclein+/Nrf2+/+). Using this novel model, we are examining whether and how the increased or decreased expression of Nrf2 affects a-Synuclein pathology and PD-related motor behavior in the mice. Firstly, we characterized control animals (a- Synuclein+) in terms of their behavior, via several specific motor tasks, at different time-points (5.5, 8, and 10 mos) during aging. We have found that a-Synuclein+ mice show significantly reduced performance in the open field, nest building, cylinder, and challenging beam tasks. Specifically, what we found was the a-Synuclein+ mice have significantly decreased grooming time, nest building ability, hind limb mobility, and ability to traverse challenging terrains. These deficits are exaggerated with age. Secondly, we are comparing the control a-Synuclein+ animals with the a-Synuclein+/Nrf2-/- and a-Synuclein+/Nrf2+/+ animals. Here, early data indicate that the a-Synuclein+/Nrf2-/- animals display reduced lifespans (3-4 mos), and increased morbidity with age compared to control animals. Given this, we are currently analyzing the behavior of these animals via the open field, nest building, cylinder, and challenging beam tasks at an early age (3 mos). In parallel, we have also begun a comparative histological analysis of a-Synuclein pathology in these animals. These studies will contribute to the understanding of Nrf2’s ability, and the mechanisms involved, to counteract a-Synuclein toxicity in the PD.
Funding provided in part by UBRP with funds from UA College of Medicine.
DOXORUBICIN TEMPORARILY MODULATES CYCLOOXYGENASE-2 LEVELS IN MALE AND FEMALE HUMAN VASCULAR SMOOTH MUSCLE CELLS TRINNY TAT, M. SO, R. BARTEL, K.L SWEAZEA, R.J. GONZALES
Doxorubicin (Dox) is a chemotherapeutic agent that is highly effective at reducing recurrence and mortality in breast cancer patients. However, this anti-cancer drug elicits dose- and time-dependent cardiovascular toxicity. The pathogenesis of Dox is multifactorial. One involves the development and progression of inflammation mediated by activation of the TLR4/NFκB pathway. In a recent study, we demonstrated that intermittent bouts of Dox administered to mimic chemotherapeutic administration clinically attenuated protein levels cyclooxygenase-2 (COX-2), a downstream proinflammatory mediator of the TLR4/NFκB pathway, in cerebral and peripheral vasculatures isolated from female ovariectomized rats. These findings were surprising and not in agreement with previously published data. Therefore, to address whether this Dox-induced attenuated
62 inflammatory response is time- and dose-dependent, we exposed cultured primary human female coronary and male aortic vascular smooth muscle cells to vehicle or Dox (0.3, 1.0, and 5 μM) for 6 and 24 hr. Following Dox treatment, cells were isolated, homogenized, and whole cell lysates analyzed for COX-2 levels via immunoblotting. We also assessed NFkappaBp65 protein levels and/or activation. Dox dose dependently decreased levels of COX-2 without altering NFkappaBp65 protein levels at the 6-hr time point. However, at the 24-hr, Dox increased COX-2 levels at only the highest Dox dose, 5 μM. NFkappBp65 translocation, measured indirectly using nuclear/cytosolic fractionation, was increased, at 6-hr. At the 24-h time point, NFkappaBp65 levels (whole cell lysate) were decreased only at the highest dose (5 μM) where COX-2 protein levels were elevated. Dox had no effect on cell morphology or density determined by light microscopy. Using trypan blue, live vs. dead cell percentages were also not affected by Dox, suggesting that the anticancer drug did not alter cell death at 6 and 24-hr time points. In conclusion, studies demonstrate a possible novel action for the anticancer agent eliciting both a dose and time- dependent effect on selective downstream mediators of inflammation such as COX-2 in the female and male vasculature. Future studies are planned to evaluate the impact of acute vs. chronic Dox treatment on COX-2 at the transcriptional or translational level to determine the mode of molecular regulation and to assess if sex differences exist.
Funding: University of Arizona Sarver Heart Center (RG and MS), the Valley Research Partnership P1 Grant (RG and MS), and the ASPET SURF grant (TT).
DEVELOPMENT OF A PROSTATE-ON-A-CHIP MEAGAN TRAN, YITSHAK ZOHAR, LINAN JIANG
Prostate cancer remains the second-leading cancer killer of men due to the inability to cure hormone-resistant metastasis. Furthermore, drugs which initially show promise in laboratory settings fail in clinical trials because the existing models for prostate cancer fail to adequately address the role of the tumor microenvironment. Not all prostate cancer cases progress to a lethal stage; however, doctors currently lack the ability to predict which tumors will progress resulting in over-diagnosis and unnecessary treatment. Clearly, current cell monolayer and animal models do not adequately represent the environment of human prostate tissue.
The development of a microfluidic-based model of a human prostate gland will allow researchers to mimic the human prostate tissue in an environment similar to in vivo processes including cell signaling among different cell types. Following standard microfabrication techniques, the microfluidic device is fabricated using a clear, flexible, and inert polymer PDMS (polydimethylsiloxane). In order to replicate a prostate gland, prostate epithelial and stromal cells are grown on either side of a porous polyester membrane. This separation membrane is sandwiched between two microchannels stacked on top of each other. Thus, each cell type is exposed to its own culture media flow while allowing communication between the two cell monolayers through the porous membrane. Utilizing these microfluidic devices, we demonstrated prostate epithelial differentiation induced by introducing androgen to the stromal layer. This approach has the potential to revolutionize how researchers study prostate cancer initiation and progression as well as environmental or pharmaceutical effects on a human prostate gland.
This project was generously funded by UBRP with funds from the Office of the Provost.
63 PHASE II CLINICAL STUDY OF METFORMIN FOR BREAST CANCER PREVENTION ROXANNE VANN, J. TRUJILLO, D.E. VILLA-GUILLEN, J.A. MARTINEZ, P. CHALASANI, C.A. THOMSON, D. ROE, M. ALTHBATCH, J.- P. GALONS, A.M. ALGATOR, H. H.CHOW
Women who are premenopausal with a BMI ≥ 25 kg, have a higher breast density, and have metabolic abnormalities are at higher risk for postmenopausal breast cancer. Metformin is an insulin-sensitizer drug used to treat type II diabetes, and previous research suggests that it could be beneficial in the prevention of breast cancer. Our lab is conducting a Phase II Clinical Trial to test the chemopreventive effects of metformin. The clinical trial begins with the recruitment of patients who meet the inclusion criteria. Patients are assigned to random permutated blocks to either a metformin group or a placebo group. The patients undergo a 6 to 12-month intervention and are monitored by FWR-MRI to measure the fat and water ratio by using MRI imaging to assess changes in breast density. Each patient also undergoes an optional core breast biopsy and then later examined under a microscope to observe the adipocytes of the tissue. Women having a higher BMI are prone to having inflamed adipocytes. Perspectives for this work are to provide clinical evidence of the chemopreventive activities of metformin in women with elements of metabolic syndrome at high-risk for postmenopausal breast cancer.
R.V. supported by the National Cancer Institute, Grant #2U54CA143924, through the Partnership for Native American Cancer Prevention (NACP) and Western Alliance to Expand Student Opportunities (WAESO) Louis Stokes Alliance for Minority Participation (LSAMP) National Science Foundation (NSF) Cooperative Agreement No. HRD-11017.
MUC1 AFFECTS CD1’S ABILITY TO REGULATE CELL QUIESCENCE IN TRIPLE NEGATIVE BREAST CANCER NEERAJ VIJ, JOYCE SCHROEDER, DAWN GEISER
Triple Negative Breast Cancer is an aggressive and invasive breast cancer in which estrogen, progesterone, and HER2 receptors are not present on the cell membrane. It effects roughly 1/40 women worldwide and has a high rate of recurrence among the breast cancers. It is well known that Epidermal Growth Factor Receptor (EGFR) travels to the nucleus upon ligand (EGF) binding where it acts as a transcription factor. Bitler et al. 2010 demonstrated that non-cancerous cells (MCF10A’s) in which MUC1 has been silenced show significantly less EGFR in the nucleus. Bitler et al. 2010 also demonstrated that the presence of MUC1 allows EGFR to bind to chromatin and act as a transcription factor for Cyclin D1 (CD1.) CD1 is of interest in triple negative breast cancer because it acts as a cell cycle regulator by helping cells cross the G0 checkpoint. In this study, we aim to determine whether the presence of MUC1 is correlated with CD1 levels in a cancerous cell line (+/- MUC1 BT20’s.) We hypothesize that MUC1- cells will show significantly lower levels of CD1 than control (MUC1+) cells. We also aim to explore CD1’s potential relationship to cell quiescence in the cancerous cell line, BT20’s. We demonstrate that control (MUC1+) cells are significantly more responsive to serum concentration than MUC1- cells, indicating that CD1 may assist cells in avoiding quiescence.
Funding provided by UBRP with funds from the Office of the Provost.
SEMI-AUTOMATED ANALYSIS OF MICROEMBOLIC LESIONS IN BRAIN DIFFUSION WEIGHTED MRI GREG WHEELER, LOI DO, WEI ZHOU, THEODORE TROUARD
Carotid endarterectomy and carotid artery stenting are used to treat high-grade carotid artery stenosis (≥70% blockage) and reduce the risk of stroke. These procedures, however, risk the creation of microemboli that can result in brain lesions. These lesions present as bright spots in diffusion weighted magnetic resonance imaging (DW-MRI) and the volumes of these lesions
64 have been shown to have a negative correlation with cognitive ability. The objective of this work was to develop a semi- automated computer program that will quantify the volume of microembolic lesions quickly while maintaining accuracy and consistency. The semi-automated analysis developed in MATLAB opens a patient’s DW-MRI images and displays them in a graphical user interface (GUI). From this GUI, the user manually selects all the lesions on a slice by slice basis. The program automatically grows a region of interest from that seed point. The region grows until the intensity of all adjacent voxels falls outside a user-defined signal intensity threshold and calculates the volume of each of the lesions. The program was evaluated on DW-MRIs obtained from patients (n=6) shortly after carotid artery intervention. Three users utilized the program at the same threshold on each set of images. The number of lesions selected and the total lesion volume were automatically calculated by the program to evaluate inter-rater reliability. The average difference in total volume obtained by different users on the same set of images was 16.3%. The main contributor to inter-user variability was differences in lesion selection. When more lesions were selected, greater volumes were obtained. When identical lesions were selected, there were no differences in the volumes calculated. This semi-automated quantitative computer analysis provided comparable results to manual methods used previously with a reduction in analysis time and should minimize inter- and intra-user variabilities. Future work will include quantification of the time savings, using the program to determine the effect of lesion volume on cognitive outcomes, and registering the lesion maps created to T1-weighted images and a brain atlas to determine relationships between lesion location and cognitive outcomes. This research was funded in part by UBRP with funds from the University of Arizona College of Medicine.
EFFECTS OF AN ENVIRONMENTALLY-ACQUIRED BURKHOLDERIA SYMBIONT ON THE DEVELOPMENT AND FITNESS OF AN INSECT HOST SHAIRA MARIE WHITAKER, RYAN COMELLA, CATHERINE GAVIN, DANA JOHNSON, ALISON RAVENSCRAFT, MARTHA S. HUNTER
Bacteria in the genus Burkholderia are common soil and plant associates that also participate in symbiotic relationships with insects (Kikuchi et al., 2011). Many true bugs, including the stilt bug Jalysus wickhami, house Burkholderia in a specialized mid- gut pocket. This relationship is unusual because the bugs do not inherit the symbiont from their parents, but instead acquire it from the environment every generation (Kikuchi et al., 2007). Environmental symbiont acquisition may provide unique benefits to a host, but researchers have largely overlooked the phenomenon.
To understand the role these bacteria play, we asked how Burkholderia affects the development and fitness of J. wickhami. We hypothesized that individuals lacking this bacterium would exhibit a decrease in survival, slower growth, and a decrease in production of offspring compared to insects colonized by the bacteria. We tested this hypothesis by rearing individual J. wickhami in a lab environment with or without Burkholderia. We mixed a Burkholderia cell suspension into the food of the experimental group and plain water into the food of the control (aposymbiotic) group and then measured survival, growth rate, and reproduction rate of these insects. Our results will clarify the benefits provided to this insect by Burkholderia, forming a necessary baseline for future research in the system.
Ultimately we are interested in the possibility that environmental symbiont acquisition may allow a host to acquire bacteria that perform different functions that are matched to local environmental conditions. The next step of the experiment will therefore be to determine whether different strains of Burkholderia have different effects on Jalysus wickhami.
This work was funded by an NSF grant to MSH (IOS-1256905) and an NIH Postdoctoral Excellence in Research and Teaching grant to AR (K12GM000708).
65 THE EFFECTS OF AGING ON STRESS GRANULE FORMATION BRITTANY WILLIAMS, DAVID DURON, BHAVANI BAGEVALU SIDDEGOWDA, DANIELA C. ZARNESCU
As the aging population continues to grow, the incidence of age-related neurodegenerative diseases has become more evident. A common factor among neurodegenerative diseases is the presence of protein aggregates. These protein aggregates are a hallmark of age-related neurodegenerative diseases, and they are thought to “evolve” from RNA stress granules. However, their connection to aging is still unknown. The purpose of this study is to analyze the dynamics of stress granule formation in Drosophila melanogaster during aging. The presence of stress granules is ascertained using Drosophila Fragile X Mental Retardation Protein (DFMRP) as a marker. To determine age dependent changes in stress granules we examined the distribution of FMRP in Drosophila brains at five different age points. Preliminary experiments using Drosophila show a decrease of FMRP distribution with aging. The anticipated outcome of this project is the dynamic expression of stress granule components during the aging process. The findings may be useful in understanding how the cell’s ability to handle environmental stress decreases with age. This work was suported by the NIH MARC Training Grant (T34 GM08718) and NIH R01 (NS091299 to DCZ).
REGULATION OF EPITHELIAL POLIFERATION PATHWAYS, YAP AND WNT, BY ENDOSOME MENG-HAN WU, CHRISTOPHER M. COX, JEAN M. WILSON
The transcriptional coactivator, Yes-associated protein (YAP), is essential in the regulation of cell proliferation, and regulates organ growth through contact-mediated inhibition. YAP is not only controlled by phosphorylation-dependent regulation through the Hippo pathway, but the integral membrane protein angiomotin (AMOT) and AMOT family members also control YAP translocation through direct binding. In addition, β-catenin, through the Wnt pathway, also regulates cell proliferation. The YAP and Wnt pathways have been shown to be linked in epithelial cells. Previous work in the lab has shown that YAP activity is controlled through interactions between the endosomal protein endotubin (EDTB) and AMOT. Based on these results, we are testing how EDTB on endosomes could impact β-catenin activity. Over-expression of EDTB decreases binding of β-catenin to AMOT. Furthermore, in the intestine of EDTB knock-out mice, there is decreased β-catenin in the nucleus. These results suggest that signaling from endosomal membranes is important in these proliferative pathways.
EXPRESSION OF RNA STRESS GRANULE COMPONENTS IN AGING: FLIES TO RATS STEPHEN YAO, B. BAGEVALU SIDDEOGOWDA, R. ECK, M.K. CHAWLA, C.A. BARNES, D.C. ZARNESCU
RNA stress granules (SGs) are known to assemble in times of cellular stress. These SGs function to protect RNAs and other resources from harmful conditions ultimately resulting in translation inhibition. Following stress removal, RNA SGs dissociate and translation can be reinitiated. RNA SGs have been associated with age-related neurodegenerative diseases suggesting a possible role in the process of aging. However, it is not known whether RNA stress granules undergo changes as organisms age. Therefore, we aim to profile expression of RNA binding proteins that are associated with RNA SGs such as PABP, TIAR, FMRP, Gcn2, eIF2a. Analysis of transcript and protein expression will shed light on changes that occur in aging fly and rat brains. We also aim to profile the global translational status, and expression of proteins that associate with SGs in aging flies. Currently, our findings support the hypothesis that RNA SGs undergo dynamic changes within aging. Future experiments include further elucidating the role of cellular stress response in aging organisms. We plan to continue fly experiments to validate our findings as well as continue mRNA distribution profiling. We also hope to continue elucidating the role of cellular stress in aging brains in the context of RNA SGs.
This work was supported by the Jim Himelic Foundation and NIH NS078429 (DCZ). Support was also provided by the University of Arizona Undergraduate Biology Research Program (UBRP) and the Arnold and Mabel Beckman Foundation (SY).
66 THE EFFECTS OF DIFFERENCES IN RELATIVE STIMULUS IN TEMNOTHORAX RUGATULUS DIVISION OF LABOR SAMUEL YEN, NICOLE LEITNER, ANNA DORNHAUS
Temnothorax rugatulus do not use a central system of communication (i.e. no central individual directing all other individuals). However, they are able to complete necessary functions for the survival of a colony. We are interested in how Temnothorax complete these functions in the absence of a central communication.
A theory used to explain their behavior is called the Response Threshold Model. It states that a creature will respond to a stimulus once a certain threshold is reached, and each individual creature possesses a unique threshold for different stimulus. An example: a college student may be able to go a few days without a shower and feel comfortable (High Threshold). Another student may need daily showers to be comfortable (Low Threshold). In other words, individuals with low thresholds to a stimulus will react more quickly or frequently to that stimulus than an individual with a high threshold. The Response threshold model suggests that ants focus their roles towards stimulus which they have a low threshold to. This project received funding from NSF (grant no. IOS-1455983 to AD).
ORGAN-ON-A-VINE: SPINACH LEAVES USED AS CANCER MODEL RYAN ZENHAUSERN, JEROME LACOMBE, FREDERIC ZENHAUSERN
The prevalence and development of organ-on-a-chip (OOC) systems has increased drastically in an effort to accurately model human physiology for application in studying cancer. Unfortunately, current models have issues in accuracy in that the 2D structures and (often rare) cell co-culture technologies that exist, lack many features or characteristics found In Vivo. The use of “Plantimals,” or decellularized plant structures re-cellularized with human cells, aims to overcome these issues by taking advantage of the innate vascular structure, natural surface texture, and decreased vascular/surface membrane size.
This project was comprised of three basic steps: the spinach leaf decellularization, leaf characterization, and recellularization. The decellularization process focuses on the spinach leaf itself and serves to create the basis of the Plantimal mode. In order to characterize the efficiency of the decellularization process, the rigidity of the leaves was assessed by Atomic Force Microscope (AFM) and DNA and protein quantification was performed. Finally, human prostate (PC3) and breast (MCF7) cancer cells were seeded onto leaf sections treated with EDTA and left to attach for 24 hours. Seeding efficiency was assessed by optical microscopy and viability was tested by trypan blue and MTT assay. Cell-seeded leaves were irradiated and the expression of five well-known radiation-responsive genes were assessed in MCF7. Additionally, DNA damage levels in PC3 cells were evaluated by γ-H2AX foci measurement using fluorescence microscopy.
The decellularization process was successful, showing a protein quantity of 0.31 and 1.4 μg/mg tissue respectively, compared to the fresh leaf at 14.4 μg/mg tissue. The DNA quantity was similarly disparate between fresh and decellularized leaves. Interestingly, we also noticed that temperature could affect the decellularization process. Indeed, mechanical testing with AFM showed a difference between the leaves decellularized at 25? and 37?, with Young’s modulus values of 2.81 MPa and 1.50 MPa respectively, compared to 197 MPa for fresh leaves. Microscopy showed that PC3 and MCF7 cells were well attached to the leaf surface after 24h incubation. Viability assays confirmed that cells were alive at least 72h post-seeding. γ-H2AX immunofluorescent imaging showed DNA damage repairs are induced after 5Gy of X-ray irradiation in PC3 cells. Finally, the gene expression assay showed changes in expression levels between 5Gy-irradiated and sham-irradiated MCF7 cells. Together, these results suggest radiation response is active in cancer cells attached to the leaf scaffold. Overall we were able to create a radiation responsive tumor model using decellularized spinach leaves as a scaffold.
Funding sources: UBRP with funds from the UA College of Medicine.
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