SQ Volume 16 (2018-2019) by Saltman Quarterly

Undergraduate Research Journal Volume 16 2018â&#x20AC;&#x201C;19 sqonline.ucsd.edu

Generously underwritten by

THE SALTMAN FAMILY & Supported by

The views expressed in this publication are solely those of Saltman Quarterly, its principal members and the authors of the content of this publication. While the publisher of this publication is a registered student organization at UC San Diego, the content, opinions, statements, and views expressed in this or any other publication published and/or distributed by Saltman Quarterly are not endorsed by and do not represent the views, opinions, policies, or positions of the ASUCSD, GSAUCSD, UC San Diego, the University of California and the Regents or their offices, employees, or agents. The publisher of this publication bears and assumes the full responsibility and liability for the content of this publication. In an effort to engage the UC San Diego Community, Saltman Quarterly holds an annual photo contest. The winners of this contest have their images featured on the cover and interior pages of the journal. FRONT COVER: A hummingbird (Trochilidae) resting on a branch in the Encinitas Botanical Gardens. Photo by Sam Zilberman INSIDE COVER: Aerial view of the Torrey Pines Reserve Slat Marsh, off the Pacific coast. Intersected by a local railroad. Photo by Katie Clark

LETTER FROM

THE EDITOR

Dear Reader, Every year, a group of UC San Diego undergraduates try to crack the ultimate cold case: how do we make people care about biology? More specifically, how do we inspire people to care about the crucial research that’s being conducted on our campus? The pursuit of research in the biological sciences is often grounded in a curiosity about how our world works and a deep commitment to the advancement of human health. Therefore, a substantial majority of people should naturally have an interest in biology research. Science, however, has historically been inaccessible to people not already in the field, with structural inequality being a key component to this. Moreover, scientific literacy in a traditional science education is not typically emphasized. This hinders an ongoing dialogue between scientists and the public, and researchers’ work can be shrouded by misunderstanding and distrust. The impacts of this cannot be understated: we are living in an era where established scientific facts are distorted and questioned, and this has very real and detrimental consequences on the well-being of our communities and our planet. To bridge this crucial gap between labs and our readers, this year’s Saltman Quarterly staff not only asked, “How does life work?” but also “Why do we care?” To answer these questions, our writers, illustrators, and photographers have crafted narratives showcasing specific biological studies. In doing this, we seek to tell the stories that inspire the work of researchers and to communicate their findings with clarity and depth. Volume 16 of SQ exemplifies the passion and talent of our staff and the UC San Diego undergraduates who work in labs on campus. It also testifies to the sheer breadth of topics that can be explored in the biological sciences. In this issue, you will find pieces on concepts like phage therapy and autism spectrum disorders, as well as manuscripts on animal biodiversity in Costa Rica and the effects of antiretroviral drugs on blood vessels. This

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variation is what makes biology such an exciting and dynamic field to be in: anyone can create their own space in the field, no matter how niche their interests may be. In my three years at SQ, I’ve learned that we not only investigate a plethora of subjects in biology, but we also have the capacity to communicate these ideas in a variety of ways. Our community outreach team visits local elementary schools and hosts engaging activities that teach students about the science behind nutrition. SQ’s publicity team uses social media platforms to give bite-sized information about the latest biological breakthroughs for our audience members on the go. SQ Online pieces range from a feature article on the latest Ebola outbreak in West Africa to a blog discussing the health benefits of organization. The SQ Insider, our quarterly newsletter, effectively describes humanity’s greatest challenges, such as sustainable consumerism and climate change. Through covering these topics, we not only aim to empower our readers, but also to strengthen our writers’ ability to tell stories about biology. Working with such incredible undergraduates who care so deeply about SQ’s mission has been a unique and humbling experience. This journal is the result of hours of work by insightful and dedicated students who have pushed me to think about my chosen field in ways that I would have never considered. I hope that Saltman Quarterly Volume 16 inspires you as well. Best,

Sharada Saraf

Editor-in-Chief, Saltman Quarterly 2018-19

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table of 3

SALTMAN DEDICATION

FEATURES

CONTENTS

Saltman Quarterly thanks the Saltman family for their generosity and support. Their contributions have allowed SQ to continue to spread Dr. Paul Saltman’s ideals of science communication and education not only to the student body at UC San Diego, but also to surrounding communities. BY CASSIDY LAM AND EMMA HUIE

Understanding Autism Spectrum Disorders

BY NIKHIL JAMPANA

Life, Death, and an Obligate Intracellular Parasite

BY CHRIS HAGAN

Self vs Non-self: How Cancer Slides Under the Immune System’s Radar

BY MAYA GOPALAKRISHNAN

Investigating Our Metabolism's Chemical Fingerprints

BY SNEHA GANGULY

RESEARCH & REVIEWS A Comparison of Vertebrate Animal Biodiversity Along the Vertical Strata of Strangler Figs (Moraceae, Ficus spp.) in Monteverde, Costa Rica Analyzing Arctostaphylos: Evaluating Viability of Seed Collections and Comparing Effectiveness of Germination Testing Methods

BY KATHERINE HERNANDEZ BY JULIA GAUDIO

The Effects of Antiretroviral Drugs on Microvascular Cells: Tenofovir Induces Cell Senescence in 3D Multi-layer Co-culture

BY RACHEL C. CHANG

The Gut Microbiome: the Key to Unlocking the Secret of Rheumatoid Arthritis

BY CHRIS MATTHEW CYRIL

Social Pack Dyanmics of the African Painted Dog, Lycaon pictus, Promote Allee Effects and Population Decline

BY CHARLOTTE BRUGGEMAN

SENIOR HONORS THESES

STAFF

Students in the Biology Honors program are required to complete a written thesis detailing their scientific research progress. The Senior Honors Theses section, which presents the abstracts of their individual theses, highlights the achievements of the accomplished undergraduate researchers in the class of 2019.

Meet the members of the 2018-2019 Saltman Quarterly staff who worked throughout the year to bring you this issue, as well as our online content, quarterly newsletters, and community outreach initiatives.

PAUL SALTMAN

ou open Instagram on your phone and among the lively pictures of friends, cute animals, and funny cartoons, you see a post of a fashionably dressed model advertising one of her favorite products: a detox tea meant to cleanse toxins, encourage weight loss, and boost your immune system in just 28 days. However, a quick Internet search will dispatch hundreds of results disproving these flashy health benefits, including numerous empirical studies from reputable scientists. Yet somehow, companies continue to scam their customers with questionable pseudoscience, health-oriented buzz words, and empty promises that make it difficult to see the truth. Detox teas and similar "fad diet" phenomena are not new to the world of nutritional science, and no one understood the importance of trusting science over marketing claims more than UC San Diego professor Dr. Paul Saltman. Dr. Saltman graduated from Caltech with a bachelor’s degree in chemistry. He then spent the next 14 years as a professor at USC, and later joined UC San Diego’s faculty in 1967, where he was later appointed provost of Revelle College. During his career, he constantly advocated for science education and nutrition research, emphasizing the importance of essential trace metals in daily consumption. He published The California Nutrition Book, which addressed everyday nutrition as a philosophy of balance based on biochemistry concepts, and claimed that “There is no junk food, there is no health food, there is no magic diet.” Dr. Saltman held a strong media presence on television and radio; while he found pleasure in debunking diet myths he did so respectfully and patiently, simplifying difficult biochemical concepts to audiences and journalists who lacked a science backsqonline.ucsd.edu

Saltman Quarterly is dedicated to the same pursuit of scientific honesty, education, and passion that Dr. Saltman encouraged in his lifetime, as well as his vision of bridging the gap between science and the broader community. ground. Combining his incredible knowledge of science and a compassionate approach to teaching, Dr. Saltman made complicated topics accessible to everyone and shared his enthusiasm for science to inspire others to do the same. He created a curriculum called the “Frontiers of Science,” encouraging non-science majors to engage in the scientific field, and he also taught extracurricular programs geared towards cultivating science knowledge at the high school level. His instructional skills were received with tremendous praise, earning him the first Career Teaching Award from the UC Academic Senate in 1994, as well as Excellence in Teaching Awards from four colleges at UC San Diego. His passion for teaching and communicating science inspired students to think critically about science and be greater arbiters of truth. Dr. Saltman not only wanted students to learn, but also to be able SALTMAN QUARTERLY

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“ I really think the real reason that I’m here is because I want to somehow communicate to you the joys of teaching… these moments that we have together will be catalytic moments.”

—Dr. Paul Saltman

to teach what they know. He said, “I really think the real reason that I’m here is because I want to somehow communicate to you the joys of teaching… these moments that we have together will be catalytic moments.” As this publication’s namesake, Dr. Saltman and his outstanding legacy have inspired future students in biology to uphold his mission of tackling misinformation with science, and encouraging others to become their healthiest selves using proven methods. Saltman Quarterly is dedicated to the same pursuit of scientific honesty, education, and passion that Dr. Saltman encouraged in his lifetime, as well as his vision in bridging the gap between science and the broader community. This year marks the twentieth anniversary of his passing, but we at Saltman Quarterly continue to do our part in upholding his legacy and mission of inspiration. Our organization utilizes outreach programs and a partnership with the YMCA to encourage scientific literacy to our local community, including grade school students and underserved neigh4

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borhoods. Every year we spotlight the work of UC San Diego’s aspiring undergraduate scientists to help make their research accessible to all. Education and the active pursuit of knowledge are key elements to understanding the world around us. We owe this central tenet of Saltman Quarterly’s mission to people like Dr. Saltman, who dedicated his life to highlighting the importance of remaining curious and critical. His legacy will continue to inspire students for generations to come.

CO-WRITTEN BY CASSIDY LAM EMMA HUIE Cassidy is a Social Psychology major from Sixth College. She will graduate in 2019.

Emma is a Human Biology major from Earl Warren College. She will graduate in 2020.

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FEATURES Rescued loggerhead sea turtle (Caretta caretta) at the Birch Aquarium. Photo by Anne Marie Berry

As a hub of biological research, UC San Diego is constantly at the forefront of scientific discovery and exploration. The Features section highlights some of the ground-breaking work accomplished by researchers affiliated with the UC San Diego campus.

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understanding autism spectrum disorders

WRITTEN BY

NIKHIL JAMPANA ILLUSTRATED BY

CRISTINA CORRAL

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Increasing awareness of Autism Spectrum Disorder (ASD) in recent years has allowed for accurate, timely diagnosis. While many of the symptoms associated with ASD are treatable with medication and therapy, its underlying causes are largely unknown. Researchers at UC San Diego, working on both the behavioral and molecular fronts, are progressing towards understanding ASD and intervening on more fundamental levels. Every success of theirs carries deep implications for the quality of life and well-being of a significant number of people. Dr. Jeanne Townsend and Dr. Leanne Chukoskie at the Research on Autism and Development Laboratory (RADLab) at UC San Diego use gaze-driven video games that they developed to treat autism. To a research subject at the RADLab, the idea of gaze-driven video games would probably be as new and fascinating as it was to me. The eye sensor would calibrate to their gaze, and they would probably start off with Shroom Digger—a game in which the subject has to fix their gaze on a mushroom house until it enlarges enough to explode. Even if they moved their eyes away for a quick second, the mushroom house would return back to its original size and would not explode, drawing their attention. They would continue with Space Race, in which they were to guide a spaceship with their eyes through a defined path, making sure not to shift their focus or dart their eyes. More games would follow. In recent years, we have seen rising rates of ASD in children. According to the latest data from the Centers for Disease Control and Prevention (CDC), about 1 in 68 children in the United States have ASD (Wright 2017). Although experts say that the increasing prevalence of ASD is due to

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improved diagnosis, rather than an actual increase in the number of cases, looking beyond the numbers is important to grasp the importance of studying ASD (Wright 2017). Our biological understanding of ASD is not at a point where effective treatments can directly target the condition itself; current treatments such as behavioral therapy and social skills training only manage to address symptoms associated with the disorder (Osborn 2018). ASD operates on two levels: while defined behaviorally, it has genetic and neurodevelopmental underpinnings (Mash et al. 2018). Hence, molecularlevel biological research supports behavioral research so as to allow interventional and clinical innovations. With the understanding that ASD can only be effectively treated at its roots, researchers at UC San Diego are investigating the disorder on both the behavioral (macro) and molecular (micro) levels. Dr. Townsend and Dr. Chukoskie study the neurodevelopmental underpinnings of ASD by looking at early childhood differences in attention, movement, and sensory reception. As part of another research group, Dr. Alysson Muotri studies ASD’s molecular underpinnings by modeling it in organoids (“mini-brains”) derived from induced pluripotent stem cells (iPSCs). Such studies on ASD are only possible because of a recent expansion of ideas surrounding the condition.

SHIFTING DEFINITIONS

Leo Kanner, an American psychiatrist, revolutionized the clinical psychiatry field and the existing understanding of autism in 1943. One of his landmark papers centered around observational studies on a group of children with symptoms of social withdrawal, impaired communicative ability, and obsessive behavior. Through these evaluations, Kanner recognized autism as a

pervasive developmental disorder. At about the same time in Germany, Hans Asperger identified similar symptoms in a group of children who did not show cognitive and communicative deficits. He termed this condition “Asperger’s” (Townsend et al. 2012). Today, we can differentiate between autism and Asperger’s, but they fall on the same diagnostic continuum of associated conditions called the Autism Spectrum Disorder. Formal diagnosis of an ASD requires impaired behavior in two areas: social communication and restricted patterns of behavior. In addition, symptoms must be persistent early on in childhood (before two years of age) and cause significant impairment without being caused by some other condition (Mash et al. 2018). The micro-level mechanistic studies in ASD research serve as the theoretical basis for macro-level behavioral studies.

FOUNDATIONAL RESEARCH ON ATTENTION

UC San Diego’s RADLab conducts behavioral studies of autism and aims to translate its findings to large-scale interventions. Headed by Dr. Townsend and Dr. Chukoskie, the RADLab recently developed a series of gaze-driven video games to train attention in children on the spectrum. While the RADLab is currently in an interventional stage of research, their work began with studies of attention, which rested on the belief that targeting the elements of attention that are impaired in ASD can have therapeutic benefits. Dr. Townsend’s work over the years has revealed that focused, conscious attentional ability (“static” attention) is relatively normal in cases of ASD, whereas there were significant deficits in dynamic attention—those rapid, reflexive switches of attention that are taken for granted as we move through the world every day. These studies examined various eleSALTMAN QUARTERLY

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ments of attention and were complemented by functional imaging techniques like EEG, fMRI, and MRI to monitor brain activity during various attention tasks. Dr. Townsend’s research subsequently took on a tripartite focus: non-spatial attention, spatial attention, and functional imaging. In studies of non-spatial attention, she and her colleagues found that adolescents with ASD were significantly slower at shifting between visual and auditory attention when compared to the control group because they took time to reorient their attention (Townsend et al. 2012). This cross-modality shift is something that is required in a dynamic environment—and no situation is more dynamic than a social setting, where one is required to pick up on subtle changes in facial movements and recognize how to respond. In these studies of spatial attention, Dr. Townsend and her colleagues designed a simple task: to press a button whenever a circle appeared in a highlighted box. Their functional imaging studies revealed that participants with ASD had a higher sensory response when the circle appeared in the area of focus. Interestingly, the response dropped significantly when the circle appeared even slightly away from the area of focus. Their studies had clear backing, as radiology reports showed that 40% of the subjects in the ASD group had an abnormality in the parietal lobe in the form of widened sulci (grooves that give the brain its folded appearance) and increased cerebrospinal fluid volume (Townsend et al. 2012). Dr. Townsend also studied attention in terms of orientation, engagement, and disengagement. They used a similar test model of central fixation on a screen coupled with simultaneous action in the periphery. The fMRI reports showed that all subjects with autism had cerebellar abnormalities and some had parietal abnormalities. Overall, the results demonstrated that “attentional orienting” is impaired in cases of ASD and that subjects with an added parietal abnormality were slow to disengage once they engaged (Townsend et al. 2012). This means that rapid attentional switching, 8

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FIGURE

This schematic shows the way Dr. Muotri and his colleagues used iPSC technology to reprogram somatic cells from someone with autism back to the embryonic stage, allowing them to then push these embryonic stem cells to develop into “mini brains.” These brain organoids recapitulate early stages of neural development, which allows for manipulation and comparison to controls to understand early developmental differences in ASD.

controlled by the cerebellum, is impaired in ASD and that more controlled, deliberate attention is unaffected (Townsend et al. 2012). This is significant, because the things we associate with autism—difficulty with social engagement, delayed speech, or heightened sensory response—are consequences of impaired dynamic attention.

INTERVENTIONAL STAGE AND IMPLICATIONS

The above research paved the way for the interventional work that the RADLab currently focuses on. Targeting eye movements, they have conducted two small feasibility trials. Dr. Townsend explained the reason for their targeting eye movements via games, saying “that the eye movement system and attention are highly intertwined.” These quick, darting eye movements—called saccades—are disrupted in autism. In collaboration with professional game developers, the RADLab helped develop a series of attention training games by targeting fixation, speed, and accuracy in eye movements. The participants in the small feasibility trial who stayed with the games showed significant improvement in at least one of many outcome measures— including: orienting, disengagement, eye movement, fixation, saccade latency, and

saccade accuracy (Chukoskie et al. 2018). As for future plans, the RADLab hopes to continue in the interventional direction, building games for younger age groups.

STUDYING THE MOLECULAR BASIS OF ASD

While the RADLab is tackling ASD by understanding and addressing attentional and sensory differences, Dr. Alysson Muotri at the UC San Diego School of Medicine is seeking to uncover the molecular and cellular pathways that lead to conditions like ASD by using induced pluripotent stem cells (iPSCs). On a broad level, Dr. Muotri is directing his research towards answering a fundamental question: what is the neurological basis of human sociability? Dr. Muotri chose to investigate ASD because it is a condition in which human sociability is impaired. The main challenge that comes with this type of research is the lack of reliable human models with which to study ASD. So far, he says, “most of the biomedical research has focused on animal models. But the human brain and human behavior are so different from that of a mouse, for example, that these models don’t fully recapitulate the conditions.” As a means of addressing this issue, the Muotri Lab reprogrammed somatic cells from ausqonline.ucsd.edu

FEATURES

tistic subjects back to the embryonic stage. They then developed protocols to differentiate these embryonic cells into brain cells. Eventually, they created “mini-brains” to reproduce early stages of neurodevelopment. Dr. Muotri was a key researcher in a field-opening study that pioneered this technology in 2010. He and his colleagues used Rett syndrome, a rare condition that was formerly on the spectrum, as a genetic model for ASD to develop iPSC-derived neurons and study their characteristics. The iPSCs were generated from fibroblasts collected from patients with Rett Syndrome. They retained the capacity to successfully differentiate into functional neurons and underwent X-inactivation in females, which validated their effectiveness (Marchetto et al. 2010).

RESULTS

The researchers found some interesting differences between the iPSC-derived neurons and their controls (iPSCs derived from healthy subjects). These neurons had a reduced number of dendritic spines, membranous projections on dendrites that receive and transmit signals to the body of the neuron. They also observed fewer synapses and a lower frequency of calcium spikes, which are necessary to depolarize the neuron and allow it to fire action potentials. It was previously known that Rett syndrome is caused by a mutation in an X-linked gene that encodes a protein called MeCP2, which is involved in the regulation of genes important to synaptic connectivity. In a series of gain-of-function and lossof-function studies on iPSC neurons, Dr. Muotri and his colleagues concluded that some of the differences observed were due to MeCP2 expression levels. Since MeCP2 is an X-linked gene, its aforementioned X-inactivation is important for examining the ability of RTT-iPSC clones to reset the X chromosome and inactivate it again after differentiation. The last element of this study involved testing drug delivery mechanisms targeting the synaptic deficiencies in Rett syndrome. sqonline.ucsd.edu

2 FIGURE

A child playing a gaze-driven video game. These games train different aspects of basic attention and eye movement skills, which were found to be lacking in cases of ASD. Hence, targeting these basic differences has the potential to improve many clinical symptoms of ASD.

Overall, their data indicate that iPSCs are a reliable system to model Rett syndrome and other neurological disorders to both understand the workings of the condition and test out pharmacological treatments (Marchetto et al. 2010). Research in the RADLab and the Muotri Lab shows us that part of the purpose of science, especially in ASD research, is to understand and hopefully better the human condition. The quest to understand ourselves can be approached through many disciplines, from literature to philosophy. However, in almost all cases, the next step—the betterment and application step—requires science. Psychological studies can help us understand behavior, and molecular studies can be used to form the link between specific behavioral outcomes and human cellular networks. And when, for example, the brain organoids are able to mature enough to allow comparative studies with real brains, we may be able to account for emergent properties that can effectively link cell structure to psychology, in which all of ASD is defined. Finding effective solutions for this challenge would allow an entire group of people with ASD to become better integrated into their communities and have a better quality of life.

WRITTEN BY NIKHIL JAMPANA Nikhil is a Human Biology major from Eleanor Roosevelt College. He will graduate in 2021.

REFERENCES

1. Wright J. 2017. The Real Reasons Autism Rates Are Up in the U.S. [Internet]. Scientific American; [cited 2019 Feb 8]. Available from: https://www. scientificamerican.com/article/the-real-reasonsautism-rates-are-up-in-the-u-s/ 2. Osborn CK. 2018. Autism Treatment Guide [Internet]. Healthline; [cited 2019 Feb 8]. Available from: https://www.healthline.com/ health/autism-treatment 3. Mash LE, Reither MA, Linke AC, Townsend J, Müller R. 2018. Multimodal approaches to functional connectivity in autism spectrum disorders: An integrative perspective. Developmental Neurobiology. 78(5):456-473 4. Townsend J, Keehn B, Westerfield M. 2012. “Abstraction of Mind”: Attention in Autism. In: Posner MI. Cognitive Neuroscience of Attention. 2nd ed. New York (NY): The Guilford Press. p. 357-373. 5. Chukoskie L, Westerfield M, Townsend J. 2018. A Novel Approach to Training Attention and Gaze in ASD: A Feasibility and Efficacy Pilot Study. Developmental Neurobiology. 78(5): 546-552. 6. Marchetto MCN, Carromeu C, Acab A, Yu D, Yeo GW, Mu Y, Chen G, Gage FH, Muotri AR. 2010. A Model for Neural Development and Treatment of Rett Syndrome Using Human Induced Pluripotent Stem Cells. Cell. 143(4):527-539.

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life, death, and an obligate intracellular parasite

WRITTEN BY

CHRISTOPHER HAGAN ILLUSTRATED BY

SHAE GALLI

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Antibiotic resistance has become one of the world’s most serious public health emergencies as harmful drug resistant bacteria appear around the globe. As the race to fight the rapid progression of “superbugs” continues, one organization at the UC San Diego School of Medicine is re-discovering the incredible effectiveness of a treatment to bacterial infections that is just over one hundred years old — bacteriophage therapy. Originally discovered decades before the beginning of the antibiotic era, these natural predators of bacteria may be the fighting chance critical patients of infectious diseases need to survive.

With missile-lock precision and a resounding thud, the tail fibers of the virus firmly affix glycolipids on the surface of the bacterial cell membrane. A thick, protein coated sheath is lowered until the base plate nears the bacterial surface, after which six small, razor sharp spikes deploy and form an irreversible bond between the virus and the bacteria. If the virus successfully injects its DNA into the bacterial cell, a bacterial polymerase will transcribe that first set of viral genes. From that point forward, it’s all over. The next set of viral genes is transcribed and the systematic takeover of bacterial cell functions commences (ViralZone 2011; Feiss 2011). This is welcome news for a patient with a severe, multidrug resistant bacterial infection, which will have progressed into a life-threatening illness. As the infection progresses, a continuously stimulated immune system and the constant release of virulence factors will cause serious harm to healthy cells (McAdam et al. 2015). If the infection isn’t stopped soon, overall conditions will deteriorate into the failure of multiple organ systems which usually results in death. Antibiotics haven’t been effective, as the bacteria is resistant to everything the hospital has on its shelves. In a last-ditch effort to save the patient’s life, doctors have pushed a solution intravenously containing roughly one billion viruses, called bacteriophage, which travel through a patient’s circulation to the site of infection. There, the viruses can invade the bacterial cells, replicate themselves and then destroy the bacteria in the process of escaping. At this point, it’s a waiting game to see how the patient responds. One organization at the UC San Diego School of Medicine is on the front lines of this battle against antibiotic resistant infections — and they believe bacteriophage are the solution. It’s called the Center for Inno-

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vative Phage Applications and Therapeutics (IPATH), and quite amazingly, it is the first facility of its kind in the United States. Founded in June 2018, its mission involves using the natural predator of bacterial pathogens against them to treat critically ill patients of infectious diseases. “We’re trying to develop a better understanding of what the opportunities and challenges are and bring phage therapeutics to people with serious infections,” says Dr. Robert Schooley, an Infectious Disease Specialist at the UC San Diego School of Medicine and Co-Director of IPATH, “and we want to stimulate the development of rigorous clinical trials, both here and other places. In the meantime, we’re working to help patients who have the need for these kinds of interventions when traditional antibiotics aren’t working.” That being said, the development of clinical trials hasn’t been easy for a therapeutic approach that has had such a tumultuous history.

BACTERIOPHAGE DISCOVERY & EARLY THERAPEUTIC USE

It all began with the discovery of a mysterious biological agent by a French-Canadian scientist named Félix d'Herelle during his research in 1917. “He knew that there was a filterable agent that went through a Pasteur filter that killed bacteria,” explains Dr. Steffanie Strathdee, an Infectious Disease Epidemiologist at UC San Diego School of Medicine and Co-Director of IPATH, “he deduced that it was smaller than a bacteria and attacked bacteria, so he figured it must be a virus.” Proving the efficacy of novel medications in the 1920s was a challenging feat and, at times, could be inherently risky. To that point, d’Herelle began demonstrating the safety of his bacteriophage by volunteering to be the first trial patient. As explained by SALTMAN QUARTERLY

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d'Herelle himself in his 1926 publication called The Bacteriophage and Its Behavior, “Before undertaking experiments on man I had to assure myself that the administration of suspensions of the Shiga-bacteriophage caused no reaction. First, I ingested increased quantities of such suspensions, aged six days to a month, from one to thirty cubic centimeters, without detecting the slightest malaise. Three persons in my family next ingested variable quantities several times without showing the least disturbance” (D’Herelle 1926). Aside from rivaling the level of trust displayed by even the most cohesive families of modern times, these trials gave d’Herelle the confidence he needed to proceed with the treatment of patients stricken by bacillary dysentery. From there, he pressed forward to successfully treat multiple different bacterial pathogens, including Staphylococcus, cholera and even four patients infected by Yersinia pestis, feared widely by its common name—the Bubonic plague (D’Herelle 1926). All of this begs the question: why didn’t phage therapy hit the ground running and spread like wildfire in the United States? The discovery of penicillin essentially cast a pall on the whole field of phage therapy, along with a complex geopolitical bias affecting relations between the United States and Europe. From that point forward, the Western world as we knew it diverged from phage therapy in favor of antibiotics, while Eastern Europe continued to develop and use phage with some success, with phage therapy centers opening up in countries like Georgia and Poland.

THE GROWING THREAT OF ANTIBIOTIC RESISTANCE

As the years progressed past the World War II era, new antibiotics were being discovered at a relatively slow rate. Following the development and approval of each new antibiotic drug, the medical community faced the inevitable appearance of clinical cases in which patients’ infections were unresponsive to these antibiotic drugs (Spellberg and Gilbert 2014). This resistance to antibiotic drugs develops through several different mechanisms. The basic principle is that using antibiotics 12

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FIGURE

Upon first contact, a bacteriophage binds to the bacterial surface and injects its DNA into the bacterial host cell. As the host cell begins to transcribe the early viral genes, proteins are created which begin the process of synthesizing new copies of viral DNA and other proteins. After assembling new viral components, the new progeny phage lyse the membrane to escape, destroying it in the process.

creates a selective pressure on bacteria that kills susceptible bacterial cells and leaves behind resistant strains to proliferate (Ventola 2015). If a concentration of antibiotics is used that is not effective at killing susceptible bacteria, the accumulation of resistance genes can occur (Laureti et al. 2013). Resistance acquired by one bacterial cell can spread to neighboring bacteria, most commonly in the form of resistance “R” plasmids (Madigan et al. 2015). These plasmids are small, circular DNA molecules that can be passed along from one bacterium to the next. This horizontal gene transfer, in a process called “conjugation,'' allows a resistant bacterium to directly pass its R plasmid to another bacterial cell upon contact. These R plasmids have been found to exist in bacteria well before antibiotics were commercially used; as a result, the widespread use of antibiotics today simply helps these resistant strains proliferate by eliminating non-resistant competition while further passing along resistance to new cells. This mechanism is largely responsible for resistant strains of bacteria isolated from patients in hospitals across the globe (Madigan et al. 2015).

While scientists are locked in a continual effort to produce novel antibiotics that can defeat resistant bacterial strains, a series of health complications have emerged from their use. As Dr. Schooley explains, these complications are unavoidable side effects of the administration of antibiotic drugs. Antibiotics are unable to distinguish between pathogenic and normal gut bacteria, and so their use results in widespread killing of good and bad bacteria. These effects can contribute to numerous health problems, including immune system disorders, like hypersensitivity reactions and autoimmune disorders, and the destruction of important bacteria that regulate metabolic function which can lead to obesity (Langdon et al. 2016).

BACTERIOPHAGE TAKE A DIFFERENT APPROACH

This is where bacteriophage really begin to diverge from antibiotics. Whereas antibiotic drugs are bound to destroying a wide range of bacteria that are susceptible to their mechanism of killing, phage viruses are specific to just one or several bacterial species (Koskella and Meaden 2013). This sqonline.ucsd.edu

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high specificity for phage to attack their target organism — and only their target organism — makes phage a fantastic candidate to fight a bacterial infection where resistant strains are unresponsive to antibiotics and where collateral gut microbiome damage is undesirable. According to Dr. Schooley, the lytic cycle of phage is key for use as a therapeutic approach. This describes the cycle that a phage enters when it attaches to and enters a host bacterium, uses host machinery to build new phage, and then bursts out of the bacterial cell, destroying it in the process (Feiss 2011). Phage will multiply and spread rapidly until there are essentially no bacteria left over to infect. At this point, phage cease replicating and, according to the most current clinical data available, are excreted from the body without notable side effects. Specific phage have also been found to attack specific parts of bacterial cells, including cell surface components. “We know that there are a number of combinations of phage and antibiotics that are synergistic,” says Dr. Schooley, referring to the concept that these treatments can work together to produce a desired effect. “One of the host defenses that bacteria have with antibiotics is that when they get inside, they just pump [the antibiotic] out really quickly. This particular phage shuts that pump down and basically poisons the organism because it can’t get rid of the antibiotic.” In this way, bacteria that were once resistant to a particular antibiotic can become susceptible once again because of the synergistic treatment with phage. One of the opportunities with bacteriophage is the potential to use the cutting edge molecular techniques of today to develop phage that are better suited for clinical treatment scenarios. In fact, it is anticipated that in 2019 the first case of genetically modified phage to treat multidrug resistant bacteria will be reported. “In addition, there is this whole field called meta-genomics,” says Dr. Strathdee, “where you can actually find segments of DNA and RNA and piece them together” to create a phage of your choosing. By doing so, we’ve reached the beginning of an era sqonline.ucsd.edu

FIGURE

During conjugation, the R plasmid of a resistant bacteria disconnects a strand of its double stranded circular DNA and passes it into the recipient bacterial cell. This is facilitated through a passage between the two cells. As the cell receives the new DNA, it begins building a complementary strand while the donor plasmid begins building a replacement for its own complementary strand that it has donated.

where we can create synthetic phage. This could be vitally important for some patients who, for example, are infected with bacteria like Clostridium difficile. The environmental phage for this particular bacteria like to enter a lysogenic cycle, which results in the phage harmlessly inserting itself into the genome of the bacterial host cell, causing no lysis of the membrane and doing nothing to fight the infection. Genetically engineering these phage to undergo a lytic cycle would be a huge step towards creating an effective treatment for bacterial infections by organisms like C. difficile. According to Dr. Strathdee, genetically modified or synthetic phage can be patented. This has created a lot of interest from industry and the entire field is expanding rapidly. “The clinical trials we want to see done at IPATH are the ones that help us understand what the shortcomings are of the phage approaches we’re using and how to optimize things that are working, including modifying phage in ways that makes them more effective antimicrobial agents,” says Dr. Schooley. “Now, having said that, we have to have a little humility. They’ve been working on this for 300 million years and they’re pretty good at it. We have to be a little careful about thinking we’re smarter at this than they are.”

WRITTEN BY CHRISTOPHER HAGAN Christopher is a Microbiology and Sensation & Perception Psychology major from Sixth College. He will graduate in 2019.

REFERENCES

1. ViralZone [Internet]. 2011. SIB Swiss Institute of Bioinformatics; [updated 2018 Dec; cited 2019 Feb 10]. Available from: https://viralzone.expasy. org/504 2. McAdam AJ, Milner DA, Sharpe AH. 2015. Infectious Diseases. In: Kumar V, Abbas AK, Aster JC. Robbins and Cotran Pathologic Basis of Disease. 9th ed. Philadelphia (PA): Elsevier Saunders. p. 350. 3. D’Herelle F. 1926. Specific Therapy with Bacteriophage Suspensions. In: D’Herelle F, Smith GH. The Bacteriophage and Its Behavior. 1st ed. Baltimore (MD): The Williams &amp; Wilkins Company. p. 540-576. 4. Spellberg B, Gilbert D. 2014. The Future of Antibiotics and Resistance: A Tribute to a Career of Leadership by John Bartlett. Clinical Infectious Diseases. 59(S2):S71-S75 5. Ventola C. 2015. The Antibiotic Resistance Crisis. Pharmacology and Therapeutics. 40(4):277-283. 6. Laureti L, Matic I, Gutierrez A. 2013. Bacterial Responses and Genome Instability Induced by Subinhibitory Concentrations of Antibiotics. Antibiotics. 2(1):100-114.

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self vs non-self:

how cancer slides under the immune systemâ&#x20AC;&#x2122;s radar

WRITTEN BY

MAYA GOPALAKRISHNAN ILLUSTRATED BY

MICHAEL KALISZ 14

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While treatments like chemotherapy and radiation damage healthy tissue in the process of fighting cancer, the immune system’s ability to attack only “non-self” molecules and leave the body’s own cells intact makes it a key area of research for cancer treatment. Because cancer does not fit into self and non-self categories, it is able to evade the immune system while spreading disease. UC San Diego researchers aim to understand how this self versus non-self mechanism evolved, a crucial step in reprogramming the immune system to recognize cancer as a foreign molecule.

The immune system, the security detail of the cell, has been honed through evolution to recognize and attack pathogens, while leaving healthy tissue unharmed. This self vs non-self mechanism is crucial to the immune response. However, cancer does not easily fit into either of these categories. Cancer arises from the body’s own cells multiplying and functioning abnormally. When problems in the cell cycle go unchecked, cancer becomes malignant and begins to spread. The immune system’s ability to recognize cancer is complex but limited, as it is programmed to only attack “non-self ” tissue and protect the body’s own cells. By disguising itself as healthy tissue, cancer is able to evade the immune system and spread throughout the body. Immunotherapy, more specifically immuno-oncology, works to fight cancer by reprogramming the immune system to attack what it perceives as the body’s own cells. Although immuno-oncology has been a relevant subject of research since the 1980s, significant advancements have only been made recently (Cancer Research Institute). The 2018 Nobel Prize in Physiology and Medicine honored research on how the immune system is blocked from attacking tumor cells. An understanding of how

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the immune response to pathogens evolved in contrast to its response to cancer is necessary for immunotherapy to overcome the obstacles that keep the immune system from recognizing cancer cells as foreign.

HOW THE SELF VS NON-SELF RESPONSE EVOLVED

The ability of the immune system to differentiate between self and non-self molecules and cells is essential to its function, but this simultaneously allows cancer cells to be camouflaged and spread throughout the body. Dr. Matt Daugherty at UC San Diego is investigating mRNA modifications in the innate immune system that contribute to the differentiation between host and foreign cells. With the help of his team, Daugherty hopes to uncover the mechanisms by which the immune system has evolved to make this distinction. Daugherty thinks of the immune system as a carefully orchestrated process that must be altered by immunotherapies in order to unveil the cancer that is hiding under the guise of a host cell. The Daugherty lab’s research has shown that evolving mRNA modification and recognition systems are key to the development of antiviral specificity of the immune system. Modifications allow the host to mark mRNA as its own, as well as prevent the translation of viral mRNA. Transcription of foreign mRNA is prevented by interferon-induced-with- tetratricopeptide repeats (IFIT) proteins, which bind specifically to viral mRNA and initiation factors, allowing the host to stay one step ahead of the pathogen. Daugherty’s investigation of IFIT proteins revealed that they are evolutionarily highly dynamic, meaning they rapidly evolve and play an important role

in recognition of foreign molecules. However, little is known about how this system works to physically make these modifications to mRNA. This is a gray area the Daugherty lab is looking to explore by means of comparative genomics, evolutionary data, and inspecting these proteins at a molecular level. The deep evolutionary roots of specific immune recognition of foreign molecules makes it difficult for cancer to be recognized as harmful to the body. Cancer does not easily fit into the category of “self ” or “non-self ” because it is a disease that originates in the body’s own cells. Therefore, to expand the body’s understanding of non-self tissue to include cancer, the self versus non-self paradigm that has evolved over time must be disrupted. Investigating how the immune system has evolved to only target foreign molecules is the first step in finding ways to intervene in the recognition process with immunotherapies.

INNATE ANTI-TUMOR PROPERTIES OF THE IMMUNE SYSTEM

Although it is primarily programmed to only recognize “non-self ” molecules, the innate immune system does have properties that allow for the identification of some cancer cells, though they are limited. The innate immune system is the first line of defense against disease and is not specific to the type of antigen. Natural killer (NK) cells are white blood cells that are a component of the body’s innate immune system. These cells detect the first few tumor cells through ligand-specific binding, leading to lysis of the transformed cells (Finn 2012). The fragments are taken up by dendritic cells and macrophages that can then present molecules from the tumor cells to SALTMAN QUARTERLY

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A cancer cell evades the immune response by blocking T-cell recognition of an antigen.

T cells and B cells, allowing for more tumor-specific T cells and B cells to target the cancer. In this process, the innate immune system activates the adaptive immune system, generating immune memory to prevent recurrence of tumors. Other components of the adaptive immune system that are able to recognize tumor cells are: CD8+ cytotoxic T cells, CD4+ helper T cells, and antibodies. These target antigens that can only be expressed on tumor cells which result from mutation or post-translational modifications (Finn 2012). Although the immune system can utilize these properties to identify tumor cells, cancer is able to evade these safeguards in ways that pathogens cannot.

HOW CANCER EVADES THE IMMUNE SYSTEM’S ATTACKS

Dr. Irving Weissman, director of the Stanford Institute for Stem Cell Biology and Regenerative Medicine, is working to locate the hiding spots that facilitate the spread of cancer cells without activating the immune system. Weissman identified two methods by which cancer cells disable the macrophage's ability to eliminate it, thereby inhibiting the immune system's attack. The first is by a signaling protein expressed by some tumor cells called CD47. CD47 is

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a transmembrane protein that binds to the protein SIRP-alpha on macrophages, disabling the macrophage’s ability to engulf and break down the cancer cell. Weissman’s team used anti-CD47 antibodies as a treatment in mice with cancer, and have found a significant increase in the macrophages’ ability to identify and destroy tumor cells. Anti-CD47 antibodies are being studied in clinical trials as a potential form of treatment in humans as well (Newman 2017). The second mechanism being studied by Weissman is the class of MHC Class 1 molecules. These are surface membrane proteins that display portions of intracellular molecules on the cell’s surface to provide an indicator of the cell’s health. Weissman’s group found that the protein LILRB1 on macrophages binds to the MHC Class 1 component on cancer cells, preventing macrophages from killing the cell. It was found that MHC Class 1 proteins on cancer cells made it significantly more difficult to block the CD47 pathway, but when both the CD47 and LILRB1 pathways were successfully blocked, tumors grew at a significantly slower rate (Newman 2017). Weissman’s research highlights the importance of understanding how cancer silences the immune response, all in order to develop immunotherapies that prevent cancer cells from doing so.

TRICKING THE IMMUNE SYSTEM INTO RECOGNIZING CANCER

Immuno-oncology seeks to treat cancer with the body’s own immune system by disrupting the self versus non-self barrier that allows cancer to evade the immune response. Chemotherapy and radiation can harm healthy cells in the process of fighting cancer, but because of key characteristics of the immune system — self versus nonself distinction and long term memory of the adaptive immune response — immunotherapy specifically attacks cancer cells without harming healthy tissue, while retaining memory of the cancer. Reprogramming the immune system with immunotherapy can be done through various treatment methods, two of which are monoclonal antibodies and adoptive cell transfer. Monoclonal antibodies (MABs) are many copies of one antibody synthesized in a lab. They work by either triggering the immune system or helping it to attack cancer cells. To induce an immune response, certain types of MABs bind to the surface of cancer cells. Acting as a flag or marker, they make it easier for the immune system to identify and attack the cell. Other MABs help by binding to immune cells themselves to attack cancer.

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FEATURES CAR T cell therapy is an example of adoptive cell transfer that is gaining the most traction in immunotherapy. An adoptive cell transfer involves collecting the patient’s immune cells and engineering them to treat cancer (National Cancer Institute 2017). By transforming ordinary T cells into CAR T cells, the antigens on cancer can be recognized and attacked just as a virus would be attacked by a normal T cell. Specifically, CAR T cell therapy begins with taking a sample of the patient’s T cells. These are then treated with an inactivated virus that inserts genes into the cell, which allows chimeric antigen receptors (CARs) to sprout from the surface, thus becoming CAR T cells (Cancer Research UK 2017). When these cells are injected into the patient, the newly added receptors are attracted to antigens on the surface of cancer cells (Leukemia and Lymphoma Society). When the CAR T cells recognize cancer cells with specific antigens, they are able to attack them in the same way that a normal T cell might attack viral pathogens. This is because CAR T cells combine the antigen specificity of MABs with the cytolytic properties of T cells. Clinical trials using CAR T cell therapy have shown complete response rates of 50-90%, a success that lead to FDA approval for the first CAR T cell therapies in 2017 (Labanieh et al. 2018). While other cancer treatments harm healthy cells and are often ineffective, utilizing the evolutionary means of the immune system in attacking foreign molecules and ensuring the safety of the host makes it an ideal tool in treating cancer. However, this very characteristic of the immune system also serves as an obstacle to perfecting immunotherapies, as cancer cannot regularly be categorized as self or non-self. Therefore, understanding how immune specificity evolves, as well as the mechanisms by which cancer evades the immune system disguised as a healthy cell, is key to the development of immunotherapies that disrupt this game of hide-and-seek and expose cancer to the proper immune response.

WRITTEN BY MAYA GOPALAKRISHNAN Maya is a Molecular Biology major from Thurgood Marshall College. She will graduate in 2021.

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FIGURE

An NK cell recognizes a cancer cell by ligand-specific binding, causing it to lyse.

REFERENCES

1. Cancer Research Institute. What is Immunotherapy? [Internet]. [cited 2019 Jan 26]. Available from: www.cancerresearch.org/ immunotherapy/what-is-immunotherapy.  2. Finn OJ. 2012. Immuno-Oncology: understanding the function and dysfunction of the immune system in cancer. Annals of Oncology: Official Journal of the European Society for Medical Oncology. 23(Suppl 8):viii69.  3. Newman T. 2017. How Does Cancer Evade the Immune System? New Mechanism Revealed [Internet]. Medical News Today; [cited 2019 Feb 6]. Available from: www.medicalnewstoday.com/ articles/320177.php.  4. National Cancer Institute. 2017. CAR T Cells: Engineering Immune Cells to Treat Cancer [Internet]. [updated 2017 Dec 14; cited 2019

Feb 6]. Available from: www.cancer.gov/aboutcancer/treatment/research/car-t-cells.  5. Cancer Research UK. 2017. General Cancer Information. Monoclonal Antibodies (MABs) [Internet]; [updated 2017 Nov 6; cited 2019 Feb 10]. Available from: www.cancerresearchuk. org/about-cancer/cancer-in-general/treatment/ immunotherapy/types/monoclonal-antibodies.  6. Labanieh L, Majzner R, Mackall C. 2018. Programming CAR-T Cells to Kill Cancer. Nature Biomedical Engineering. 2(6):377-391.  7. Leukemia and Lymphoma Society. Chimeric Antigen Receptor (CAR) T-Cell Therapy [Internet]; [cited 2019 Feb 10]. Available from: www.lls.org/treatment/types-of-treatment/ immunotherapy/chimeric-antigen-receptor-cart-cell-therapy.

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investigating our metabolismâ&#x20AC;&#x2122;s chemical fingerprints

WRITTEN BY

SNEHA GANGULY ILLUSTRATED BY

VARSHA RAJESH

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Metabolomics provides a way to rapidly test for all the biomolecules in our blood and give us the identity of the disease. Since the concentration of metabolites in different diseases varies, UC San Diego researcher, Dr. Shankar Subramaniam, has taken upon the task to identify all the known metabolites in our bodies and assess their levels in different diseases. His research has helped medical professionals understand pathologies better and look for various individualized therapies to treat them. Since 150 CE, pathologists have been responsible for diagnosing diseases such as diabetes. Ancient Greek pathologists, known as "water tasters", would diagnose diabetes by tasting the urine of those suspected to have it. Fortunately for modern pathologists, simple chemical tests now exist that facilitate easy determination of the presence of sugar in a person’s urine. Moreover, the latest technologies have enabled pathologists to not only detect whether a person is suffering from diabetes— by profiling the glucose, glucagon, and insulin levels in a person’s blood —but also to note the severity of the disease. These new tests are revolutionary as they have made it easier and more accessible for people to understand whether or not they are suffering from diabetes; this, in turn, allows patients to get treated before the disease progresses extensively.

METABOLITES AS CHEMICAL FINGERPRINTS

The method of systematically analyzing metabolites—low molecular weight biochemicals including sugars, amino acids, organic acids, nucleotides, and lipids—in a biological sample to understand a pathology is known as metabolomics. Metabolomics involves the analysis of the

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total number of metabolites in the blood and their interactions within a biological system at any point in time. Metabolites are produced as by-products of metabolic reactions that take place in our body. To conduct metabolomic studies, saliva and blood are used because they are more easily obtained from the body and don’t involve invasive procedures, unlike tissue analysis. A regular blood test consists of measuring a person’s fasting blood sugar, hemoglobin A, creatinine, lipid, and cholesterol content to determine the endpoints of their physiological response, allowing doctors to assess if the person is healthy. These biomolecules or metabolites in the blood can be used to determine the precursor mechanism that led to their formation and look for possible defects. By doing so, pathologists can consequently examine whether one is suffering from a disease or not. The non-invasive aspect of metabolomics is what makes it a popular topic of research.

PLAN FOR THE FUTURE OF METABOLOMICS

Metabolomics carries the potential to transform healthcare and could make the diagnosis of complex diseases like cancer, Alzheimer’s, and coronary heart disease as simple as a blood test (once metabolic profiles for these diseases are found). One of the groups studying the discovery of metabolic markers for certain diseases in the blood is the Metabolic Workbench, led by the chair of UC San Diego’s Bioengineering Department, Dr. Shankar Subramaniam. Dr. Subramaniam is one of the pioneers in the field of developing data and analysis programs for metabolomics. His research group focuses on systems biology and systems medicine, for diseases of the liver, muscles, brain, and vascular system. He recently received a $12 million grant from the National Institute of Health to expand his research on human metabolism, allowing his group to discover metabolites

in the bloodstream and create an international clinical database of metabolites for researchers and physicians to access. This database could be used to drastically improve and accelerate the diagnosis of diseases through blood sample analysis.

LIPID MAPS PROJECT AND METABOLIC WORKBENCH

When Dr. Subramaniam recognized the lack of a comprehensive metabolomic database, he formed the Metabolic Workbench with a vision to provide the international community with a resource that would will not only store data, but do so in a format that would allow users, to understand the ongoing biochemistry in a mammalian cell. Dr. Subramaniam’s Metabolomic Workbench initially began as the Lipid Maps Project in 2003. It was one of the first efforts in metabolomics that primarily focused on creating a database of lipid structure and nomenclature to assist the international lipid research community. The resources that were utilized in the Lipid Maps Project were the forerunners for the Metabolic Workbench. Dr. Subramaniam’s team created a database that was available at any time for processing and analysis to aid scientists in reconstructing the metabolic pathway networks. They solved the challenge of homogenizing and harmonizing the diversity in metabolites by developing a comprehensive classification and nomenclature system (Fahy et al. 2009). This system’s simple web interface would allow people to access it and add their own data according to the specified guidelines and structure. The metadata (age, gender, ethnicity, tissue sample, pathology) was then classified into groups and studied. Besides the data collected for the research at UC San Diego, the Metabolic Workbench has already collected data from 2000 studies worldwide—each of which consists of thousands of subjects. Scientists and people in the clinical field can easily and quickSALTMAN QUARTERLY

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Our blood sample contains all the metabolites that are a result of biochemical reactions. Certain levels of lipids, creatinine, blood sugar and cholesterol are associated with specific pathologies; metabolomics helps us identify them with relative speed.

ly obtain high volumes of specific and related information on metabolites and their protein sequences from this database to utilize for their own research.

COMPLEX AND INDIVIDUALIZED METABOLOME STUDIES

Dr. Subramaniam aims to take this concept of metabolomics as useful evidence of an individual’s functioning metabolism and extend the analysis to understanding the physiological pathways of a huge repertoire of metabolites. He also aims to extend this analysis to include those metabolites that come as constituents of drugs or medicines to understand how effective drugs are in treating a pathology. Looking at these metabolites also provides a glimpse into the transcriptional and proteomic changes and inquires whether or not these changes were the consequence of metabolic regulation or other mechanisms. But to make this comparison, knowledge of the steady-state metabolome is necessary to notice any changes during a pathology. The main hurdle of this task lies in the fact that the steady-state metabolome differs in every person based on age, ethnicity, race, and even region of residence. Unlike genes and proteins, metabolite interactions are heavily dependent on their quantity in the biological environment, making the process of determin20

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ing someone’s metabolome composition an individualized process. For example, by titrating against a standard, one can determine the quantity of cholesterol in one’s body. Additionally, collecting and grouping terrabytes of metabolome data can help group individuals within a certain range based on similar characteristics. These groups provide us with general information on how the aforementioned characteristics affect the metabolome. For example, it was found that Southeast Asians have high levels of Haemoglobin A1c (HbA1c) caused by a mutation in the gene responsible for producing HbA1c (Sabanayagam et al. 2015). This causes them to be diagnosed with insulin resistance by foreign metabolome standards, even though the levels are considered normal by regional metabolome standards. This knowledge helped healthcare providers in Southeast Asia to modify treatment for diabetes mellitus by increasing resources to treat patients with an inherently high HbA1c threshold.

IMPLICATIONS OF ADVANCED TECHNOLOGY ON METABOLOME RECONSTRUCTION

Dr. Subramaniam credits the rapid advance of metabolomics in recent times to improvements in the range and precision of measurement instruments. Current

mass spectrometric methods have become so sophisticated and sensitive that they allow scientists to determine a dynamic range of measurements. This range allows scientists to notice microscopic changes in metabolites and connect these changes to physiological responses. They then use these detailed reports to map the mechanisms back to the origin of the metabolites by reconstructing the physiological mechanisms that lead to them. One of the first mapping projects which was successfully completed by Dr. Subramaniam’s team was the construction of the mammalian lipidome and lipid metabolic map for inflamed macrophages (Dennis et al. 2010). They traced the lipid metabolic reactions in a macrophage from when an immune cell underwent a bacterial invasion, which caused the macrophage to get inflamed and produce cytokines, until the completion of phagocytosis. They then built the wiring diagrams by making systematic longitudinal measurements of the macrophage since the time of infection. These diagrams gave rise to the first complete lipidome. The lipidome helped scientists understand the cellular metabolism that is critical in shaping the macrophage polarization states and immune functions (Teng et al. 2017). This information is helping scientists understand how modification of the lipidome can alter the way the macrophage manipulates a host-pathogen towards its own advantage and consequently develop ways by which this feature can be used to advance pathogen recognition and phagocytosis (Pradervand et al. 2006).

HOW METABOLOMICS RESEARCH IS IMPROVING HEALTHCARE

Dr. Subramaniam has done extensive work with tissue-specific metabolites as well. "The reason tissues need to be looked at separately," Dr. Subramaniam says, "is that sometimes specific metabolites inside the tissue are not available to a vascular sqonline.ucsd.edu

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FIGURE

The applications of metabolomics research are versatile. The metabolomics database can be used to identify tissue-specific metabolites present only in the blood as well as link certain diseases to specific ethnicities or geographic regions.

system like a blood vessel and hence go undetected in the metabolome in a blood sample.” While doing research on Nonalcoholic Fatty Liver Disease and Steatohepatitis, his team looked at liver tissue and blood; they observed metabolite concentrations in them and found that there were several metabolites in the liver which were excluded from the blood (Gorden et al. 2015). Currently, his Systems Biology lab is working on biliary atresia in collaboration with a lab from Pittsburgh (PI: Ningappa, So). Biliary atresia is the failure to form a bile duct in a child which results in the eventual death of the child from liver failure. His team is doing a systems level analysis on what causes the liver to fail to form the bile duct. This research could possibly help find a therapy to this life-threatening disease. Researchers in Dr. Subramaninam’s lab are also working on the metabolome of the neurons in those affected with Alzheimer’s Disease (AD). Early-onset familial AD (eFAD) is caused by mutations in APP, PS1 and PS2 genes which shows symptoms of dementia and AD (Tang & Gershon 2003). Besides this, old age also makes a person more susceptible to dementia and AD. His team noticed that the metabolic endpoints, as well as the MRI images, appeared to be very similar in both AD and dementia; this

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available for precise metabolomic measurements, he decided to switch. He believes that a considerable number of diseases can be detected and investigated using metabolomics, as it has a unique opportunity to impact discovery-driven science in a way that genomics, transcriptomics, and proteomics cannot fully exploit.

led them to conclude that the mechanisms that trigger cases of eFAD and old age AD are similar as well. In AD, neurons become dedifferentiated into a non-neuron state which causes them to lose their respective cognitive functions. Thus, the team is studying how metabolite concentrations vary through the dedifferentiation process in neurons, as well as trying to reconstruct mechanisms that are apparent in initiating very early AD and discover ways to address the disease. Additionally, his team is using machine learning methods to integrate metadata obtained from cancer cell profiles in colorectal cancer. They identified the different markers in the colorectal tissues and are trying to establish the effect and activity of drugs and therapies on the tissues from stage 1 of cancer all the way to stage 4 (Subramaniam). This will allow them to track the mechanisms of these drugs and make improvements to current treatments. When asked why he chose metabolomics as his area of research, Dr. Subramaniam excitedly answered, “I get a thrill when I finally get to look at the big picture, understanding the cell as an integrated unit and not as genes, proteins, and metabolites.” Previously, he worked with proteomics, transcriptomics, and regulatory genomics, but when advanced technology became

WRITTEN BY SNEHA GANGULY Sneha is a Biochemistry and Cellular Biology major from Revelle College. She will graduate in 2021.

REFERENCES 1. Fahy E, Subramaniam S, Murphy RC, Nishijima M, Raetz CR, Shimizu T, Spener F, van Meer G, Wakelam MJ, Dennis EA. 2009. Update of the LIPID MAPS comprehensive classification system for lipids. J Lipid Res. 50(Suppl):S9-14 2. Sabanayagam C, Khoo EYH, Lye WK, Ikram K, Lamoureux EL, Chen CY, Tan MLS, Salim A, Lee J, Tavintharan SLS, et al. Diagnosis of Diabetes Mellitus Using HbA1c in Asians: Relationship Between HbA1c and Retinopathy in a Multiethnic Asian Population. 2015. The Journal of Clinical Endocrinology &amp; Metabolism. 100( 2):689–696 3. Dennis EA, Deems RA, Harkewicz R, Quehenberger O, Brown HA, Milne SB, Myers DS, Glass CK, Hardiman G, Reichart D, et al. 2010. A mouse macrophage lipidome. J Biol Chem. 285(51):39976–85 4. Teng O, Ang CKE, Guan XL. 2017. Macr phage-Bacteria Interactions-A Lipid-Centric Relationship. Front Immunol. 8(1836):1-17 5. Pradervand S, Maurya MR, Subramaniam S. 2006. Identification of signaling components required for the prediction of cytokine release in RAW 264.7 macrophages. Genome Biology. 7(2):R11.1-14 6. Gorden DL, Myers DS, Ivanova PT, Fahy E, Maurya MR, Gupta S, Min J, McDonald JG, Kelly SL, Duan J, et al. 2015. Biomarkers of NAFLD progression: a lipidomics approach to an epidemic. J Lipid Res. 56(3):722–736 7. Tang Y, Gershon ES. 2003. Genetic studies in Alzheimer's disease. Dialogues in Clinical Neuroscience. 5(1):17-26 8. Subramaniam S. unpublished

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RESEARCH A Shore Crab (Pachygrapsus crassipes) at La Jolla Shores between tides. Photo by Francesca Hummler

As one of the most prestigious public schools for the biological sciences, UC San Diego offers a unique undergraduate experience with enriching opportunities, such as hands-on laboratory experience. Through original research manuscripts and review papers, the Research section showcases the current understanding of various fields in biology.

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RESEARCH

A COMPARISON OF VERTEBRATE ANIMAL BIODIVERSITY ALONG THE VERTICAL STRATA OF STRANGLER FIGS (MORACEAE, FICUS SPP.)

IN MONTEVERDE, COSTA RICA

Katherine Hernandez1 Dr. Federico A. Chinchilla Romero2 Tropical rain and cloud forests, like those of Monteverde, Costa Rica, contain a vertical plane that constitutes multiple tree layers, which vary in plant and animal composition. Strangler fig trees (Moraceae, Ficus spp.) have a unique anatomy that may allow them to serve as structures for vertebrates to travel between and across a forest’s vertical layers in a way that they otherwise would not be able to. In this study, camera traps were used to observe vertebrate behavior around strangler figs. Behavior was analyzed according to time of day, location, and use of the tree. Photographs revealed many animals of different species and orders actively climbing the tree, with some appearing to use it as a means of access to neighboring tree canopies. There is a considerable amount of evidence for vertebrates using strangler figs as vertical strata bridges. This study is purely observational and further research should be done to investigate how the vertebrate activity on strangler figs compare to other tree species in Central American cloud forests.

UC San Diego, Warren College, Ecology, Behavior & Evolution, 2019 2

The Monteverde Institute, Monteverde, Puntarenas, Costa Rica

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INTRODUCTION

Monteverde, Costa Rica is home to some of the world’s most diverse and well-protected tropical cloud forests. The trees in these forests create a considerable difference in height between the forest floor and tree canopies, providing the conditions for a vertical gradient to emerge. Vertical gradient refers to the assemblage of forest layers, or strata, that differ from one another in plant and animal composition as a result of forest depth—ranging from the understory to canopy layers—and light accessibility (Kricher 1975). Plants and animals have adapted to such physical factors at varying degrees. Thus, as depth and light changes, so does the community of species living within each stratum (Whittaker 1975). Further animal variation may exist depending on the tree species. Some plant species have evolved to discourage native animal species from interacting with them (Janzen 1980). This affects the animal composition of vertical forest layers, not only for each tree, but for the local ecosystem overall. However, while some tree species may discourage interaction, others have evolved forms that are advantageous for animals as places of refuge and accessible food sources (Janzen 1980). It is important to note that the existence of this kind of interaction between a tree species and any number of animals does not necessarily mean that the interaction is beneficial for the tree. A captivating example of a canopy-reaching tree commonly found in Monteverde is the strangler fig (Ficus spp., Moraceae), easily recognizable by its skeletal, braid-like appearance and, in the case of older specimens, almost completely hollow trunk (Forsyth and Miyata 1984; Gentry 1993). Strangler fig trees are primary hemiepiphytes: they begin their lives attached to a host tree and later send roots down to the forest floor (Lüttge 1997). The vines of the young fig will eventually grow over the host tree in its entirety and deprive it of light and vital nutrients. The host tree is subsequently “strangled” and dies, decaying from within the trunk of the strangler fig and leaving it hollow (Gentry 1993). Hollow fig trees serve as shelter, nesting spots, and sites of refuge for various animal species (Lomáscolo et al. 2010). Fig trees also fruit independently of the season of other fig trees, meaning that they can produce fruit all year long and provide a vital food source to all seed predators in the area (Knightbridge and Ogden 1998). Arboreal vertebrates have evolved specific adaptations to arboreal life, such as prehensile tails and long, grasping claws. These adaptations improve the species’ movement and communication amongst tree canopies; consequently, adaptations vary depending on where along the vertical strata an animal species lives (Kays and Allison 2001; Emmons 1983; Malcolm 1991). To access the canopy layer of any particular tree, non-flying vertebrates must either climb the tree itself or access it through surrounding trees (Emmons 1983; August 1983). The woody vines and overall trunk structure of mature, hollow strangler figs are much easier to maneuver up and down through the vertical gradient of the forest than other native tree species and have the potential to act as strata “bridges”—the means through which vertebrates can access and move between vertical strata, including the canopy layer. SALTMAN QUARTERLY

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Site

Strata

Initial Placement

Final Retrieval

Santuario Ecologico

Canopy

8:55 am; 5/17/17

8:56 am; 5/25/17

Santuario Ecologico

Understory

9:01 am; 5/17/17

8:48 am; 5/25/17

Curi Cancha, Crandell Reserve

Canopy

11:00 am; 5/18/17

8:34 am; 5/26/17

Curi Cancha, Crandell Reserve

Understory

11:20 am; 5/18/17

8:24 am; 5/26/17

Curi Cancha, Crandell Reserve

Canopy

11:35 am; 5/18/17

8:56 am; 5/26/17

Curi Cancha, Crandell Reserve

Understory

11:45 am; 5/18/17

8:49 am; 5/26/17

Curi Cancha, Crandell Reserve

Canopy

12:07 pm; 5/18/17

9:25 am; 5/26/17

Curi Cancha, Crandell Reserve

Understory

12:11 pm; 5/18/17

9:20 am; 5/26/17

Tree

Table 1. Log of all eight cameras by tree, area in Monteverde, strata of focus/in view/placed in, initial time and date of placement, and time and date of retrieval.

Accessing the canopy layer of an established tropical forest can be very labor- and time-intensive, not to mention dangerous. In the late twentieth century, scientists learned to use typical alpine and rockface climbing techniques to access the upper layers of rainforests; even so, experiments that encompass a forest’s vertical gradient in its entirety are especially rare because of the logistical difficulties (Lowman 2009). This study attempts to fill the gap in knowledge relating to rainforest vertical strata, animal behavior, and the strangler fig by asking whether or not arboreal vertebrates use the strangler fig as a vertical strata bridge. It does not attempt to determine the intensity of traffic via strangler figs, or how good of a bridge they are, but rather if they are being used as a bridge at all. If true, then this finding would expand our knowledge of the strangler fig and how animals have evolved behaviors to overcome the challenges of terrestrial vertical strata.

MATERIALS AND METHODS

Data was collected between May 17, 2017 and May 26, 2017. Four strangler fig trees were chosen for this study. All except one of the trees were decades old, tall and hollow; the other was substantially shorter and was known to have been planted relatively recently in comparison to the other three. This study focused on strangler figs because of the tree’s unique form and because of the foundational science showing that many animal species already interact with strangler figs for shelter and food. Camera traps were utilized over other observation methods for several reasons. Camera traps are mostly non-intrusive and allow experimenters to observe vertebrates remotely. This is especially vital for capturing elusive animal species that would not approach the study site with a human presence nearby. Additionally, camera traps can record for extended periods of time without end, so long as the battery allows (Whitworth et al. 2016; Gregory et al. 2014). As discussed in a study by Schipper (2007), this method is not al24

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ways non-intrusive; cameras using flash-photography during surveys of nocturnal environments caused nocturnal vertebrates to actively avoid traps, and consequently, the study site. To avoid this issue, infrared only camera traps were used in the experiment. Two cameras were attached to each tree and strategically placed so as to maximize the chance of capturing animals descending and ascending the tree. Half of the cameras faced the understory tree base while the rest were tied to the inside of the trunk structure and positioned to see as much of the middle and upper strata layers of each tree as possible (Table 1). Cameras were functional 24 hours a day for every day of the study, capturing diurnal and nocturnal life in the area (Jayasekara et al. 2007). Three picture frames were taken every time a camera was triggered by movement. Initially, camera dates and times were incorrect due to mechanical error. Corrections to the data sheet were hastily made at the beginning of the experimental period, and all cameras after the fact were fixed to show the true time and date. The displayed pictures occurred before this correction and were twelve hours behind the time they were actually taken. For documenting data, each photographed animal was categorized by the camera that captured it, species, time and date of photograph, and observed behavior occurring in frame. The observed animal behavior in each set of photographs were labeled according to how and if they interacted with the strangler fig. If the animal(s) showed no interest in climbing or interacting with the tree, they were labeled as “Indifferent”. If the animal(s) in frame were not Indifferent, then they were labeled as either “Ascending” or “Descending”, along with the direction from which the individual(s) came—from either “Higher” or “Lower” on the photograph. These labels were chosen as a means of organizing animal behavior to movement alone and to movement relative to the strangler fig. Camera positions, batteries, and sensitivity settings were adjusted as needed. sqonline.ucsd.edu

RESEARCH It was important to know where and when certain behaviors were performed and by what species. Without this data, it would be difficult to identify the purpose of each animal’s behavior. Multiple photograph sets could be of the same individual. It may be that the individual was spot descending a tree it had just ascended, and so was captured by both the canopy and understory camera traps. An animal might also be a shelter resident—an individual using the strangler fig as a regular place of rest, rather than as a place to facilitate movement between and across the vertical strata. To determine which scenario was occurring, photographs were carefully analyzed with great attention to time and location of sighting on the tree. A large time gap between photographs of the same species with an incompatible path of movement reduces the likelihood that the photographs were of the same individual(s). If the photographs of a species were only taken at the times of day that species is known to leave or enter shelter, and the animal failed to return into view for a time period typical of a foraging or sleeping period, then the photographs could be of a single shelter resident. This analysis was done on a case by case basis in order to be sure of what exactly was being observed and its relevance to the study.

Image 1. Camera 1A. At least six different coatimundis (Nasua narica) can be seen in front of tree one: two adults and four juveniles. Captured at 6:17:16-17 am, 5/22/17.

RESULTS

The overwhelming majority of documented animals for both the canopy and forest floor camera traps were coatimundis (Nasua narica), which had 63 three-frame photograph sets (Figure 1). No amphibians were photographed. There was only one reptile seen by the preliminary camera, a small lizard climbing up tree two. As expected, the vertebrates spotted by the canopy camera traps were species that are well adapted to arboreal and semi-arboreal lifestyles. The species with the most individuals recorded was the coatimundi—21 in the canopy, 45 at the forest floor. There was no Indifferent behavior from species photographed by canopy cameras (Figure 3a). However, Indifferent was the most common type of behavior documented by the forest floor camera traps (Figure 3b). All other behaviors and actions found for both the photographed canopy and forest floor individuals were similar in quantity (Figure 3). Coatimundis were the most active climbers of the recorded species. Coatimundis were twice as likely to be photographed in the “forest floor” (Image 1) than the “canopy” (Image 2; Figure 2), but were equally as likely to ascend and descend the tree in either strata (Figure 3). Camera 4A spotted a Mexican tree porcupine climbing up and down the middle of the trunk during the evenings and late night of May 18 to May 20, 2017. The camera was placed about mid-way up the center of tree four’s hollow trunk and pointed upward. Photographs of the porcupine show that it was making use of the ladder-like structure of the strangler fig and emerged several times from both the very top and very bottom of the screen (Image 3). Likewise, small rodents—excluding squirrels—were found in the canopies of trees two and three, always climbing through the trunk with the apparent purpose of reaching a different vertical stratum. Images 4a and 4b show a coatimundi climbing up the center of tree four, but, like many of the spotted mice and rats, it was not later seen climbing down. sqonline.ucsd.edu

Image 2. Camera 1A; two juvenile coatimundis (Nasua narica) climbing up a strangler fig limb. Captured at 5:57:14-16 am, 05/20/17.

Image 3. View from camera 4A, pointing up and inside of the tree trunk, on 5/20/17. A porcupine was captured climbing down the tree at 3:15:39 am. In the photograph below a porcupine, likely the same individual as captured prior, is climbing up the tree at 4:30:44 am.

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Number of Picture Sets

60 50 40 30 20 10 0

Coati

Porcupine

Opossum

Mice/Rats

Squirrel

Agouti

Bird

Reptiles

Amphibians

Vertebrate Species Seen Figure 1. Total number of picture sets taken per species seen across all cameras. The amount of picture sets taken of each species does not represent the number of total individuals seen, since there were multiple picture sets that had several individuals photographed at a time.

DISCUSSION

Out of all the species captured by the cameras, the mammal activity was the most consistent and abundant. While reptiles, birds, and amphibians were caught around and moving within the tree, it is unclear what behavior was actually being performed. Furthermore, their appearances were rare with respect to the timeline of the project, and so cannot be considered as reliable, common behavior. The Mexican porcupine was only ever captured in tree four, often at the beginning and end of the night, and was seen multiple times within a relatively short time period. From this, one can infer that the Mexican porcupine photographs were all of one resident individual, living in tree four. Thus, these photographs cannot be used as supporting evidence for vertebrates using strangler figs as vertical strata bridges. Likewise, agouti and opossum appearances were infrequent with little Ascending or Descending behavior. On the other hand, squirrels, mice, and rats were seen actively climbing the subject trees, providing evidence that these species are regularly using strangler fig trees, possibly as vertical strata bridges.

The coatimundi was captured with a high frequency across tree subjects, at different cameras, and without a significant preference in time. This suggests that the observed coatimundis were not the same individuals exiting a tree the camera had just witnessed them enter, nor were they residents of any particular tree like the porcupine was for tree four. The abundance of coatimundis is likely due to how common they are in the area. Several of the coatimundi photographs show individuals ascending a tree with no additional photographs from their sister camera to show an individual descending within a time period where one could consider it to be the same individualâ&#x20AC;&#x201D;or descending and ascending respectively. Special attention was given to forest floor stratum photographs that showed individuals ascending from the tree base but not descending that same tree later within a reasonable time period for that species. Special attention was also given to canopy stratum photographs that showed individuals descending from the canopy strata of a tree that had not been seen ascending earlier by either camera. The presence of such photographs suggests that observed coatimundis have access to the canopies of

Images 4a & 4b. Camera 4A. Center of image 2a, a coatimundi (Nasua narica) is climbing and reaching into the trunk of the strangler fig. Top left corner of image 2b, the same coatimundi is climbing up the strangler fig by use of the web-like hollow center. Captured at 11:06:55-57 am, 05/20/17.

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sqonline.ucsd.edu

Number of Individuals

RESEARCH

Coati

Porcupine

Opossum Mice/Rats

Squirrel

Agouti

Bird

Reptiles

Number of Individuals

Vertebrate Species Observed in Canopy Stratum

Coati

Porcupine Opossum Mice/Rats

Squirrel

Agouti

Bird

Reptiles Amphibians

Vertebrate Species Observed in Forest Floor Stratum Figure 2. Number of individuals per species photographed by a) canopy and b) forest floor camera traps. Coatimundis (Nasua narica) had the most individuals photographed by canopy cameras (21) and forest floor cameras (45).

Amphibians Reptiles Bird Agouti Squirrel Mice/Rats Opossum Porcupine Coati

!! Indifferent " #$ % From Lower " #& From Higher ' Descending Ascending

Amphibians Reptiles Bird Agouti Squirrel Mice/Rats Opossum Porcupine ! Coati

" ## Indifferent $ %& ' From Lower $ %( From Higher ) Descending Ascending

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Frequency of Observed Activity in Forest Floor Stratum

Vertebrate Species Seen

Figure 3. Observed behaviors of each documented individual per species from a) canopy and b) forest floor camera tramps. The legend denotes the action or apparent intention of each individual.

Vertebrate Species Seen

Frequency of Observed Activity in Canopy Stratum

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other trees surrounding the strangler figs and are using them as additional points of entry and exit. This is vital when it comes to determining whether or not a tree is being used as a vertical strata bridge. To reiterate, to be a “bridge,” the tree must allow animal species to climb between and across vertical strata. In order to move across the vertical strata, an animal would need to be able to access neighboring trees’ canopies by way of the fig’s canopy. Thus, to be a vertical strata “bridge”, a tree must allow animals use both the tree’s canopy and its base as points of entry and exit. The photographs suggest that multiple species, coatimundis especially, have such access; thus, there is evidence that vertebrates use strangler fig trees as vertical strata bridges. The majority of coatimundi photographs were taken at tree one, the shortest and least mature of the four trees. There appears to be no significant difference in activity between tree one and trees two through four. This suggests that the hollow, braid like structure of a mature strangler figs is not the reason strangler figs may be vertical strata bridges. Their value as a good vertical strata bridge is independent of this unique anatomy. On later inspection, tree one had also been fruiting within the experimental period while all others were not. The presence of fruit may have skewed the data by providing further encouragement for local animals to interact with it. Furthermore, tree one was in a separate reserve from the other three trees, and this may have allowed unknown factors to significantly skew results. For future research and recreations of this study, experimenters should ensure that they have longer study period, more tree sites, and a single location to do their research.

CONCLUSION

In conclusion, there is substantial evidence that vertebrates use strangler figs (Moraceae, Ficus spp.) as vertical strata bridges. Fruiting trees appear to get more use than non-fruiting strangler figs; however, this is inconclusive due to sample size and the fact that the one fruiting tree in question was at a different location from all other trees. This study is descriptive in nature and cannot comment on why this behavior may be or how activity at strangler figs compares to other tree species in this location. Further research can be done to explore such unknowns and expand the scientific community’s knowledge of tropical forests’ vertical strata and vertebrate behavior. Studies focused on vertical strata and the upper canopy layer have proven difficult, especially where animal behavior is concerned, seeing as such studies often demand constant monitoring and involve a heightened level of danger for scientists. As technology improves and becomes more accessible, more studies can be performed to expand the community’s understanding of tropical forests and the effect of their three-dimensionality.

ACKNOWLEDGEMENTS

I would like to thank the Monteverde Institute for their support, lab space, and equipment. Thank you to Mauricio Valverde, Jorge Mora, and 28

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the Salazar family for allowing me to use their property for my study. I would like to also thank my advisor and co-advisor, Federico Chinchilla and Andres Camacho for their guidance throughout this project, and my host family for housing and caring for me during my time in Costa Rica. None of this would have been possible without the aide and interest of everyone listed here, and for that I am truly grateful.

REFERENCES 1. August PV. The Role of Habitat Complexity and Heterogeneity in Structuring Tropical Mammal Communities. Ecology 64(6), 1495–1507, doi:10.2307/1937504 (1 Dec. 1983). 2. Emmons, LH, Gentry, AH. Tropical Forest Structure and the Distribution of Gliding and Prehensile-Tailed Vertebrates. The American Naturalist 121(4), 513-524 (Apr., 1983). 3. Forsyth, A, Miyata, K. Tropical Nature: Life and Death in the Rainforests of Central and South America. Scribners/MacMillan, New York (1984). 4. Gentry AH. A Field Guide to the Families and Genera of Woody Plants of Northwest South America (Colombia, Ecuador, Peru) with supplementary notes on herbaceous taxa. Washington, DC: Conservation International (1993). 5. Gregory, T, Carrasco Rueda, F, Deichmann, J, Kolowski, J, Alonso, A. Arboreal camera trapping: taking a proven method to new heights. Methods in Ecology and Evolution 5(5), 443-451 (2014). 6. Janzen, Daniel H. When Is It Coevolution? Evolution 34(3), 611-12 (1980). doi:10.2307/2408229. 7. Jayasekara, P, Weerasinghe, UR, Wijesundara, S, Takatsuki, S. Identifying diurnal and nocturnal frugivores in the terrestrial and arboreal layers of a tropical rain forest in Sri Lanka. Ecotropica 13, 7-15 (2007). 8. Kays, R, Allison, A. Arboreal tropical forest vertebrates: current knowledge and research trends. In: Linsenmair, K.E.; Davis, A.J.; Fiala, B.; Speight, M.R. (eds) Tropical Forest Canopies: Ecology and Management. Forestry Sciences vol 69, 109–120. Springer, Dordrecht (2001). 9. Knightbridge, PI, Ogden, J. Establishment patterns and host tree preferences of the emergent hemi-epiphytic tree Metrosideros robusta in northern New Zealand. New Zealand Journal of Botany 36, 203–212 (1998). 10. Kricher J. Tropical Ecology. Princeton, NJ: Princeton University Press (2011). 11. Lomáscolo, SB, Levey, DJ; Kimball, RT; Bolker, BM; Alborn, HT. Dispersers shape fruit diversity in Ficus (Moraceae). Proc. Natl Acad. Sci. USA 107, 14668–14672 (2010). 12. Lowman, MD. Canopy research in the twenty-first century: a review of arboreal ecology. Tropical Ecology 50(1), 125–136 (2009). 13. Lüttge U. Epiphytes, Lianas and Hemiepiphytes. In: Physiological Ecology of Tropical Plants, 139-196. Springer, Berlin, Heidelberg (1997). 14. Malcolm, JR. Comparative Abundances of Neotropical Small Mammals by Trap Height. Journal of Mammalogy 72(1), 188-191 (1991). doi:10.2307/1381995. 15. Schipper, J. Camera-trap avoidance by Kinkajous Potos flavus: rethinking the “non-invasive” paradigm. Small Carnivore Conservation 36, 38-41 (2007). 16. Whittaker, RH. Communities and Ecosystems. 2nd ed. New York, NY: Collier Macmillian Publishers (1975). 17. Whitworth, A, Braunholtz, LD, Huarcaya, RP, MacLeod, R, Beirne, C. Out on a limb: arboreal camera traps as an emerging methodology for inventorying elusive rainforest mammals. Tropical Conservation Science 9(2), 675-698 (2016). doi:10.1177/1940082916009002.

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RESEARCH

ANALYZING ARCTOSTAPHYLOS: EVALUATING VIABILITY OF SEED COLLECTIONS AND COMPARING EFFECTIVENESS OF GERMINATION TESTING METHODS Julia Gaudio1 Stacy Anderson2 Joyce Maschinski, Ph.D.2 This experiment evaluated the germination potential and the effectiveness of germination trial pretreatments on seed collections of Arctostaphylos, commonly known as the manzanita. The three species tested in this project are found in San Diego County, including A. otavensis, an endemic subspecies which is the rarest in the county. The first part of this experiment, through a series of baseline germination tests, evaluated the viability of the three subspecies of Arctostaphylos. Results indicated that all the seed collections had adequate germination percentages under baseline conditions, so they can be frozen and banked for long-term storage. The second focus of this project explored the effectiveness of different germination methods by implementing different pretreatments designed to imitate natural germination triggers. We found that a gibberellic acid (GA3) pretreatment may increase germination percentages and/or speed up germination, while a smoke water pretreatment may not have a beneficial effect. Additionally, within each species, accessions responded differently to the treatments, possibly due to the varying storage states of the seed collections. The third part of the project examined the possibility of simplifying and speeding up the seed processing by attempting to simulate germination by soaking seeds in sulfuric acid (H2SO4) then imposing a cold stratification. Results from this part of the experiment resulted in minimal germination and were inconclusive.

1 2

UC San Diego, Muir College, Ecology, Behavior & Evolution, 2019

Plant Conservation Department, San Diego Zoo Conservation Institute

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INTRODUCTION

Seed banking contributes to global conservation efforts by conserving genetic diversity and protecting species from extinction (Peres 2016). To evaluate the viability of seed collections before placing them in a freezer for long-term storage, germination tests are performed on the seeds. This project, conducted at the San Diego Zoo Global (SDZG) Native Plant Seed Bank, involved germination tests on seed collections of three subspecies of manzanita: Arctostaphylos pungens, Arctostaphylos glandulosa ssp. glandulosa, and Arctostaphylos otayensis. Standardized germination tests, processing methods, and storage protocols are commonly used for preserving a variety of seeds at seed banks around the world. A simple “baseline” germination test involves disinfecting the seeds, soaking them in water, then placing them on agar plates in a temperature controlled germination chamber (Davies et al. 2015). However, these methods may not stimulate germination in seeds with hard, thick casings such as observed with Arctostaphylos seeds. Each red “fruit” or “stone” of the Arctostaphylos contains a resinous endocarp surrounding multiple smaller segments called “nutlets.” The individual nutlets have thick, woody casings that protect the embryo or “naked seed” within (Rundel and Gustafson 2005). Previous experiments have found that Arctostaphylos typically exhibits seed coat dormancy, meaning that in its nutlet form, the seed coat is impermeable to oxygen and water, and requires additional treatment to expose the naked seed (Emery 1995). Findings from previous studies on Arctostaphylos germination and guidelines on general germination techniques directed the choice of the pretreatments used in this experiment. For part 1, a commonly used baseline germination protocol was used as a control treatment. This involved sterilizing the naked seeds and soaking them in reverse osmosis (RO) water, a type of purified water that lacks ions and large particles (Davies et al. 2015). For parts 2 and 3, alternative pretreatments were selected to replicate conditions the seeds likely encounter in their natural environment, acting as natural catalysts for germination. In part 2, a solution of gibberellic acid (GA3) and a solution of smoke water were used as alternatives to the RO water soak. GA3 is a naturally found hormone that weakens seed cover and aids with germination, often used in seed germination tests on various species (Emery 1995; Gupta and Chakrabarty 2013). Research suggests that smoke water should also aid with germination by exposing the seeds to the compound karrikin (KAR1), a seed germination stimulant naturally released as plant tissue burns. Due to the frequent occurrence of fire in the natural chaparral environment of Arctostaphylos, KAR1, present in charate (char red plant matter) may act as a natural and common germination stimulant (Emery 1995; Lightner 2011; Meyer 2008). Part 3 involved chemical scarification of the nutlets by soaking them in a solution of sulfuric acid (H2SO4). Multiple sources suggest this as the optimal method for germinating Arctostaphylos seeds because H2SO4 chemically breaks down the thick seed coat and allows water to imbibe the nutlet (Emery 1995; Carlson and Sharp 1975; Deno 1993). This is meant to simulate the acidic conditions of animal stomachs if the seeds are eaten. Ideally, enough sulfuric acid penetrates the seed to enlarge the micropyle, an opening on the surface of the ovule. This allows water to imbibe without acid damaging the cotyledon, the embryonic first leaf of a seedling (Radchuck and Borisjuk 2014). After the H2SO4 treatment, cold stratification can be imposed, SALTMAN QUARTERLY

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Table 1. Initial State of Collections and Tests Performed. Outlines the state of the six collections of Arctostaphylos seeds before the processing and testing performed for this project. States the number of seeds or nutlets used for each test. ERH = Equilibrium Relative Humidity.

mimicking conditions a seed may experience during winter before germination in spring and the release from physiological dormancy (Davies 2015). For each subspecies, the SDZG Seed Bank possesses two separate collections from populations of the species that were collected in different locations in San Diego County (Table 1). Of the three focal manzanita species, the main objective was to process and store seeds from A. otayensisâ&#x20AC;&#x201D;a rare and endemic subspecies found only in San Diego County. For the A. otayensis seeds, only the baseline pretreatment was used, due to limited quantities of seed. Banking the seeds of this rare manzanita contributed to SDZG Seed Bankâ&#x20AC;&#x2122;s overall mission to store seeds for research and enable possible future restoration of the natural populations, thus preserving local genetic diversity. Ultimately, this project addressed the following research questions: (1) Are the Arctostaphylos seed collections still viable? (2) Will germination pretreatments such as gibberellic acid and smoke water increase the germination percentage and/or speed up the germination process? Are certain treatments more effective on different Arctostaphylos subspecies? (3) Is it possible to stimulate germination without removing the seed coat, by using sulfuric acid scarification combined with cold stratification?

MATERIALS AND METHODS

Part 1: Testing viability of naked seeds with baseline method: Prior to starting the germination tests on the naked Arctostaphylos seeds, the nutlets were processed using a mortar and pestle to remove the outer casing and extract the embryo. The viability of the naked seeds was tested using standard SDZG Seed Bank germination testing protocol. All embryos were soaked in a solution of 1% bleach (sodium hypochlorite) for 10 minutes then transferred to petri dishes for the pretreatment. The baseline pretreatment consists of soaking the seeds for 24 hours at room temperature in reverse osmosis (RO) water. Then, the seeds were placed on agar plates in a stable growth chamber. The seeds were monitored for germination (emergence of a radicle) every few days for a total of 22 days (Davies 2015). 30

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Part 2: Comparing germination pretreatment methods on naked seeds, using GA3 or smoke water solution: The same processing and bleaching methods were used as in the standard baseline pretreatment, but the pretreatment differed in the solution for the 24-hour soak. The GA3 pretreatment substituted the RO water soak with a soak in GA3 solution. This solution was created by dissolving 1.125 grams of GA3 in 30 mL of 80% ethanol, heating the solution to burn off most of the ethanol, then dissolving it into 50 mL of RO water (Davies 2015). The smoke water pretreatment substituted the RO water soak with a soak in 50% concentration smoke water. This solution was created by filtering boiling water through charate. After each respective pretreatment, the seeds were placed on agar plates in a stable growth chamber and monitored for germination for 22 days (Davies 2015). Part 3: Testing germination potential of seeds in nutlet form, using H2SO4 solution and cold stratification: The stones were separated into nutlets, soaked in concentrated H2SO4 for either three or six hours, soaked in RO water for 88 hours, then placed in moist vermiculite and stratified at 5.5 Â°C for 17 days. Half of the stratified nutlets (400) were then placed on agar and monitored for emergence of a radicle. The other 400 nutlets were left to stratify for 35 days. They were plated and continually monitored beyond the timeline of this project. Separate trials involve soaking the seeds in sulfuric acid for two different time periods because sources differ on the recommended soak time. Exposing the seeds to acid for a longer time increases the chance of acid penetrating the seed through the micropyle and damaging the cotyledon (Emery 1995; Carlson and Sharp 1975). Statistical methodology: We used a generalized linear mixed-effects model for three analyses: (1) to compare final germination percent in baseline trials among all three Arctostaphylos subspecies, (2) to compare the effect of germination pretreatments (baseline, smoke water, and GA)3 on final germination percent of processed seed in A. pungens and A. glandulosa ssp. glandulosa, and (3) to evaluate the effect of sulfuric acid treatments on nutlets on germination of unprocessed nutlets of A. pungens and A. glandulosa ssp. glandulosa. We used a binomial error distribution because each seed in the trials has two possible sqonline.ucsd.edu

RESEARCH outcomes: germinated or un-germinated. The dependent variable in these models was the proportion of seeds germinated at the end of the trial weighted by the number of seeds in the trial. The baseline model included species as a fixed effect and the two treatment models included a species versus treatment interaction effect. Both models included seed accession as a random effect, to account for variability due to the source population. We used a type II Wal chi-square test to evaluate the statistical significance of the species effect.

this species, germination was 15% higher on average in the GA3 trials relative to the baseline trials; however, the effect of smoke water differed between accessions. The accession from Banner Grade, which had been stored at room temperature, displayed higher germination in the smoke water trial (43%) relative to the baseline trial (27%). In contrast, the accession from Smith Ranch, which had been frozen, displayed very low germination (14%) in the smoke water trial compared to the baseline (32%) (Figure 2; Table 3). (3) Sulfuric acid treatment results inconclusive: After two weeks of measurement, only 17 out of 400 plated nutlets treated with H2SO4 showed signs of germination. This low level of germination was significantly lower than the baseline treatment and did not result in enough data to compare results between accessions and/or treatments.

RESULTS

In total, 1,000 seeds and 800 nutlets were tested. For each pretreatment, batches of 100 or 50 seeds were used and batches of 100 nutlets were used. Typical germination trials involve batches of 100 seeds, but this was reduced to 50 seeds for certain accessions (#0168 and #0617) due to the state of the collections causing increased processing difficulty. Also, batches of 50 were used for the A. otayensis baseline tests due to the limited quantity of seed (Table 1). (1) Baseline comparisons show highest percent germination among A. otayensis seeds: After 22 days of measurement, the three Arctostaphylos species differed significantly in percent germination in baseline trials (Figure 1). A. otayensis, the rare subspecies, exhibited the highest percent germination, A. glandulosa ssp. glandulosa showed the next highest percent germination, and A. pungens showed the lowest percent germination. Percent germination did not differ among accessions of the same species, indicating that there may not be strong population effects on germination success. Thus, the germination percentages for each species reflect the average percentage between the two accessions per species (Figure 1). (2) Germination pretreatment comparisons results show highest germination with GA3 pretreatment: Across both species and accession, seed exposed to GA exhibited 14% greater germination success than baseline seed and 12% greater germination than seed treated with smoke water (Table 2). The effect of treatment differed between the two accessions of A. glandulosa, as demonstrated by the significant treatment versus accession interaction (X2 = 7.53, df = 2, P = 0.02). For the Noble Canyon accession, both gibberellic acid (36%) and smoke water (32%) trials had lower germination success than the baseline trial (44%). In contrast, for the accession from El Cajon Mountain, the gibberellic acid trial had substantially higher germination (75%) than the baseline (51%) or smoke water trials (57%) (Figure 2; Table 3). There was also a significant treatment versus accession interaction in A. pungens germination trials (X2 = 13.06, df = 2, P < 0.001). In

DISCUSSION

Part 1: Baseline treatment comparison: The germination percentages indicate that all Arctostaphylos collections are viable, with A. otayensis exhibiting the highest germination percentage. The significantly higher germination percentages observed with A. otayensis (P < 0.001) may be attributed to the age of the collections compared to the collections of the other species. The A. otayensis seeds were collected in 2016, while the other species were collected from 2006 to 2012. Some of the older collections had never been fully processed and have been stored in the low humidity, room-temperature environment of the seed bank for many years. If not frozen, seeds are more likely to deteriorate at a faster rate due to the effects of temperature, humidity, and seed-parasitic insects (Australian National Botanic Gardens 2011). When frozen, the lower humidity and temperature slows degradation, and kills any seed-parasitic insects. However, even frozen seeds deteriorate over time, so it is important to check the viability of collections every few years to determine if a new collection should be made. Confirming the viability of all seed collections means the accessions that have not yet been frozen can now be fully processed and stored. The relatively low germination percentage for A. pungens (29%) suggests that the current seed collections have not been well processed or stored. Although many seeds remain viable, it may be wise to make new collections of the subspecies within the next few years.

Species Baseline Comparison 100

Figure 1. Baseline Pretreatment Germination Comparison Between Species. A. otayensis (ARCOTA) is shown in green, with 79% germination. A. glandulosa ssp. glandulosa (ARCGLA) is shown in red, with 49% germination. A. pungens (ARCPUN) is shown in blue, with 29% germination. The two lines of data points for each color represent results from the two separate accessions tested for each species. All species were grown in â&#x20AC;&#x2DC;baselineâ&#x20AC;&#x2122; conditions. (X2 = 53.9, df = 2, P < 0.001).

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Germination (%)

75 Species ARCGLA ARCOTA ARCPUN

0 0

Days Since Start of Trial

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Treatment Comparison

Figure 2. Final Germination Percentages from Pretreatment Comparison Test. A. glandulosa (ARCGLA) results are on the left, A. pungens (ARCPUN) results are on the right.

Germination (%)

Treatment Baseline Gibberellic Acid Smoke Water

Nonetheless, the significant difference between germination percentages for the pretreatments indicates that GA3 may be an effective aid to germination. The high germination percentages from GA3 align with germination studies on other plant species, as the plant hormone is shown to naturally aid germination in various plants by inEl Cajon Mtn Noble Canyon Banner Grade Smith Ranch creasing the growth potential of the embryo and Seed Accession Locations stimulating production of hydrolytic enzymes (Gupta and Chakrabarty 2013). In contrast, smoke water did not noPart 2: Pretreatment comparison with naked seeds: The comparticeably improve germination percentages compared to the baseline, ison of pretreatments on naked seeds provides information on the relsuggesting that a smoke water treatment may not be a practical method ative state of the seed collections as well as the effectiveness of different for increasing germination potential in Arctostaphylos seeds. The smoke pretreatments. The differing success of pretreatments and distinctions water pretreatment did result in the highest germination percentage for between germination timelines may be partially explained by the varythe A. pungens accession from Banner Grade, but when considering the ing state of the collections. During the cleaning and sorting process, it average germination results across all species and accessions, the smoke became apparent that certain accessions were in a more degraded state water did not result in improved germination. than others; these accessions had high percentages of empty or broken Although GA3 resulted in significantly different final germination nutlets before any processing. The results confirm this, as certain colpercentages when compared to both baseline and smoke water treatlections have significantly different germination percentages when comments (P = 0.02 for A. glandulosa and P < 0.001 for A. pungens), the paring results from all pretreatments to results from other collections. other treatments show higher germination percentages at different For example, the A. glandulosa collection from Noble Canyon was the stages of monitoring for some collections (Figure 3). Due to time conoldest collection tested, and during processing it became evident that straints for this project, trials ended when low levels of new germinamore than half the nutlets had holes, were empty, or had shriveled emtion were still occurring. Further research needs to be done to explore bryos. It was not surprising that this collection had much lower germiwhether the other pretreatments have the potential to produce higher nation success across pretreatments compared to the other A. glandulosa germination percentages if monitored over a longer experimental pecollection (from El Cajon Mountain), which appeared to be in a much riod. Therefore, within the time frame of this project, GA3 proved to better state before testing. Another notable difference was the smoke wabe the most effective at promoting germination overall, but a longer ter pretreatment, which resulted in lower germination percentages for experimental period may not result in as significant of a difference bethe two collections that had been frozen compared to the non-frozen tween germination methods. The other methods may promote greater collections of their respective subspecies (Figure 2; Table 3). This indigermination percentages if given a longer time to germinate. cates that smoke water may act as a more effective germination stimPart 3: Germination potential of seeds in nutlet form: The sululant for collections that have only been stored at room temperature. furic acid treatment resulted in very few nutlets germinating within the experimental period, but the germination trials are ongoing and Germination will likely show increased germination over time. Compared to naPercent Treatment ked seeds, seeds in nutlet form do not as readily germinate because it 39% Baseline takes longer for water and nutrients to reach the embryo (Emery 1995; 53% Gibberellic Acid Carlson and Sharp 1975; Deno 1993). Due to the time constraints of 41% Smoke Water this project, only the preliminary results can be reported, but continued germination trials may provide valuable information on the effecTable 2. Germination Results Across Accessions for tiveness of this treatment. If this treatment reveals comparatively high Pretreatment Comparison Test. This table shows the germination percentages, it may be used as the treatment of choice average germination percent for each pretreatment across when testing viability of Arctostaphylos seeds in the future. Using this accessions, while the graphs show the germination percent germination method would eliminate the time-consuming process of separated by accession and pretreatment. cracking open nutlets to expose the naked seeds, which is required

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RESEARCH Treatment Comparison

Germination (%)

Figure 3. Timeline of Germination from Pretreatment Comparison Test. A. glandulosa (ARCGLA) results are shown in the top two graphs, A. pungens (ARCPUN) results are shown in the bottom two graphs.

for all other tested methods of germination. Future directions for related research may include experimentation with the specifics of the H2SO4 treatment to optimize germination and reduce processing time.

Treatment Baseline Gibberellic Acid Smoke Water

CONCLUSION

This project evaluated the viability of the Arctostaphylos seed collections and determined the relative effectiveness of germination methods. Testing various germination methods may increase the chances of successful germination if the baseline method results in minimal germination. Furthermore, determining the most effective germination method (highest germination percentage) may accelerate the process of periodically checking the viability of the stored seed collections. The results suggest that a GA3 pretreatment may be used to produce higher levels of germination and/or speed up germination compared to the normal baseline method. Future experiments should implement a longer germination trial period to further study the comparison between a baseline and GA3 pretreatment. Additionally, future experiments should explore the feasibility of H2SO4 pretreatments by varying the acid soak times as well as implementing a longer germination trial period. If this pretreatment produces similar or improved germination percentages when compared with the baseline test, it may prove to be the optimal method for testing viability of Arctostaphylos seeds because it bypasses the time-consuming process of extracting naked seeds. Ultimately, this project provided valuable information on the state of Arctostaphylos collections at the SDZG Native Plant Seed Bank. The pretreatment comparison results also added to the minimal research previously done on germination methods for Arctostaphylos. A. glandulosa

A. pungens

El Cajon Mountain

Noble Canyon

Banner Grade

Smith Ranch

Baseline

51%

44%

27%

32%

Gibberellic Acid

75%

36%

42%

48%

Smoke Water

57%

32%

43%

14%

Table 3. Final Germination Percentages from Pretreatment Comparison Test. Results from each accession shown.

Days Since Start of Trial

ACKNOWLEDGMENTS

I am incredibly grateful for the guidance and assistance of my mentor, Stacy Anderson, as well as the other members of the seed bank, Joe Davitt and Tobin Weatherson. Special thanks to Katie Heineman for undertaking statistical analysis of my data. I would also like to thank the family of Henry and Winnifred Frabotta for their support of my summer fellowship.

REFERENCES 1. Peres, S. “Saving the Gene Pool for the Future: Seed Banks as Archives.” Studies in History and Philosophy of Science Part C: Studies in History and Philosophy of Biological and Biomedical Sciences, vol. 55, 96–104, (2016). 2. Davies, R, Di Sacco, A, Newton, R. Germination testing: procedures and evaluation. Royal Botanic Gardens, Kew, (2015). 3. Rundel, PW, Gustafson R. Introduction to the Plant Life of Southern California, Coast to Foothills, University of California Press, 77-79, (2005). 4. Emery, DE. Seed Propagation of Native CA Plants, Santa Barbara Botanic Garden, (1995). 5. Gupta, R, Chakrabarty SK. Gibberellic acid in plant: still a mystery unresolved, Plant Signaling and Behavior, (June 2013): doi: 10.4161/psb.25504. 6. Lightner, J. 201. San Diego County Native Plants, San Diego Flora, 103-105, (2011). 7. Meyer, SE. Arctostaphylos Adans. Manzanita, Woody Plant Seed Manual, 266-270, (2008). 8. Carlson, JR, Sharp WC. Germination of High Elevation Manzanitas, Tree Planters’ Notes, Vol. 26, No. 3, (1975). 9. Deno, NC. Seed Germination Theory and Practice, USDA National Agricultural Library, 99, (1993). 10. Radchuck, V, Borisjuk L. Physical, metabolic and developmental functions of the seed coat, Frontiers in Plant Science, Vol. 5, 510, (October 2014): doi: 10.3389/fpls.2014.00510. 11. Australian National Botanic Gardens, Seed Bank Procedures, (2011).

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THE EFFECTS OF ANTIRETROVIRAL DRUGS ON MICROVASCULAR CELLS: TENOFOVIR INDUCES CELL SENESCENCE IN 3D MULTI-LAYER CO-CULTURE

Rachel C. Chang1 Benchawanna Soontornniyomkij, Ph.D.2 Anya Umlauf, M.S.2 Virawudh Soontornniyomkij, M.D.2

Antiretroviral (ARV) therapy is recommended for all adults with HIV infection, regardless of their CD4+ T-cell count. With the advent of ARV drugs, HIV infection has become manageable and plasma HIV-1 RNA load can be suppressed to undetectable levels, resulting in a drastic increase in life expectancy. With this success, clinical attention has shifted towards the improvement of treatment that optimizes long-term safety. A clinicopathological study of HIV-infected persons by our group revealed an association between the use of ARV drug emtricitabine (FTC) and occurrence of cerebral small vessel disease. FTC is commonly prescribed with tenofovir (TFV) in ARV regimens. Here, we explored in vitro the cytotoxic effects of FTC, TFV, and FTC+TFV at clinically relevant concentrations (versus vehicle) on primary human brain microvascular endothelial cells, smooth muscle cells, and pericytes grown in 3D multi-layer cocultures. β-Galactosidase activity was used as a marker of cell senescence. Using two-way ANOVA, we found that there was no significant interaction effect of cell types and ARV drugs, whereas the main effect of ARV drugs was observed. ARV drugs affected all cell types. Compared with vehicle, TFV exposure increased β-galactosidase activity, whereas exposure to FTC alone did not show any significant effect (Dunnett’s post hoc t-test). In conclusion, TFV exposure induced cellular senescence of primary human brain microvascular cells in 3D multi-layer co-cultures. The chronic effects of long-term exposure to TFV should be studied in animal models. 1 UC San Diego, Roosevelt College, Biochemistry & Cell Biology and Music, 2020 2 Department of Psychiatry, UCSD School of Medicine, La Jolla, CA, USA

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INTRODUCTION

Human immunodeficiency virus (HIV) is a retrovirus that alters a person’s immune system by destroying white blood cells that fight infections (Al-Jabri 2003). Individuals with HIV disease suffer from a chronic decrease in CD4+ T-cell count, a measure of white blood cells present in the bloodstream that coordinate immune response. This can be caused by a number of mechanisms, including programmed cell death. A progressive decrease in CD4+ T-cell counts to levels below normal range (<500 cells/µL) significantly increases the risk of serious illnesses (Lundgren et al. 2015). Though there is no current cure, antiretroviral (ARV) therapy (ART) is effective in suppressing HIV replication. As part of proper medical care, ART is given to all persons living with HIV, regardless of their CD4+ T-cell count, and is continued throughout life (Department of Health and Human Services 2019). Due to widespread implementation of ART, the life expectancy of HIV-infected persons has drastically increased (Teeraananchai et al. 2017). It is therefore important to understand the long-term consequences of ART. A clinicopathological study of HIV-infected persons by our group revealed an association between the use of ARV drug emtricitabine (FTC) and occurrence of cerebral small vessel disease (Soontornniyomkij et al. 2018). Cerebral small vessel disease is a degenerative vascular change commonly associated with aging, hypertension, and diabetes mellitus (Cysique and Brew 2019). We further study this association through aging-related cerebral degenerative changes, which is characterized by cellular senescence. Due to a variety of stressors, including ARV drug use, cellular senescence has been found to occur prematurely (Cohen and Torres 2017). FTC is a component of most first-line ARV regimens as recommended by the Department of Health and Human Services. FTC belongs to the nucleoside reverse transcriptase inhibitor (NRTI) class of ARV drugs. NRTIs stop HIV from making new viral copies of itself in host cells by blocking the reverse transcriptase enzyme. FTC is typically prescribed with tenofovir (TFV, another NRTI drug) in a fixed dose combination, because of their additive anti-HIV activity (Department of Health and Human Services 2019). In the present study, we explored the cytotoxic effects of FTC and TFV exposure both individually and in combination on primary human brain microvascular cells in 3D multi-layer co-culture. Previous studies on the toxic effects of ARV drugs on vascular cells in vitro used traditional 2D cultures of one or two cell types (Herbert et al. 2004; Jiang et al. 2006; Lefevre et al. 2010). Our present study mimicked the in vivo small vessel wall using 3D multi-layer co-cultures of primary human brain microvascular cells with the Alvetex polystyrene scaffold system. The Alvetex disk is a dynamic and versatile structure that allows cell-cell interaction and microvascular cells can produce their own native extracellular matrix. We assessed cellular senescence by measuring β-galactosidase activity levels. Increased β-galactosidase activity is a phenotype generally shared among senescent cells (Dimri et al. 1995). Results will allow us to further assess the long-term consequences of ARV drugs FTC and TFV.

MATERIALS AND METHODS

Cell culture: Primary human brain endothelial cells (EC, #ACBRI 376) were obtained from Cell Systems (Kirkland, WA). Primary human brain vascular smooth muscle cells (SMC, #1100) and pericytes (PC, #1200) were obtained from ScienCell Research Laboratories (Carlsbad, CA). The cells were cultured in 5% CO2 incubators with complete classic medium with serum and CultureBoost (CSC, #4Z0-500, Cell Systems) until Passage 7-8 for EC, Passage 5-6 for SMC, and Passage 8-9 for PC. EC and SMC were individually seeded on 24-well Alvetex Scaffold disks (#AVP006, REPROCELL USA, Beltsville, MD) and PC on 6-well Alvetex Scaffold disk inserts (#AVP004). sqonline.ucsd.edu

RESEARCH 3D multi-layer co-culture and ARV drug exposure: For 3D multi-layer co-cultures, EC- and SMC-grown disks were removed from their wells and placed on the PC-grown Alvetex 6-well disk inserts system in the order of EC (top), SMC (middle), and PC (bottom) to mimic the in vivo small vessel wall (Figure 1). The multi-layer co-cultures were treated with CSC for 1 day (Figure 2) before being exposed to ARV drugs (prepared in CSC) at the clinically relevant unbound drug concentration of 2.5 μg/mL for FTC (#10071, NIH AIDS Reagent program) and 0.386 μg/mL for TFV (#10199) (Department of Health and Human Services 2019). The microvascular cell co-cultures exposed to either ARV drugs or vehicle were maintained for 8 days with 50% medium changes made every 2 days. Vehicle (CSC) was the control experiment, 3D multi-layer co-cultures with no ARV drug exposure. β-Galactosidase activity assay: EC-, SMC-, and PC-grown Alvetex Scaffold disks were removed from the inserts. To extract protein, lysis buffer (#T1012, Applied Biosystems) was added to the disks individually and stored on ice for 10 minutes, with 15-second vortex every 3 minutes. Samples were then centrifuged at 13.2K rpm for 5 minutes at 4°C. β-Galactosidase activity was quantitatively assayed in cytosolic lysates by using the chemiluminescent Galacto-Star System (#T1012, Applied Biosystems) in 96-well plates according to the manufacturer's protocol. Chemiluminescent signal was measured using the microplate reader Synergy HTX Multi-Mode Microplate Reader (BioTex) at 60 minutes after adding the reaction buffer at 25°C. A standard curve was generated from serial dilutions of β-galactosidase (#G-4635, Sigma-Aldrich). Levels of β-galactosidase activity were normalized by the corresponding protein concentrations (BCA, Pierce Biotechnology) in each lysate sample. Normalization was performed to minimize variation in protein content concentration that would have varied during extraction. Statistical Analysis: In each of the ARV drug exposure conditions (FTC, TFV, FTC+TFV, vehicle), four or five biological replicates were included (18 in total). For all cell types (EC, SMC, PC), a total of 54 protein lysate samples were assayed for β-galactosidase activity. The average of two technical replicates of β-galactosidase activity measurement was used. Data were analyzed using two-way ANOVA that included the interaction term (cell types by ARV drugs), followed by Dunnett’s post hoc t-test. Interaction term refers to the interaction between independent variables that affects the dependent variable; in this case, specific cell types responding differently to each ARV drug exposure condition. Main effect refers to the effect of an independent variable on a dependent variable; in this case, each ARV drug exposure condition having an effect on the microvascular cells. A two-tailed P value of less than 0.05 was considered statistically significant.

RESULTS

ARV drugs contribute to a difference in β-galactosidase activity: Data on β-galactosidase activity analyzed using two-way ANOVA showed no

Figure 1. The Alvetex 6-well inserts system for 3D multi-layer co-cultures of primary human brain endothelial cells (EC), vascular smooth muscle cells (SMC), and pericytes (PC).

significant interaction effect of cell types and ARV drugs (F(6,42) = 0.53, P = 0.78). Statistics concluded that EC, SMC, and PC were not affected differently by ARV drugs. The combination ARV drugs FTC and TFV do not appear to affect each other either. Since the interaction term was not significant, we went on to look at the main effects. The main effect of cell types was not statistically significant (F(2,42) = 2.33, P = 0.11). The main effect of ARV drugs was statistically significant (F(3,42) = 11.01, P < 0.001, partial-eta squared = 0.44). ARV drugs contributed to a difference in β-galactosidase activity. TFV contributes to increased β-galactosidase activity: Since the main effect of ARV drugs was statistically significant, we went on to do the Dunnett’s post hoc t-test. Compared with vehicle, FTC exposure was not statistically significant (P = 0.86), i.e. FTC did not significantly contribute to a difference in β-galactosidase activity. TFV exposure (P < 0.001, Cohen’s d = 1.73) and FTC+TFV exposure (P = 0.010, Cohen’s d = 1.67) were statistically significant in contributing to an increase in β-galactosidase activity. Overall, TFV exposure contributed to a higher increase in β-galactosidase activity than FTC+TFV exposure.

DISCUSSION

We found that TFV exposure for 8 days induced an increase in β-galactosidase activity in primary human brain microvascular cells in 3D multi-layer co-culture, indicating cellular senescence. In contrast, there was no evidence suggesting that FTC exposure alone induces cellular senescence in microvascular cells. Combination FTC+TFV exposure also contributed to an increase in β-galactosidase activity, but at lower levels than TFV exposure alone.

Figure 2. Microvascular cells in multi-layer co-cultures. Primary human brain endothelial cells (EC), vascular smooth muscle cells (SMC), and pericytes (PC) were cultured in 3D multi-layer Alvetex Scaffold disks and stained with Neutral Red (#N6264, Sigma-Aldrich). Cells were examined on bright-field light microscopy at the original magnification x200.

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Beta-galactosidase activity by ARV exposure 1.6 1.4

Mean Beta-galactosidase

Figure 3. ARV drug exposure induces β-galactosidase activity in microvascular cells. 3D multi-layer microvascular cell co-cultures (EC, SMC, PC) were exposed to ARV drugs (FTC, TFV, or FTC+TFV) or vehicle for 8 days. β-Galactosidase activity was quantitatively assayed. Error bars represent standard error of the mean (SEM).

1.2 1 EC

0.8

SMC PC

0.6

There might be an interaction effect of FTC and 0.4 TFV exposure. If the two drugs were not interacting with 0.2 each other, FTC+TFV exposure induced β-galactosidase activity would be expected to be comparable to that of 0 TFV exposure alone. The lowered β-galactosidase activNo drug FTC TFV FTC + TFV ity observed in FTC+TFV exposure suggests that FTC counteracted the Antiretroviral drug treatment TFV-induced increase in β-galactosidase activity. Among prior studies that explored the association between ARV drug exposure and premature aging, Nacarelli et al. (2016) exposed human lung and cardiac fibroblast cells to FTC+TFV combination for 7 days at conREFERENCES centrations within 10-20 μg/mL in a 2D cell system. They demonstrated 1. Al-Jabri, AA. How does HIV-1 infect a susceptible human cell?: Current that the drugs induced cellular senescence in agreement with our present thinking. Journal for scientific research. Medical sciences 5, 31-44 (2003). study. Nonetheless, our experiments might better reflect in vivo conditions 2. Cohen, J, Torres, C. HIV-associated cellular senescence: A contributor because clinically relevant concentrations of FTC and TFV were used and to accelerated aging. Ageing research reviews 36, 117-124, doi:10.1016/j. the 3D multi-layer co-culture system better mimicked the dynamic and arr.2016.12.004 (2017). versatile environment of human brain microvascular cells. 3. Cysique, LA, Brew, BJ. Vascular cognitive impairment and HIV-associated neurocognitive disorder: a new paradigm. Journal of NeuroVirology, The present study had limitations. The experimental setup was dedoi:10.1007/s13365-018-0706-5 (2019). signed to mimic the natural course of circulating ARV drugs in vivo in 4. Dimri GP, Lee X, Basile G, Acosta M, Scott G, Roskelley C, Medrano EE, the small vessel lumen, exposing the drug to EC first, then SMC, then PC. Linskens M, Rubelj I, Pereira-Smith O. A biomarker that identifies senescent However, due to structural limitations of the Alvetex inserts well plate, it human cells in culture and in aging skin in vivo. Proceedings of the National Academy of Sciences. 1995;92(20):9363–9367. doi:10.1073/pnas.92.20.9363 was not possible to distribute the drug in this order. Instead, all cell types 5. Hebert, VY, Crenshaw, BL, Romanoff, RL, Ekshyyan, VP, Dugas, TR. Effects were exposed to ARV drugs simultaneously. Secondly, the microvascular of HIV drug combinations on endothelin-1 and vascular cell proliferation. cells were co-cultured for a total duration of 8 days. It would be ideal to Cardiovascular toxicology 4, 117-131 (2004). observe cellular senescence over an extended period of time that would 6. Jiang, B, Hebert, VY, Zavecz, JH, Dugas, TR. Antiretrovirals induce direct result in significant changes in β-galactosidase activity. Based on our pilot endothelial dysfunction in vivo. Journal of acquired immune deficiency experiments, it was not possible to maintain primary human brain microsyndromes (1999) 42, 391-395, doi:10.1097/01.qai.0000228790.40235.0c (2006). vascular cells for longer than 8 days. Lastly, no HIV live particles or derived 7. Lefèvre C, Auclair M, Boccara F, Bastard J-P, Capeau J, Vigouroux C, CaronDebarle M. Premature Senescence of Vascular Cells Is Induced by HIV proteins were added into cell cultures. However, our experiments focused Protease Inhibitors. Arteriosclerosis, Thrombosis, and Vascular Biology. only on the cytotoxic effects of ARV drugs in the context of systemic viral 2010;30(12):2611–2620. doi:10.1161/atvbaha.110.213603. suppression in HIV-infected patients. 8. Lundgren JD, Babiker AG, Gordin F, Emery S, Grund B, Sharma S,

CONCLUSION

Using 3D multi-layer co-cultures of primary human brain microvascular cells, we found that TFV exposure induced cellular senescence, whereas there was no evidence suggesting that FTC exposure induced cellular senescence. To better reflect the clinical course of HIV infection and ART in humans, the cytotoxic effects of long-term exposure to TFV should be studied in animal models. Further studies can also explore the mechanistic pathways of TFV-induced cellular senescence in microvascular cells.

ACKNOWLEDGMENTS

Research reported in this publication was supported by the National Institute on Aging of the National Institutes of Health (NIH) under Award Number R56AG059437. The following reagents were obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAD, NIH: Emtricitabine [(-) FTC] and Tenofovir. 36

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Avihingsanon A, Cooper DA, Fätkenheuer G, Llibre JM, et al. Initiation of Antiretroviral Therapy in Early Asymptomatic HIV Infection. The New England Journal of Medicine. 2015 Aug 27 [accessed 2019 May 3]. https://www. ncbi.nlm.nih.gov/pubmed/26192873.

9. Nacarelli, T, Azar, A, Sell, C. Mitochondrial stress induces cellular senescence in an mTORC1-dependent manner. Free radical biology & medicine 95, 133-154, doi:10.1016/j.freeradbiomed.2016.03.008 (2016). 10. Panel on Antiretroviral Guidelines for Adults and Adolescents. Guidelines for the Use of Antiretroviral Agents in Adults and Adolescents Living with HIV. Department of Health and Human Services. Available at http://www.aidsinfo. nih.gov/ContentFiles/AdultandAdolescentGL.pdf. Accessed [January 2019]. 11. Soontornniyomkij V, Umlauf A, Soontornniyomkij B, Gouaux B, Ellis RJ, Levine AJ, Moore DJ, Letendre SL. Association of antiretroviral therapy with brain aging changes among HIV-infected adults. AIDS (London, England). 2018 Sep 10 [accessed 2019 May 3]. https://www.ncbi.nlm.nih.gov/ pubmed/29912063 12. Teeraananchai, S, Kerr, SJ, Amin, J, Ruxrungtham, K, Law, MG. Life expectancy of HIV-positive people after starting combination antiretroviral therapy: a metaanalysis. HIV medicine 18, 256-266, doi:10.1111/hiv.12421 (2017).

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REVIEW

THE GUT MICROBIOME: THE KEY TO UNLOCKING THE SECRET OF RHEUMATOID ARTHRITIS Chris Matthew Cyril

UC San Diego, Sixth College, Neurobiology, 2022

The etiology of rheumatoid arthritis (RA) is complex and not yet fully understood. However, researchers have identified changes in gut flora in RA patients not observed in healthy controls. The identification and characterization of the microorganisms involved could enhance efforts to better understand the prognosis of the disease.

AUTOIMMUNE DISEASE

Autoimmune diseases occur when the immune system attacks self molecules following a breakdown of immunologic tolerance to autoreactive immune cells (Smith and Germolec 1999). Autoimmune diseases affect 3% of the population in the Americas and Europe, and more than 75% of these autoimmune disease patients are women (Smith and Germolec 1999). While the exact reasons for increased susceptibility in women are unclear, increased estrogen and estrogen-like hormone production in females compared to males may are thought to might be involved (Smith and Germolec 1999). In experiments, male autoimmune mice are observed to have low testosterone levels, and testosterone administered to lupus-prone autoimmune mice had a strong suppressive effect on the disease (Smith and Germolec 1999).

RHEUMATOID ARTHRITIS, AND CURRENT TREATMENTS

Caused by a combination of genetic and environmental factors, rheumatoid arthritis (RA) is a systemic autoimmune disease that can lead to joint disability (Scher et al. 2013; Picchianti-Diamanti et al. 2018). Its etiology is unknown. The onset of the disease typically occurs during mid-adulthood (35–60 years) (Wilsdon and Hill 2017). Currently, treatment involves the use of Disease-Modifying Antirheumatic Drugs (DMARDs) to reduce chronic inflammation, with methotrexate being the most popular among such medications. However, DMARDs elevate the risk of infection by suppressing the immune system, and vaccines are recommended before beginning sqonline.ucsd.edu

therapy using DMARDs (Wilsdon and Hill 2017). Methotrexate serves as the backbone of treatment and is effective in 40% of patients when used alone (Wilsdon and Hill 2017). Treatment is found to be more effective when combined with other DMARDs, most commonly sulfasalazine, hydroxychloroquine, and prednisone (Wilsdon and Hill, 2017). Increased drug use, however, can result in many side effects such as liver and kidney damage as well as skin rashes, thus requiring patient monitoring (Wilsdon and Hill, 2017).

THE GUT MICROBIOME AND RHEUMATOID ARTHRITIS

The gut microbiome is composed of over 1000 bacterial species, archaea, fungi and other microorganisms (Ursell et al. 2014). Although its diversity remains mostly constant throughout an individual’s lifetime, its composition varies among individuals (Shreiner et al. 2016). The gut microbiome has co-evolved with its host over millennia and provides many benefits to its host including, but not limited to, digestion, production of nutrients, detoxification, protection against pathogens and regulation of the immune system (Wu and Wu 2012). An alteration in gut communities, dysbiosis, is involved in the onset and maintenance of autoimmune disease (Picchianti-Diamanti et al. 2018). Dysbiosis could lead to alterations in the epithelial cell layer; this causes increased exposure to antigen stimulators of the immune system, leading to T cell activation (Asquith et al. 2014; Ciccia et al. 2010). A study by Picchianti-Diamanti et al. compared the composition of the gut microbiomes of RA patients against those of healthy controls, using 16S sequencing to identify species. While the relative abundance of microbial phyla was nearly unchanged, some significant differences were also observed (Picchianti-Diamanti et al. 2018). The class Bacilli and the order Lactobacillales showed a fivefold and seventeen-fold increase in RA patients compared to the controls, respectively (2.89 ± 3.19 vs. 0.58 ± 0.39; p = 0.035 and 2.69 ± 3.15 vs. 0.15 ± 0.34; p = 0.021) (Picchianti-Diamanti et al. 2018). Lactobacillus is the largest genus of the lactic acid bacteria group, and it aids in the production of cheese and fermented milks (NIH). Lactobacillus Acidophilus produces lactic acid and is known to help the gut maintain natural microbial composition (NIH). Tumor Necrosis Factor alpha (TNF-α) is a cell signaling protein involved in systemic inflammation (Wang and Lin 2008). Etanercept (ETN) is one example of a TNF-α protein (Picchianti-Diamanti et al. 2018). In patients treated with ETN, an increase in cyanobacteria, which produce secondary metabolites with anti-inflammatory and immunosuppressant properties, was observed. (Picchianti-Diamanti et al. 2018).

FUTURE DIRECTIONS

In the study by Picchianti-Diamanti et al. mentioned previously, the researchers found that ETN treatment was effective in partially restoring the natural state of the gut microbiome. Not only did ETN treatment increase beneficial Cyanobacteria, it also decreased Clostridia, which was previously enriched in RA patients (Picchianti-Diamanti et al. 2018). Thus, drug therapy may alter gut microbiome composition. If so, ways to fully restore the gut microbiome must be investigated. Furthermore, future studies need to focus on obtaining more conclusive evidence confirming a reduction in symptoms, especially inflammation, due to the alteration of the composition of the gut microbiome. SALTMAN QUARTERLY

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B Unclassified Caldithrix Chloroflexi Thermotogae Fusobacteria Nitrospirae Euryarchaeota Synergistetes Cyanobacteria Actinobacteria Tenericutes Verrucomicrobia Proteobacteria Bacteroidetes Firmicutes

100 90 80 70 60 50 40

90 80 70 60 50 40

Healthy

Naive

ETN

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ETN + MTX

C 90 80 70 60 50 40 30 20 10 Healthy

Healthy

Naive

ETN

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Unclassified Synergistales Turicibacterales Nostocales Pasteurellales Erysipelotrichales Acholeplasmatales Desulfauromonadales Rhodospirillales Lactobacillales Aeromonadale Enterobacteriales Actinomycetales Sphingobacteriales Bifidobacteriales Desulfovibrionales Verrucomicrobiales Burkholderiales Natranaerobiales Coriobacteriales Flavobacteriales Bacteroidales Clostridiales

100

Unclassified Synergistia Nostocophycideae Erysipelotrichi Mollicutes Alphaproteobacteria Gammaproteobacteria Sphingobacteriia Actinobacteria Verrucomicrobiae Betaproteobacteria Deltaproteobacteria Bacilli Flavobacteriia Bacteroidia Clostridia

100

Naive

ETN

MTX

ETN + MTX

Figure 1. Microbiota composition of healthy controls and different treatment groups of RA patients at the phylum (A), class (B), and order (C) levels. Naive: naive to immunosuppressants. ETN: etanercept treatment. MTX: methotrexate. (Picchianti-Diamanti et al., 2018).

6. Shreiner, AB, Kao, JY, Young, VB. The gut microbiome in health and disease. Curr. Opin. Gastroenterol. 31(1). 2016.

REFERENCES 1. Smith, DA, Germolec, DR. Introduction to immunology and autoimmunity. Environ. Health Perspex. 107(Suppl 5). 1999.

7. Wu, HJ, Wu, E. The role of gut microbiota in immune homeostasis and autoimmunity. Gut Microbes. 3(1). 2012

2. Scher JU, Sczesnak A, Longman RS, Segata N, Ubeda C, Bielski C, Rostron T, Cerundolo V, Pamer EG, Abramson SB, et al. Expansion of intestinal Prevotella copri correlates with enhanced susceptibility to arthritis. eLife. 2013 Nov 5 [accessed 2019 May 3]. https://www.ncbi.nlm.nih.gov/ pubmed/24192039

8. Asquith M, Elewaut D, Lin P, Rosenbaum JT. The role of the gut and microbes in the pathogenesis of spondyloarthritis. Best Pract. Res. Clin. Rheumatol. 2014;28:687–702.

3. Picchianti-Diamanti, A, Panebianco, C, Salemi, S, Sorgi, ML, Di Rosa, R, Tropea, A, Sgrulletti, M, Salerno, G, Terracciano, F, D'Amelio, R, et al. 2018. Analysis of Gut Microbiota in Rheumatoid Arthritis Patients: Disease-Related Dysbiosis and Modifications Induced by Etanercept. Int. J. Mol. Sci.

9. Ciccia F, Bombardieri M, Rizzo A, Principato A, Giardina AR, Raiata F, Peralta S, Ferrante A, Drago S, Cottone M, et al. Over-expression of paneth cell-derived anti-microbial peptides in the gut of patients with ankylosing spondylitis and subclinical intestinal inflammation. Rheumatology. 2010;49:2076–2083.

4. Wilsdon, TD, Hill, CL. Managing the drug treatment of rheumatoid arthritis. Aust. Prescr. 40(2). 2017.

10. NIH. "Lactobacillus Acidophilus." NCBI. Accessed March 10, 2019. https:// www.ncbi.nlm.nih.gov/genome/?term=Lactobacillus%20acidophilus[Organism]&cmd=DetailsSearch.

5. Ursell LK, Haiser HJ, Van Treuren W, Garg N, Reddivari L, Vanamala J, Dorrestein PC, Turnbaugh PJ. Knight R. The intestinal metabolome: An intersection between microbiota and host. Gastroenterology. 2014;146:1470–1476

11. Wang X, Lin Y. Tumor necrosis factor and cancer, buddies or foes? Acta pharmacologica Sinica. 2008 Nov [accessed 2019 May 3]. https://www.ncbi. nlm.nih.gov/pubmed/18954521

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SOCIAL DYNAMICS

Painted dogs exhibit cooperative strategies in most aspects of pack functioning, but these behaviors may promote Allee effects that threaten small populations. As obligate cooperative breeders, painted dogs are a species that rely on the entire group to ensure pup survival. Pups unable to leave the den are especially vulnerable; therefore, a pup guard remains behind while others are hunting (Courchamp et sqonline.ucsd.edu

1.0 0.6

0. 8

Pre-lion era Lion era

0. 4

The African painted dog, Lycaon pictus, is experiencing consistent decline despite reduced human impacts in recent years. Evidence suggests that this decline is attributed to their social dynamics as an obligate cooperative breeder, in which the dominant breeding pair receive care from non-breeding members of the pack, making the species more vulnerable to Allee effects (Forssman et al. 2018; Courchamp et al. 2000). An Allee effect is defined as a positive relationship between individual fitness and the population density of other individuals of the same species (Buettner et al. 2006). Painted dogs rely on other group members for survival, and smaller packs are more at risk for lower fitness, which potentially strengthens Allee effects (Groom et al. 2016). In addition to smaller packs being at risk, their group dynamic prevents them from establishing larger packs due to energetic demands (Creel and Creel 2015). This review paper analyzes research on the connections between the social aspects of painted dogs and the linkage between these dynamics and their susceptibility to Allee effects on different levels. Studies have shown that the cooperative nature of painted dogs places an upper limit on pack sizes, preventing them from establishing large populations that would alleviate the detrimental Allee effects associated with small pack sizes.

0.2

INTRODUCTION

Wild dog pup survival probability

Charlotte Bruggeman

UC San Diego, Revelle College, Ecology, Behavior & Evolution, 2020

al. 2002). However, this results in a trade-off between pup survival and hunting compensation; the guard does not contribute to hunts, which results in other members expending more energy to compensate (Courchamp et al. 2002). This is especially detrimental in small packs, which cannot afford to leave behind a pup guard, resulting in increased pup mortality (Courchamp et al. 2002). Because of their short breeding seasons, painted dogs only have one recruitment period per year, and high pup mortality reduces the overall fitness of the group (Groom et al. 2016). Pups are also extremely vulnerable to predators, with lions being the major cause of pup mortality (Figure 1) (Groom et al. 2016). The increased mortality by large carnivores has been observed to lead to fewer dispersers, strengthening the mate-finding Allee effectâ&#x20AC;&#x201D;the increased difficulty of an individual finding a mate at lower

SOCIAL PACK DYNAMICS OF THE AFRICAN PAINTED DOG, LYCAON PICTUS, PROMOTE ALLEE EFFECTS AND POPULATION DECLINE

Age of pups (months)

Figure 1. Survival probability for first-year pups: Survival probability of painted dogs from birth to 12 months in the presence and absence of lions. Both scenarios show an increase in pup mortality at 2-3 months when pups first leave the den. In the presence of lions, there is a significant decrease in the survival probability after this time compared to the survival in the absence of lions (Groom et al. 2016).

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Number of populations

Figure 2. Pack distribution model: The distribution modeled for 1000 populations, which consist of nine packs that are joined together by dispersal, for the number of packs over 100 years under IDD (shaded) and density dependence (unshaded). There are few populations consisting of a small number of packs (approximately 5) compared to greater numbers or extinction, indicating the greater extinction probability for small packs, as is observed under IDD conditions (Courchamp et al. 2000).

Number of packs per population densities (Groom et al. 2016). In theory, the strength of these Allee effects could be reduced if pack sizes were larger, but the cooperative nature of painted dogs places an upper limit on pack size (Creel and Creel 2015). A larger pack produces larger litters, which decreases the extinction risk due to predation and reduces the stress on dominant individuals for pup provisioning via regurgitation (Vucetich and Creel 1999; Forssman et al, 2018). However, larger packs expend more energy on hunting to feed all members of the pack and increased size is not necessarily more effective in defending against kleptoparasites, species that steal their kills (Creel and Creel 1995; Carbone et al. 2005). Carbone et al. (2005) found that a hunting group of 2-6 dogs performs best as this number provides a balance between kill defense and sharing costs. Furthermore, while a larger pack increases pup survival, it decreases adult survival due to pups being given first access to kills, therefore compromising the survival of adults (Creel and Creel 2015). This hypothesized upper limit on pack size forces painted dogs to remain in smaller populations, putting them at risk for Allee effects and increasing their chance of decline and extinction.

ALLEE EFFECTS

Painted dogs exhibit inverse density dependence (IDD), in which their population growth rate is constrained at higher population densities and Allee effects increase the risk of extinction for a group or population when the numbers of the group or population drop beneath a certain threshold (Courchamp et al. 2000; Sanderson et al. 2014). Models show that IDD increases pack ex40

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tinction and failure to colonize new patches of territory by dispersers, producing a population comprised of fewer, smaller packs. This increases the chance of group extinction and potentially population extinction (Figure 2) (Courchamp et al. 2000). However, contradicting studies discuss the presence of Allee effects at the population level. More recent research concludes that demographic Allee effects, which occur when the performance of the population is dependent on the performance of individual groups, are absent and that pack size has no effect on population growth (Somers et al. 2008; Angula et al. 2013). Rather a group Allee effect was observed, which occurs when the group performance depends only on group size independent of population size, thus establishing the importance of group size in pack dynamics (Figure 3) (Angulo et al. 2013). This observation has led to the hypothesis that Allee effects at the group and individual level will not lead to population extinction. A study observed an 80% group extinction rate, but continued population persistence (Angulo et al. 2013). One proposed explanation for the absence of demographic Allee effects is that independent large and small packs buffer one another, with the large groups compensating for the decline of the smaller ones (Angulo et al. 2013). While it has been hypothesized that there is a critical pack size under which a group Allee effect will generate a demographic Allee effect, no definite size has been determined (Courchamp et al. 2000; Buettner et al. 2006). However, it is likely that critical size would depend on additional factors, such as geographical area and carnivore community structure (Buettner et al. 2006). Strong kleptoparasitism could force packs to maintain an optimum pack size in order to maximize energy, sqonline.ucsd.edu

REVIEW

Figure 3. per capita productivity and pack size. The relationship between per capita productivity and annual pack size, demonstrating the connection between individual fitness and pack size. Smaller packs had lower individual productivity than medium sized packs (Angulo et al. 2013).

which would make the minimum pack size more critical (Buettner et al. 2006). Further, painted dogs choose geographic ranges with low lion and kleptoparasite densities, which may lower the demand for helpers and increase juvenile survival (Buettner et al. 2006). However, if the habitat is largely homogenous, packs have little choice and a minimum pack size may play a larger role in persistence in the presence of lions and kleptoparasites (Buettner et al. 2006).

CONCLUSION

Studies into the different scales of Allee effects in painted dogs in conjunction with aspects of the social dynamics have demonstrated the increased risk faced by this species. Due to their cooperative dynamics, painted dogs are unable to form large packs because excessive numbers have detrimental effects on their fitness. With continued human impacts, there is a greater chance of localized extinctions and declining pack numbers, reducing the viability of the entire population. A potential direction for future research is focus on the painted dog metapopulation and additional factors that may exacerbate Allee effects. It is important to consider in order to determine management plans that can ensure the existence of the painted dog in the future.

affecting juvenile survival in African wild dogs (Lycaon pictus) in Kruger National Park, South Africa. Journal of Zoology. 272:10-19. 3. Carbone, C, Frame, L, Frame, G, Malcolm, J, Fanshawe, J, FitzGibbon, C, Schaller, G, Gordon, IJ, Rowcliffe, JM, Du Toit, JT. 2005. Feeding success of African wild dogs (Lycaon pictus) in the Serengeti: the effects of group size and kleptoparasitism. Journal of Zoology. 266:153-161. 4. Courchamp, F, Clutton-Brock, T, Grenfell, B. 2000. Multipack dynamic and the Allee effect in the African wild dog, Lycaon pictus. Animal Conservation. 3:277-285. 5. Courchamp, F, Rasmussen, GSA, Macdonald, DW. 2002. Small pack size imposes a trade-off between hunting and pup-guarding in the painted hunting dog, Lycaon pictus. Behavioral Ecology. 13(1):20-27. 6. Creel, S, Creel, NM. 1995. Communal hunting and pack size in African wild dogs, Lycaon pictus. Animal Behavior. 50:1325-1339. 7. Creel, S, Creel, NM. 2015. Opposing effects of group size on reproduction and survival in African wild dogs. Behavioral Ecology. 26(5):1414-1422. 8. Forssman, KR, Marneweck, C, Oâ&#x20AC;&#x2122;Rian, J, Davies-Mostert, HT, Mills, MGL. 2018. Pup provisioning in the cooperatively breeding African wild dog, Lycaon pictus, is driven by pack size, social status, and age. African Journal of Wildlife Research. 48(1):1-10. 9. Groom, RJ, Lannas, K, Jackson, CR. 2016. The impact of lions on the demography and ecology of endangered African wild dogs. Animal Conservation. 20:382-390. 10. Sanderson, CE, Jobbins, SE, Alexander, KA. 2014. With Allee effects, life for the social carnivore is complicated. Population Ecology. 56(2):417-425.

REFERENCES 1. Angulo, E, Rasmussen, GSA, Macdonald, DW, Courchamp, F. 2013. Do social groups prevent Allee effect related extinctions?: The case of wild dogs. Frontiers in Zoology. 10:-13. 2. Buettner, UK, Davies-Mostert, HT, du Toit, JT, Mills, MGL. 2006. Factors

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11. Somers, MJ, Graf, JA, Szykman, M, Slotow, R, Gusset, M. 2008. Dynamics of small re-introduced population of wild dogs over 25 years: Allee effects and the implications of sociality for endangered speciesâ&#x20AC;&#x2122; recovery. Oecologia. 158:239-247. 12. Vucetich, JA, Creel, S. 1999. Ecological interactions, social organization, and extinction risk in African wild dogs. Conservation Biology. 13(5):1172-1182.

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SENIOR HONORS

THESES

An underwater perspective of Amphiprion percula's (clown fish) preferred habitat of choice, sea anemone. As climate changes drastically over time, this setting of loneliness becomes the ordinary. Photo by Mark Allan Jacob

BISP 196 is open to undergraduate biology majors who have an overall major GPA of 3.6 or higher, have senior standing, and commit to three consecutive quarters of research during their senior year. The program aims to increase faculty-student interactions and encourage more students studying biology to pursue independent research. During the spring quarter, students in the program participate in a research showcase to discuss their research with faculty and fellow students. These are the abstracts of all the exceptional research projects conducted by the undergraduates during the 2018-2019 academic year. 42

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THESES

LAUREN BRUMAGE

John Muir College Microbiology & Biological Anthropology Double Major

PI: Joe Pogliano, Ph.D., UC San Diego, Division of Biological Sciences

Characterizing the Antibiotic Properties of a Natural Product Library

It is imperative to find and characterize novel antibiotics in order to combat antibiotic resistance. However, in recent years, few new and clinically useful antibiotics have been discovered. In this project, I characterized the antibacterial properties of a library of natural products. I evaluated the minimum inhibitory concentration of the compounds against a panel of Gram-positive and Gram-negative bacteria to determine the spectrum of activity and potency. To elucidate the mechanism of action, I used bacterial cytological profiling, a fluorescent microscopy technique that allows visualization of the physiological response of bacteria to a given treatment. Results indicate that many of the compounds have a potency comparable to currently used antibiotics. Each of the major biosynthetic pathways is inhibited by at least one compound in the library. Some of the compounds could be clinically relevant, but further work is needed to determine the precise target of these compounds.

Sixth College Physiology and Neuroscience Major Psychology Minor

THERESA BUI

PI: Emily Troemel, Ph.D., UC San Diego, Division of Biological Sciences, Section of Cell and Developmental Biology

Nanoluciferase as a Genetic Reporter for Measuring Activation of the Intracellular Pathogen Response Pathway

The Intracellular Pathogen Response (IPR) is a newly identified transcriptional response in the nematode C. elegans that helps in tolerating proteotoxic stress, including intracellular infection. The gene pals-5 is one of the most highly induced IPR genes, making it an effective IPR reporter when its promoter is coupled to the highly sensitive bioluminescent reporter protein Nanoluciferase. Here, we used transgenic animals that express Nanoluciferase under the control of the pals-5 promoter to test triggers of the IPR, including those that perturb proteostasis and other core cellular processes. These studies should provide new insights into how animals cope with proteotoxic stress.

SELINA JUJU CHANG

PI: Miguel A. Lopez-Ramirez, Ph.D., UC San Diego School of Medicine, Division of Rheumatology, Allergy & Immunology

Earl Warren College Physiology & Neuroscience Major Business Minor

Investigating Small Molecule-Induced Blood-Flow Gene Expression Pattern in Endothelial Cells

Endothelial cells (ECs) line the interior of the entire circulatory system, and EC dysfunction is commonly associated with the development of vascular diseases. In the Lopez-Ramirez Lab, previous work has demonstrated that KRIT1 (Krev interaction trapped protein 1) and transmembrane protein HEG1 (Heart of Glass) directly interact with each other to downregulate the gene expression of transcription factors KLF2 and KLF4 (Kruppel-like factors), which are implicated in blood-flow induced gene regulation. Based on this information, we hypothesized that HKi001G, a HEG1-KRIT1 protein interaction blocker, would upregulate the gene expression of KLF2 and KLF4 when applied to ECs, thereby mimicking blood-flow gene expression with vasoprotective activities. During this experiment, we treated brain endothelial cell-line hCMEC/D3 with HKi001G at different concentrations and durations to investigate its efficacy. Our results indicate that both increasing HKi001G concentrations and durations lead to upregulation of KLF2 and KLF4 expression.

Eleanor Roosevelt College Biochemistry and Cell Biology Major Economics Minor

RUIYUAN ZHANG

PI: Nunzio Bottini, M.D., Ph.D., UC San Diego School of Medicine, Department of Rheumatology, Allergy and Immunology

Role of PTP4A1 in Systemic Sclerosis

Previous studies in our lab have shown that the expression of Protein Tyrosine Phosphatase IVA I (PTP4A1) is elevated in dermal fibroblast of patients with systemic sclerosis–a fibrosing autoimmune disease–correlating with stimulation of profibrotic genes. PTP4A1 promotes fibrosis via enhanced activation of the kinase SRC, its downstream RAS-ERK pathway and the transcription factor SMAD3 in fibroblasts. We are interested in understanding a key physical interaction between PTP4A1 and the SRC kinase at the molecular level. Co-precipitation successfully demonstrated direct binding between PTP4A1 and SRC. We have purified sufficient amounts of SRC and PTP4A1 proteins in active form and are in the process of utilizing a multidisciplinary approach to assess how different variables–including the phosphorylation status of SRC, the oligomerization state of PTP4A1 and the contribution of different domains of SRC–affect the interaction. This knowledge can potentially help in the search of novel treatments for systemic sclerosis.

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YOGITHA CHAREDDY

PI: Pradipta Ghosh, M.D., UC San Diego School of Medicine, Departments of Medicine/Cellular and Molecular Medicine

Earl Warren College Molecular Biology Major Business Minor

Using CRISPR/Cas9 Macrophage Cell Lines to Test GIVâ&#x20AC;&#x2122;s Function in Regulating Inflammation

Chronic inflammation is a key component of many diseases, including inflammatory bowel disease and cancer. Despite the importance of inflammation as a protective immune process, dysregulation in cellular signaling cascades can lead to long-term inflammation, which damages tissue and causes disease. My mentor's lab has recently identified the non-receptor guanine nucleotide exchange factor GIV/GIRDIN as a novel pivot point between pro-/anti-inflammatory signals and a possible target for therapeutics. To characterize GIV downstream of Toll-like receptor 4, I used a macrophage cell model with knockdown of GIV via short hairpin RNA and stimulated with lipopolysaccharide while attempting to rescue with different GIV constructs. Once successful, I will repeat these experiments in a macrophage model with knockout of GIV via CRISPR/ Cas9. This system will allow me to interrogate the precise mechanism by which GIV regulates the pro-/anti-inflammatory switch in macrophages and provide valuable insight into the development of a therapeutic target.

ARIANA CHIRBAN

Roger Revelle College Human Biology Major

PI: Kamil Godula, Ph.D., UC San Diego, Assistant Professor of Chemistry and Biochemistry

Development of a Human 6-O- Endosulfatase Fluorogenic Probe

Human 6-O-Endosulfatases, HSulf-1 and HSulf-2, uniquely modify the extracellular matrix through hydrolysis of 6-O sulfates on heparin sulfate proteoglycans. The study of HSPGs has become increasingly significant in the context of healthy and disease cellular states. Upregulation of Hsulf-2 has been implicated in several metastatic cancers through extracellular signaling processes that promote angiogenesis. Despite HSulfs serving in these significant roles, there has been little advancement in characterizing the hydrophilic domain of HSulfs. Previous models have used general sulfatase probes, for example, the fluorogenic substrate, 4-methylumbelliferyl sulfate (4-MUS) to detect arylsulfatases. This probe, however, lacks the specificity necessary to understand how endosulfatases recognize regions of heparin sulfate. In this study, a fluorogenic probe for endosulfatases has been modified to include various linkers and glycans that can be processed by the Hsulfs. Using MCF-7 breast cancer cells that endogenously over-express HSulf2, Michaelis-Menten kinetics will be used to determine the optimal binding substrate for Hsulfs through affinity assays. In the future, this probe may serve as a tool to image HSulf activity in healthy and diseased models to further delineate the diseased state and its role in promoting cancerous phenotypes.

BENYAMIN DADPEY

PI: Shannon M. Reilly, Ph.D., UC San Diego School of Medicine, Department of Endocrinology

Roger Revelle College Biochemistry and Cellular Biology Major

STAT3 Interaction With GPAT3 Suppresses Fatty Acid Re-esterification in Adipocytes

The sympathetic nervous system promotes the mobilization of energy-rich triglycerides from adipocytes through a process called lipolysis which results in the secretion of free fatty acids (FFA). Adipocytes reuptake some of the secreted FFA and either oxidize them for energy or re-esterify them for storage as triglycerides. The Reilly lab has found that lipolysis driven oxidative metabolism requires suppression of re-esterification by STAT3. As a consequence of this, adipocyte-specific Stat3 KO mice were found to be more sensitive to diet-induced obesity. I discovered that STAT3 interacts specifically with Glycerol-3-phosphate acetyltransferases-3 (GPAT3), which catalyzes the first step of FFA re-esterification. STAT3 is activated by phosphorylation at tyrosine 705, so I investigated the role of this residue on STAT3â&#x20AC;&#x2122;s interaction with GPAT3, and found that the Y705F STAT3 mutant does not interact with GPAT3. Therefore, the interaction of STAT3 with GPAT3 may be regulated by STAT3 phosphorylation.

Roger Revelle College Biochemistry and Cellular Biology Major Computer Science Minor

JOHN PAUL DAVIS PI: Joe Pogliano, Ph.D., UC San Diego, Division of Biological Sciences, Section of Molecular Biology

Identifying Antibiotics and Determining Their Mechanism of Action

Despite their initial effectiveness, we have nearly exhausted the capabilities of extant antibiotic treatments due to increasing numbers of resistant bacterial strains. If we wish to continue employing antibiotic treatments, it is essential that new antibiotics are identified and that their mechanism of action is determined. Bacterial Cytological Profiling is a fluorescence microscopy technique used to screen potential antibiotics by characterizing their effect on cellular pathways. Using this technique, two small molecules with low minimum inhibitory concentrations have been identified as DNA replication inhibitors and lipid biosynthesis inhibitors. Resistant mutants against both compounds have been isolated, and Next Generation Sequencing revealed that these mutations lie in yafC, which encodes a DNA binding transcriptional regulator, and in several genes relating to lipid biosynthesis. This project aims to investigate how these compounds are interacting with the proteins encoded by these effected genes, and how these interactions inhibit the growth of bacteria.

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THESES

ZHUORAN DING

Earl Warren College Bioinformatics Major

PI: Julian Schroeder, Ph.D., UC San Diego, Division of Biological Sciences, Cell and Developmental Biology

Identify Potential Genomic Variants Causing Stomatal Insensitivity in Response to Elevated CO2 level in Brachypodium distachyon

In this study, I use Unix-based software programs to identify potential genomic variants that lead to stomatal insensitivity in the grass species Brachypodium distachyon in response to elevated CO2 level. I built a genomic variant identification pipeline. The pipeline uses tools in the Genome Analysis Toolkit (GATK) to identify variants in whole-genome sequencing data of Ethyl-methanesulfonate (EMS) mutant lines that show a mutant phenotype and uses SnpEff to annotate identified variants with gene-identifier names. After filtering-out common false positive variants, by running the pipeline on six EMS mutant lines with other phenotypes, I discovered 164 and 143 potential mutations causing variants in two mutant lines with promising phenotypes. I also collected a set of common false-positive variants and regions with rich false positive variants, using eight independent genome sequences of the parent line. Additionally, I built an Arabidopsis transcriptome expression database to facilitate further studies on stomatal biology and genetics.

Roger Revelle College Physiology and Neuroscience Major Humanities Minor

BENJAMIN FINANDER PI: Satchidananda Panda, Ph.D., Salk Institute of Biological Studies, Regulatory Biology

Intra-retinal Circuit of Intrinsically Photosensitive Retinal Ganglion Cells in the Mouse Retina

Using a genetic mouse line (Opn4/Cre Knock-in mice), serial blockface electron microscopy, and a viral vector (MiniSOG), we have reconstructed the circuit structure of Intrinsically Photosensitive Retinal Ganglion Cells, or ipRGCs, within sections of the mouse retina. By studying the connections and cellular structure of ipRGCs, we increase our understanding of how ipRGCs may mediate non-image forming vision functions such as circadian photoentrainment and pupil constriction. Specifically, we studied the frequency of synaptic input along the dendrites of ipRGCs and the sources of these synaptic inputs. We report that several types of bipolar cells and amacrine cells synapse onto ipRGC dendrites with varying frequency depending on the location of the dendrite in the retina. We also investigated the localization patterns of different neural processes within the retinal volume and discovered specific patterns for ipRGC axons in the ganglion cell layer of the retina.

RAIMA GHOSH

John Muir College Human Biology & Developmental Psychology Double Major

PI: Michael Croft, Ph.D., and Chris Benedict, Ph.D., La Jolla Institute for Immunology, Department of Immune Regulation

Profiling the Unique CD4 T Cell Population Responsible for Resolving Persistent Cytomegalovirus Infection

Prior research has shown CD4 T cells are critical for resolving persistent cytomegalovirus (CMV) infection. CD4 T cells recognizing an epitope derived from the CMV m09 protein, which expand at later times compared to canonical CD4T cells, are particularly important for resolving CMV replication in the salivary gland and driving the virus into latency. These â&#x20AC;&#x2DC;late-risingâ&#x20AC;&#x2122; m09 cells display a unique phenotype, and show robust protection when induced by vaccination. Hence, we sought to compare m09-specific CD4 T cells to a canonical CD4 T cell specific for a m25derived viral epitope, using data derived from bulk-cell RNA sequencing of these two T cell populations. Our new results show that these two subsets of CD4 T cells show unique gene expression profiles that vary by time of infection and organ of isolation.

Thurgood Marshall College Physiology and Neuroscience Major

VIDIT GOYAL

PI: Matthew Banghart, Ph.D., UC San Diego, Division of Biological Sciences, Section of Neurobiology

Assembly of a Nanobody-Based Fluorescent Sensor for the Neuropeptide Substance P

The neuropeptide substance P (SP) is secreted by nerves and inflammatory cells and acts by binding neurokinin receptors. Because it has high affinity for its receptors, only low concentrations accumulate in extracellular fluids upon secretion. Thus, determining SP release sites and the timescale of signaling is very challenging. We therefore set out to develop a genetically-encoded fluorescent sensor of SP. Our design is based on a SP-binding nanobody coupled through circularly-permuated GFP (cpGFP) to a low-affinity tethered SP variant (SPv), such that fluorescence is enhanced when endogenous SP displaces SPv. The sensor scaffold and a control construct lacking SPv were constructed using a combination of Gibson assembly and restriction enzyme-based cloning. The resulting constructs were then expressed in E. coli, purified, and analyzed by fluorescence spectroscopy.

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BRENDA HUG

Roger Revelle College Physiology and Neuroscience Major Cognitive Science Minor

PI: Shauna Yuan, M.D., UC San Diego School of Medicine, Department of Neuroscience

Effect of Gamma-Secretase Modulator on the Level of Autophagy Activation in Cells with APP and Presenilin Mutations

Amyloid beta (Abeta) plaque formation, which is a hallmark of Alzheimer’s disease (AD), can be caused by autophagy and endosome dysregulation due to inefficient cleavage of amyloid precursor protein (APP) by gamma-secretase. This APP processing maintains the normal level of the endosomal and autophagic activity in cells with familial AD mutations, such as APP and presenilin (PS-1 and PS-2) mutations. Gamma-secretase modulator (GSM) allosterically increases the efficiency of APP processing by reducing the production of amyloidogenic Abeta peptides. We hypothesize that GSM treatment results in decreased endosomal and autophagic activity in familial AD neurons. In this study, neurons with APP, PS- 1, and PS-2 mutations were treated with GSM. The endosome markers, Rab5 and Lamp2 were analyzed by immunofluorescent imaging and biochemistry. The autophagy markers, LC3 and P62 were evaluated by western blot. We predict that autophagic and endosomal activity will restore to normal function with GSM treatment.

TIANA HUYNH

Roger Revelle College Biochemistry and Cell Biology Major

PI: Ju Chen, Ph.D., UC San Diego School of Medicine, Department of Medicine/Cardiology

The Role of NEDD4 Protein in Cardiac Function and Disease

Neuronal precursor cell-expressed developmentally downregulated 4 (NEDD4) is a member of the NEDD4 family of HECT E3 ubiquitin ligases. NEDD4 is highly expressed in cardiomyocytes. However, the specific role of NEDD4 in cardiomyocytes is unknown. To study the role of NEDD4 adult cardiomyocytes, we have generated a Nedd4 flox mouse model and crossed it with tamoxifen-inducible MHC-MerCreMer mice to generate an inducible Nedd4 cardiomyocyte-specific knockout mouse model (Nedd4 icKO). In this study, we performed comprehensive molecular and cardiac physiology studies on NEDD4 icKO mice. Our results show that NEDD4 icKO mice displayed cardiomyopathy after tamoxifen-induced gene deletion. Utilizing the NEDD4 icKO mice model, we will be able to gain significant insight into mechanisms by which loss of NEDD4 in adult cardiomyocytes results in cardiomyopathy. Ultimately, this will bring us closer to our long-term goal of identifying new therapeutic targets for human cardiomyopathy.

AHMED KHALIL

PI: Eric A. Schmelz, Ph.D., UC San Diego, Division of Biological Sciences, Cell and Developmental Biology

Eleanor Roosevelt College Physiology and Neuroscience Major

Cross Leveraging Maize and Sorghum to Understand Biochemical Layers of Immunity

In planning for increased production to match the increasing world population, large-scale and long-term maize (Zea mays) and sorghum (Sorghum bicolor) monocultures will predictably experience increased biotic and abiotic stresses. With a recent evolutionary divergence, these two important crops are among the worlds most closely related and are certain to share specialized metabolic pathways that play central roles in stress resilience. To elucidate conserved and unique pathways, we performed identical fungal-elicitation treatments on plant tissues of similar developmental stages and performed GC/MS and LC/MS-based metabolomics. Maize contains a diverse array of inducible oxygenated terpenoids while few have been characterized in sorghum. A candidate sesquiterpenoid present in both species was pursued using numerous analytical and informatic approaches yet yielded an alternative identification. Current efforts are focused on the regulation of sesquiterpenoids in both plants and the identification of Cytochrome P450 Cyp81-derived products in the core maize zealexin pathway underlying pathogen resistance.

NABTA LAZ

Eleanor Roosevelt College General Biology Major Political Science Minor

PI: Mark H. Tuszynski, M.D., Ph.D., UC San Diego School of Medicine, Department of Neuroscience

Enhancing Specificity of Intrathecal Gene Therapy for Alzheimer’s Disease

Numerous preclinical gene therapies for Alzheimer’s disease have been developed including proteolytic enzymes, anti-inflammatory genes, anti-tau or anti-amyloid antibodies, and growth factors such as brain-derived neurotrophic factor (BDNF). Alzheimer’s disease ultimately affects the entire cerebral cortex but widespread cortical gene delivery by direct brain injection is not possible. Intrathecal and intravenous infusion of Adeno-Associated Virus 9 (AAV9) are promising methods to drive widespread gene transfer to the cortex but off-target gene transfer may cause adverse side effects. We tested multiple strategies to target gene expression to cortical neurons including cell-specific promoters and microRNA binding sites. We found that the cortex-specific CTX-1 promoter successfully restricted gene expression to cortical neurons. Intrathecal or intravenous infusion of AAV9 carrying this promoter could achieve widespread and specific gene therapy of the cortex through a single and minimally invasive injection.

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THESES

AMY LEE

Thurgood Marshall College Biochemistry and Cell Biology Major

PI: Abraham A. Palmer, Ph.D., UC San Diego School of Medicine, Department of Psychiatry

Glyoxalase 1 Inhibition is a Potential Novel Therapeutic Target in Chronic Ethanol Withdrawal

The enzyme glyoxalase 1 (GLO1) metabolizes methylglyoxal, a competitive partial agonist at GABAA receptors. GLO1 inhibition has been linked to lower anxiety, depression, and seizure susceptibility in mice. We examined whether GLO1 inhibition can also alleviate negative changes seen during chronic ethanol withdrawal. Mice were given a chronic ethanol treatment to induce dependence; a separate control group received no ethanol treatment. During withdrawal, mice were given GLO1 inhibitor or vehicle injections and tested for handling-induced convulsions, anxiety- and depression- like behavior. Inhibitor-treated mice exhibited a trend towards lower seizure severity than vehicle mice (p=0.082), and vehicle-treated mice showed greater seizure severity than control mice (p<0.001). Inhibitor-treated mice did not differ from the control group in anxiety-like behavior, while vehicle treated mice showed significantly higher anxiety-like behavior than control mice. These data suggest that GLO1 inhibition may have therapeutic potential for treating some physiological (seizures) and affective (anxiety) changes of ethanol withdrawal.

HEI YIU LEUNG

Earl Warren College Molecular Biology Major Cognitive Science Minor

PI: Dong Wang, Ph.D., UC San Diego Skaggs School of Pharmacy and Pharmaceutical Sciences

Analysis of Biochemical Activity of Rhp26 Mutations Involved in Cockayne Syndrome

Cockayne syndrome (CS), a fatal autosomal recessive neurodegenerative disorder, is commonly associated with mutations in Cockayne Syndrome group B protein (CSB). CSB is an important protein involved in transcription-coupled DNA repair. Specific CSB mutations have been identified in CS patients, but how these mutations affect the biochemical activities of CSB remains unknown. To address this question, we compiled CSB mutations observed in CS patients from clinical reports and generated these mutations in Rhp26, the Schizosaccharomyces pombe homolog of CSB. Biochemical assays will be run to test their chromatin remodeling activity and ATPase activity. Understanding how these Rhp26 mutants function biochemically can lead to a better understanding of transcription-coupled DNA repair and the pathology of Cockayne Syndrome.

FABIAN LIM

Roger Revelle College Molecular Biology Major

PI: Emma Farley, Ph.D., UC San Diego, Division of Biological Sciences, Section of Molecular Biology and School of Health Sciences, Department of Medicine

Highly Conserved Enhancer Grammar

Enhancers are elements within the genome that act as switches to control the location and timing of gene expression. Thus, enhancers are fundamental for successful development. The functional features of an enhancer are “binding sites”, sequences that recruit specific transcription factors to allow activation of gene expression. The order, orientation, and spacing of these sites — collectively known as “enhancer grammar” — is thought to be important for the activity of an enhancer. We bioinformatically identified an enhancer for the gene Hes1 that has conserved its grammar for over 500 million years across all deuterostomes and some protostomes. We tested this enhancer from 4 protostomes, including scorpion, and 6 deuterostomes for activity in Ciona intestinalis embryos. The conserved enhancer grammar encodes a conserved expression pattern in the developing nervous system. Further studies of this rare conservation could uncover important principles governing enhancer grammar and help identify how grammar confers fitness during evolution.

Roger Revelle College Physiology and Neuroscience Major Psychology Minor

NIROSH MATARAARACHCHI PI: Farah Sheikh, Ph.D., UC San Diego School of Medicine, Department of Medicine

A Novel Platform to Uncover Pro-Arrhythmogenic Pathways Driving Arrhythmogenic Right Ventricular Cardiomyopathy

Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC) is a genetic cardiac disease that causes arrhythmias and sudden death in young athletes, often triggered by exercise. Mutations within genes of the desmosome, a mechanical cell-cell anchoring junction, account for 40% of ARVC cases. Plakophilin-2 (PKP2) is the most mutated desmosomal gene; however, there are limited insights on mechanisms driving ARVC due to PKP2 mutations. We generated a novel PKP2 mutant mouse that recapitulates the adult disease spectrum of ARVC. We hypothesize that neonates represent a pre-disease stage to mine for early pro-arrhythmogenic pathways underlying ARVC at baseline and adrenergic stress (also triggered by exercise). We show that neonatal PKP2 mutant cardiomyocytes display loss of desmosomal proteins (desmoplakin, desmoglein-2) and the calcium regulator, SERCA2. These defects may underlie the adrenergic stress-induced arrhythmias that we also observed in neonatal PKP2 mutant cardiomyocytes. We provide a novel model to uncover mechanisms driving the early electrical dysfunction in ARVC.

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CESAR NAVA GONZALES PI: Chih-Ying Su, Ph.D., UC San Diego, Division of Biological Sciences, Section of Neurobiology

Roger Revelle College Physiology and Neuroscience Major

Morphometric Analysis of Compartmentalized Olfactory Receptor Neurons in Drosophila

In Drosophila, ephaptic interaction, or direct electrical interaction, mediates lateral inhibition between olfactory receptor neurons (ORNs) housed in the same sensillum. Interestingly, the strength of such lateral inhibition is asymmetric in that the larger spike A neuron can exert greater ephaptic influence onto the smaller spike B neuron. Mechanistically, this functional asymmetry arises from differences in the passive electrotonic properties of neurons, such as input resistance. This leads us to hypothesize that compartmentalized ORNs are of different sizes. The goal of my research is to systematically characterize the morphometric features of grouped ORNs in different sensillum types. I will perform computer-based segmentation of images acquired by serial block-face electron microscopy. Based on the 3D models, I will subsequently measure the size, length and surface area of grouped ORNs. My study is expected to yield critical information for us to understand how neuronal size influences the strength of ephaptic interaction.

John Muir College Physiology and Neuroscience Major Psychology Minor

NOELLE YING

PI: Michael J. McCarthy, M.D., Ph.D., UC San Diego School of Medicine,Department of Psychiatry

Modelling Neuronal Circadian Rhythms in Bipolar Disorder Using Human Induced Pluripotent Stem Cells

Bipolar Disorder (BD) is a common psychiatric illness characterized by manic and depressive mood episodes and profound disturbances in circadian rhythms, manifesting in irregular sleep and activity cycles. The molecular mechanisms underlying BD and the associated rhythm disturbances remain mostly unknown. We differentiated pluripotent stem cells (iPSC) from BD patients and healthy controls into neural progenitor cells (NPCs) and neurons. We then recorded cellular circadian rhythms using Per2-luc, a bioluminescent reporter of circadian clock activity. Our preliminary data indicate that circadian rhythms in NPCs and neurons are weaker and damp faster in cells from BD patients vs. controls. Single cell Per2-luc imaging validated this result in NPCs. Ongoing work is investigating the ways in which neuronal circadian rhythms in BD vary, and whether pharmacological intervention can alter the BD-associated reduction in rhythm. The results of this work may inform future efforts to develop new drug treatments for BD.

KYLIE PAUL

PI: Leslie Crews, Ph.D., UC San Diego School of Medicine, Division of Medicine

Roger Revelle College Physiology and Neuroscience Major

Targeting Aberrant RNA Splicing of MCL-1 to Prevent Malignant Regeneration in Multiple Myeloma

Multiple myeloma is a form of cancer that is characterized by malignant plasma cells which produce abnormal antibodies that can cause symptoms such as renal damage, bone pain, and hypercalcemia. There is currently no known cure for multiple myeloma and knowledge is limited regarding the molecular basis of the disease as it relates to RNA splicing alterations. Splicing is a form of RNA processing in which removal, or lack thereof, of introns creates novel RNAs which can produce unique proteins. This project explored the role of aberrant splicing of the pro-survival gene MCL-1, which is correlated with disease progression and drug resistance in multiple myeloma. Cell viability and selfrenewal were evaluated through both genetic modulation of the splicing factor SF3B1 and pharmacological modulation using a small molecule targeting the spliceosome complex in human myeloma cells. These findings could provide an avenue to explore novel therapies for multiple myeloma treatment.

Earl Warren College Biochemistry and Cell Biology Major

YUANHAO QU

PI: Weg M. Ongkeko, M.D., Ph.D., UC San Diego School of Medicine, Department of Surgery

The Role of HPV in Shaping the Tumor Immune Landscape

Immunotherapy has emerged as the most promising treatment for cancers. However, failure of a large percentage of patients remains the ultimate obstacle to successful treatment. Etiology-associated dysregulation of genes involved in the immune system may be central to the differential clinical response. To understand the role of human papillomavirus (HPV) in shaping the tumor immune landscape, we conducted a comprehensive investigation of immune-associated (IA) gene expression in HPV-associated cancers. Using next-generation RNA sequencing datasets, we identified a unique panel of HPV-dysregulated IA genes. To investigate the molecular mechanism for their dysregulation, we used in silico methods to explore their potential regulation by genomic and transcriptomic alterations, aberrant microRNA expression, and DNA methylation. Finally, we validated the dysregulation of several IA genes in vitro using HPV E6/E7-transfected cell lines. The panel of IA genes we identified may elucidate the HPV-mediated molecular mechanism of tumor immune evasion, and ultimately contribute to better treatment modalities for HPV-associated cancer patients.

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sqonline.ucsd.edu

THESES

ASHLEY ROSANDER

Roger Revelle College Human Biology Major

PI: Mariana Cherner, Ph.D., UC San Diego, Department of Psychiatry

A Role for TAAR1 Genotype Associated with MA Intake in Dopaminergic Changes

Multiple factors contribute to individual susceptibility to drug addiction. Mouse lines MAHDR (High) and MALDR (Low) were bred for the amount of methamphetamine (MA) they voluntarily consume. MAHDR mice exhibit a homozygous TAAR1 gene SNP, making it non-functional. We tested the role of the TAAR1 mutation in dopaminergic and inflammatory pathway dysfunction, induced by the TAAR1 agonist MA, by measuring transcription of dopamine receptor DRD1, dopamine transporter DAT, and CCL5/RANTES chemokine in both groups at multiple time points after intraperitoneal MA. MAHDR mice showed an overall delayed response in dopaminergic regulation in major reward pathway regions such as pre-frontal cortex (PFC) and nucleus accumbens (NAc), characterized by increased DRD1 and DAT levels, detectable at 30min after MA. CCL5/ RANTES was decreased in MALDR NAc at 30min, but increased in MAHDR ventral mid-brain (VMB) at 60min. These results suggest a role for TAAR1 in the susceptibility to MA intake and addiction.

John Muir College Molecular Biology Major Mathematics Minor

EVAN SALDIVAR

PI: Eric Schmelz, Ph.D., UC San Diego, Division of Biological Sciences, Department of Cell and Developmental Biology

The Elucidation and Untangling of Maize Biosynthetic Pathway Underlying Defense

Small-molecule specialized metabolites constitute an important biochemical layer of plant immunity for pathogen defense. Maize (Zea mays) produces a complex mixture of antimicrobial isoprenoids in response to pathogen elicitation, including a genus specific family of acidic sesquiterpenes derived from β-macrocarpene, termed zealexins. Candidate zealexin biosynthetic genes are among the most dynamically regulated processes known in maize during pathogen attack. In this study we combined forward genetics, reverse genetics, bioinformatics and enzyme co-expression studies to define enzymes and catalytic promiscuity. We demonstrated the sufficiency of three cytochrome P450 enzymes located in a CYP71Z class gene cluster on chromosome 5, termed Zx5, Zx6 and Zx7, to independently catalyze oxygenation reactions of diverse olefin substrates, yielding different combinations and ratios of zealexin A1, D1, D2, β-costic acid and α-santalenoic acid. These enzymes represent an important biosynthetic node for maize defense biochemistry with many parallel defense pathways utilizing the same gene cluster.

DELLA SYAU

John Muir College Biochemistry and Cell Biology Major

PI: Randolph Y. Hampton, Ph.D., UC San Diego, Division of Biological Sciences, Section of Cell and Developmental Biology

Characterizing a Novel Endoplasmic Reticulum Membrane Stress

Endoplasmic reticulum-associated degradation (ERAD) is a form of protein quality control that degrades misfolded proteins in the cell. In ERAD, proteins are ubiquitinated, removed from the ER to the cytosol in a process called retrotranslocation, then degraded in the cytosol. Dfm1, a yeast ER membrane protein, is a key factor in retrotranslocation. We have discovered that in strains lacking Dfm1, expression of various ERAD substrates leads to a profound growth stress originating at the ER. Here we characterize this novel stress, including the molecular features of the proteins that cause it, the manner in which Dfm1 alleviates the effects, and the cellular systems that participate in both its generation and abatement.

Thurgood Marshall College General Biology & Public Health Double Major Business & Chemistry Double Minor

JESSE TAI

PI: Li-Fan Lu, Ph.D., UC San Diego, Division of Biological Sciences, Section of Molecular Biology

The Role of NLRP3 in Gut-Resident Regulatory T Cells

Regulatory T (Treg) cells are a subset of adaptive immune cells known for their immunosuppressive functions. RNA-seq analysis performed on mice during systemic autoimmune inflammation revealed high expression levels of Nod-like receptor protein 3 (NLRP3) in gut-resident Treg cells. NLRP3 is part of a well-characterized inflammasome commonly associated with innate immune cells. To determine the role of NLRP3 in gut-resident Treg-mediated immunosuppression, we obtained a mouse model with a Treg-specific, constitutively active form of NLRP3 (Foxp3cre NLRP3iCA/+). After immunologically challenging these mice via Citrobacter rodentium and Toxoplasma gondii, our data suggests that gut-resident NLRP3iCA/+ Treg cells have impaired control over Th17 responses but enhanced control over Th1 responses. Next, we plan to examine the efficacy of Treg cells in the presence of constitutive activated NLPR3 or in the absence of NLRP3 to inhibit tumor-promoting inflammation by utilizing an well-established in vivo model of colorectal cancer.

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KIMI TAIRA

Eleanor Roosevelt College Human Biology Major Dance Minor

PI: Jeffry S. Isaacson, Ph.D., UC San Diego, Division of Biological SciencesDepartment of Neuroscience

The Effects of Fear Learning on Sensory Representations in Secondary Auditory Cortex

Sensory representations in our brain are known to change with experience. During auditory fear conditioning, pairing a neutral tone with an unpleasant stimulus (i.e. mild shock) changes tone responses in the auditory cortex. However, it remains unclear what changes in cortical input underlie this plasticity. The lateral amygdala (LA) plays an important role in fear and emotion processing and sends dense projections to the auditory cortex. Thus, projection cells in LA are candidates for mediating changes in auditory cortex sound representations. To investigate this possibility, we used in vivo two photon calcium imaging in head-fixed mice to simultaneously image activity of LA projections and layer 2/3 pyramidal cells in secondary auditory cortex before and after fear conditioning. Our results show that fear conditioning leads to long term changes in sound-evoked LA activity that may contribute to learning-related changes in tone responses of cortical L2/3 principal cells.

JIALI TAN

Sixth College Biochemistry and Cell Biology Major

PI: David Cheresh, Ph.D., UC San Diego School of Medicine, Department of Pathology

Cancer stem cells (CSC) represent a small, yet aggressive metastatic sub-population within the tumor characterized by their plasticity, drug resistance, stress tolerance, tumor initiation and metastatic capacity. We found that CSC can secrete lysophosphatidic acid (LPA), a bioactive lipid that is known to induce tumor progression and metastasis. We identified integrin avb3 as a cancer stem cell marker that contributes to the synthesis of LPA by recruiting phospholipase A2 to the plasma membrane, a critical enzyme in the biosynthesis of LPA. Once secreted, LPA can reprogram surrounding cancer cells toward a more stem-like fate through its six LPA receptors. In fact, LPAR4 is selectively upregulated in pancreatic carcinoma cells after exposure to microenvironmental stress or drug treatment. LPAR4 expressing cells gain stem-like and metastatic properties. Taken together, these findings identify a mechanism whereby CSC through the secretion of LPA can enhance pancreatic cancer progression and reveal potential therapeutic targets to decrease the progression of this disease.

TIANZHI TANG

John Muir College Human Biology Major

PI: Dhakshin Ramanathan, M.D., Ph.D., UC San Diego School of Medicine, Department of Psychiatry

Optimizing Methodologies for High-Quality Single-unit Neurophysiological Recording Coupled with Optogenetic Manipulation

Measuring the activity of single neurons, or â&#x20AC;&#x153;unitsâ&#x20AC;? allows researchers to relate spiking activity in a certain brain area with behaviors of interest. By coupling single-unit recording with optogenetic perturbations, a method of activating or inhibiting spiking activity, researchers can observe how neural activity changes and infer causal relationships between neural activity and behavior. There are relatively few cost-effective and standard methods for coupling these technologies. For my honors thesis, I have developed a new optrode system capable of combined single unit recordings and optogenetic manipulations. This new system is designed by fixing tetrode-wires capable of measuring single unit activity together with a fiberoptic cannula into a metal canula. I have found that this method is effective for the collection of single unit activity. Future efforts will be made to minimize signal noise, improve the longevity of neural recording, and optimizing the system for combined optogenetic manipulations.

John Muir College Biochemistry and Cell Biology Major Computer Science Minor

NUO TIAN

PI: Milton Saier, Ph.D., UC San Diego, Division of Biological Sciences, Department of Molecular Biology

Investigation of Protein Structural Relationships Between ABC Superfamily Types

The ATP-binding Cassette (ABC) superfamily consists of transporters that use ATP as energy source. Previous studies based on primary protein sequence suggested that ABC exporters evolved independently at least 3 times. Given the increasing availability of structural data within the superfamily, we are now able to investigate whether 3D structural similarities agree with the conclusions attained by sequence analyses. In our strategy, all available protein structures in the Protein Data Bank (PDB) for the three ABC types were cut into bundles of 4 transmembrane a-helices (4HB) and the alignments within and between ABC types were examined. Clustering and phylogenetic analyses will also be performed for the membrane constituents and ATPases to determine whether they have co-evolved. Our results will provide insights on the evolutionary relationships between ABC types, and serve as a guide for future studies of the ABC superfamily.

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THESES

VINEET TUMMALA

Roger Revelle College General Biology Major Business Minor

PI: Philip Gordts, Ph.D., UC San Diego School of Medicine, Division of Endocrinology and Metabolism

Smooth Muscle Cell Heparan Sulfate Proteoglycans Mitigate Atherosclerosis Formation

Atherosclerosis is a major cause of cardiovascular disease, which kills 17 million people worldwide annually. Atherosclerosis is an inflammatory response initiated by cholesterol buildup in the artery walls. Sub-endothelial proteoglycans produced by smooth muscle cells (SMCs) in the artery wall trap low-density lipoprotein (LDL) cholesterol. This project examines how vascular SMC heparan sulfate proteoglycans (HSPGs) affect the development of atherosclerosis. To test this, we use mice lacking the HSPG biosynthetic gene GlcNAc N-deacetylase/Nsulfotransferase 1 (Ndst1) selectively in SMCs by crossing Ndst1f/f mice with SM22aCre+ mice. Ndst1 inactivation results in significantly undersulfated SMC HSPGs. Weekly injections of LDL receptor anti-sense oligonucleotides and a high fat diet were used to generate hypercholesterolemia-induced atherosclerosis in both the wild type and Ndst1f/fSM22aCre+ mice. Analysis of the lesions so far show that loss of vascular SMC HSPGs promotes atherosclerosis lesion size and remodeling. Further studies will examine how HSPGs affect SMC proliferation, migration and inflammation.

Roger Revelle College Molecular Biology

ALAN S. H. TUNG

PI: Reuben J. Shaw, Ph.D., Salk Institute for Biological Studies, Department of Molecular and Cell Biology

Bridging the Gap between Energy Sensing and Mitophagy: The regulation of mitophagy receptors by ULK1 and TBK1

AMP-activated protein Kinase (AMPK) is a highly-conserved master regulator of cellular metabolism and energy homeostasis. Previous studies have shown that AMPK regulates mitochondrial autophagy (mitophagy) when activated, yet the mechanism remains to be elucidated. Mitophagy receptors are proteins on the mitochondrial outer membrane that help the recruitment of LC3-decorated phagophore to damaged mitochondria. Previous studies have shown that Unc51-like protein kinase 1 (ULK1), an AMPK substrate and a critical component of autophagy initiation complex, phosphorylates FUNDC1 and increases FUNDC1 affinity to LC3B. Several other mitophagy receptors including Bnip3, NIX, and Optineurin have a serine adjacent to LC3 Interacting Region (LIR) motif, we are wondering whether the phosphorylation state of the serine could serve a similar regulatory function. This study aims to study how ULK1 regulates various mitophagy receptors and how ULK1 may potentially link AMPK energy homeostasis to mitophagy.

NIGEL ZHANG

John Muir College Human Biology Major Art History Minor

PI: David Salmon, Ph.D, UC San Diego School of Medicine, Department of Neurosciences

Apolipoprotein Genotype Distribution in Early vs. Late-Onset Alzheimer’s Disease

The Apolipoprotein E ε4 allele (ApoE4) is a major genetic risk factor for late-onset Alzheimer’s disease (AD), but its impact on early onset AD is unknown. To address this question, we compared ApoE genotypes in Early-Onset (EO; age ≤ 60, n=163) and Late-Onset (LO; age ≥ 70, n=629) AD cases, omitting rare cases (n=8) with known autosomal dominate mutations for EO AD (e.g., Amyloid Precursor Protein, Presenilin 1 or 2). Average ages of the EO and LO groups at clinical evaluation were 59.98 (sd=4.74; 45-72) and 78.65 (sd=4.77; 65-101) years, respectively. EO and LO groups did not differ in sex distribution (approximately 50% males in both groups). The ApoE ε4 allele (ε4,ε4 or ε4,ε3 genotype) was present in 55.8% (91/163) of EO and 52.9% (333/629) of LO cases (X2=0.278; p=.598). Similar distributions of ApoE genotypes in EO and LO AD suggest common underlying disease mechanisms.

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HEAD ADVISORS James Cooke, Ph.D. Assistant Teaching Professor of Neurobiology Hermila Torres Manager, do/bio Center

FACULTY ADVISORY BOARD Suckjoon Jun, Ph.D Carolyn Kurle, Ph. D Chih-ying Su, Ph. D Jim Wilhelm, Ph.D

Elvira Tour, Ph.D Elsa Cleland, Ph. D Jill Leutgeb, Ph. D Lisa McDonnell, Ph.D

ALUMNI ADVISORY BOARD Cade Oost Sid Ambulkar

Rithvik Shankar Yogitha Chareddy

Top Row: Alejandro Dauguet, Daniel Lutz, Anne Marie Berry, Mark Jacob, Jialin Xu, Michael Kalisz, Sam Zilberman 2nd Row: Soha Khalid, Jenny Chiem, Sneha Ganguly, Shreya Shriram, Sanjana Sharma, Shae Galli, Melody Nazarbegian, Meera Patel, Nikhil Jampana 3rd Row: Lisa Chik, Andra Thomas, Vickie Kuo, Salma Sheriff, Maya Gopalakrishnan, Alexandra Babakanian, Megan Griswold, Katie Clark, Gayathri Kalla, Andrea Pednault, Cristina Corral, Michael Endow Bottom Row: Tushara Govind, Arya Natarajan, Theresa Bui, Victoria Hoznek, Rebecca Hu, Serena Tan, Liam Huber, Cassidy Lam, Tiffany Huynh, Emma Huie, Lauren Brumage, Ashni Vora, Samreen Haque, Sharada Saraf

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