SQ Vol 15 (2017-2018) by Saltman Quarterly

Undergraduate Research Journal Volume 15 2017â&#x20AC;&#x201C;18 sqonline.ucsd.edu

Generously underwritten by

THE SALTMAN FAMILY &

supported by

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 carpet anemone, Heteractis magnifica, sits on display at the Monterey Bay Aquarium for all visitors to enjoy. Photo by Arya Natarajan. INSIDE COVER: A field of rapeseed, Brassica napus, provides the main component in canola oil. Photo by Anthony Avalos.

Dear Reader,

LETTER FROM

In your hands you hold the 15th volume of Saltman Quarterly (SQ), UC San Diego’s premier undergraduate biological research journal. This journal is the fruit of over a year’s worth of intense labor, carried out by over 100 undergraduates. It is our mission to capture and communicate some of the most exciting research happening on and around campus, and I can proudly say that this year we did just that. I began my journey in SQ as an online reporter, a role that let me bring attention to exciting research on campus, discuss my personal interests, and discover my own voice as a writer. The following year, I tried to foster that same growth in others as the online editor, and now as the Editor-in-Chief I had the distinct privilege of overseeing not just our online teams but everything that we as an organization publish and participate in. Since the majority of my background was with our website, I spent the beginning of the year gaining familiarity with everything that went into the making of this journal, and I was astounded by the countless hours it required and the relentless dedication shown by everyone on our staff. To get to the final product you’re now reading took many sleepless nights and tireless effort, but it is a final product that I wholeheartedly believe everyone in SQ can be proud of. In this journal you will find articles on topics ranging from new classes of brain cells, algae as a fuel source of the future, and even an investigation into the causes of diabetes. This journal is the pride and joy of our organization, but in past years we’ve established many other efforts to carry out our mission of accessible science communication. Our community outreach team has gone to elementary schools in the San Diego area teaching children about nutrition (a nod to Dr. Saltman’s area of expertise) and helped tutor high schoolers through the YMCA. Our review board hosted journal clubs to discuss exciting new manuscripts and help students break down complex scientific writing. Our online teams posted articles ranging from cutting-edge news like interviews with students and research spotlights to articles about autopsy case studies and even the evolutionary possibility of dragons. We also published two quarterly SQ Insider issues, the first being on astrobiology and discussing the origin of life on our planet and the possibility of it occurring on others, and the second being on the past, present, and future of genetic sequencing and its potential uses and implications. The accomplishments and improvements of this organization in the past year are humbling and inspiring, and they were a joy to be a part of. I am proud of SQ because we are an organization that stands up for what we believe in and seeks to not only spread knowledge but also excite curiosity, in a world that all too often seems to spread only rumors and fear. I firmly believe that by using science as a tool, there are few problems beyond our fixing. And by trying to give our readers that tool, we come one step closer to a world dedicated to intelligent solutions and a population with the means to find them. It was undoubtedly the most humbling experience of my undergraduate career being surrounded by the hard-working, talented, and passionate staff of SQ. They all inspired me every day and will continue to do so long after my time at UC San Diego has come to a close. I’ve made some of my best friends through my roles in SQ, and I hope that the passion and comradery that went into this journal comes through to you, the reader. With all that said, it is with great pride and honor that I present to you Saltman Quarterly Volume 15. Sincerely,

Cade Oost

Editor-in-Chief, Saltman Quarterly 2017–18

TABLE OF FEATURES Fueling the Future with Algae BY AISHWARYA VUPPALA

The Brain Reward Circuit BY GRACE SAHYOUNI

SALTMAN DEDICATION The impact of Dr. Saltman’s influence can be seen most clearly in the Saltman Quarterly journal; despite never knowing him in person, the founding members of SQ named the journal after him and continued to support and expand the undergraduate experience that he espoused... BY YOGITHA CHAREDDY & KWANG-TOU CHOU

Discovering New Types of Neurons BY ARJUNE NIBBER

Type 2 Diabetes: Obesity is Not the Full Picture BY ANOKHI SAKLECHA

RESEARCH Resting Statement Brain Dynamics Supporting Creativity

Taking a Look at Ocean Toxicology

BY SUBATHRA RAJ

BY SKYLAR RAINS

Differences in Heterospecific Pollen Deposition across Pollination Syndromes in Monteverde, Costa Rica BY KARA POWELL

BREVIAS Identification and Characterization of PACSIN-1 as an LRP1 Cell Signaling Candidate In Peripheral Nerve Injury

Effects of Anxiety, Depression, and Fatigue on Decision Making in Humans BY MICHAELA JULES, CARLY SHEINSKY, AND PAMELA REINAGEL

BY: SHAHRIAR TAHVILIAN

An Investigation of Theropithecus Gelada as a Modern Analog for Extinct Hominid Paranthropus Boisei Nerve Injury BY HERMSMEYER ET. AL.

REVIEW A Driver of Cancer Metastasis: Vasculogenic Mimicry BY RISHI N. MODI

SENIOR HONORS THESES The Division of Biological Sciences’ Senior Honors Theses Program aims to increase faculty-student interactions and encourage more students studying biology to pursue independent research.

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STAFF

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PAUL SALTMAN Few activities are as intense and rewarding as surfing. The sport requires a tenacious spirit, nimble reflexes, and an undulating passion as deep as the sea. UC San Diego professor Dr. Paul Saltman seemed to have found a profession with similar principles: teaching. Dr. Saltman approached instruction with the same mindset he practiced as a surfer, and he left a legacy of fostering undergraduate education and mentorship that has shaped the UC San Diego biology department into an innovative powerhouse. The impact of Dr. Saltman’s influence can be seen most clearly in the Saltman Quarterly journal; despite never knowing him in person, the founding members of SQ named the journal after him and continued to support and expand the undergraduate experience that he espoused. Among some of Dr. Saltman’s most enduring works are his contributions to the academic sphere in nutrition science. His drive to make the subject interesting and understandable has led to global recognition, and one of his nutrition books written for UC San Diego continues to be an academic staple. SQ reflects this mentality through our featured manuscripts and activities. We have included an engaging article in this volume of SQ related to redefining type 2 diabetes and its relationship with obesity and excess weight and have reached out to fifth-graders through our community outreach efforts to teach basic nutrition concepts in a manner that Dr. Saltman would have supported. sqonline.ucsd.edu

“What more meaningful service could we perform as academic scientists than to forge an effective and sustained relationship with our primary and secondary school teaching colleagues?” - Dr. Paul Saltman

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The invaluable work of making science accessible can further be seen in Dr. Saltman’s investment in undergraduate education. His genuine interest in his students made him a major figure in the lives of undergraduates; their appreciation allowed him to win the Excellence in Teaching award from Muir, Marshall, Revelle, and Warren colleges and the first ever Career Teaching Award in 1994 from the UC Academic Senate. He pioneered a curriculum series designed for non-science majors so that they can learn to relate science to global movements with far-reaching consequences, like social justice and environmental conservation. He maintained TV series for National Education Television, NBC, and PBS and was a firm believer in the importance of a well-supported primary and secondary education. One of his major endeavors during his time at UC San Diego was in convincing his fellow academic instructors that they have a major responsibility to teach undergraduates, not just do research and mentor graduate students. His views on education are reflected in SQ’s contributions to outreach, especially centered around elementary to high school students. To name just a few, we partner with the YMCA to teach and inspire homeless and otherwise at-risk teens, we work with underserved high schoolers in low-income areas San Diego to share strategies of scientific communication, and we help set kids up for a healthy future by teaching nutrition in elementary school classrooms. In these efforts, we encourage young minds not just to ride the waves but also to turn the tide.

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Dr. Saltman’s work has made waves on campus, the surrounding community, and the world of teaching, but he remains an accessible and familiar figure to his students. Many of them remember him fondly as a down-to-earth, charming professor who encouraged them to join him in surfing to destress and sparked in them a love for science and the sea. This affection for the ocean has continued to influence generations of students after, as can be seen in this volume of SQ in articles related to ocean pollution and the use of algae as a biofuel. Dr. Saltman’s legacy is as ever-present as the ocean next to campus, and has left a mark as deep. Saltman Quarterly endeavors to do the same, to imbue a passion for science into a publication that can help our readers ride the waves into a new tomorrow.

CO-WRITTEN BY

YOGITHA CHAREDDY Yogitha is a Molecular Biology major from Earl Warren College. She will graduate in 2019.

KWANG-TAO CHOU Kwang-Tao is a Microbiology and Marine Biology double major from Thurgood Marshall College. He will graduate in 2018.

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FEATURES 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 groundbreaking work accomplished by researchers affiliated with the UC San Diego campus.

Stalks of sunflowers, Helianthus annuus, bathe under the sun until their seeds are ready to be harvested. Photo by Elmer Steve Mejia sqonline.ucsd.edu

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FUELING THE FUTURE WRITTEN BY AISHWARYA VUPPALA â&#x20AC;¢ ILLUSTRATED BY APRIL DAMON

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FEATURES Algae are versatile organisms that offer medical solutions, alternative materials, and energy sources. They are one of the oldest organisms on this planet; however, there is still much to know about them. Current research, including crucial findings from work done at UC San Diego, demonstrates that alternatives like algae-based biodegradable foams can replace current, non-biodegradable petroleum-based materials. Increasing biodegradability has the potential to reduce or eventually eliminate the accumulation of this type of trash, which will ultimately be extremely beneficial to the environment. Algae show potential for a multitude of applications, and understanding them will inform their genetic manipulation for increased biomass and bioproduct yields.

nergy is a requirement for many components of everyday life, and without it, society as we know it would cease to function. Multiple sources of energy power vehicles, companies, homes, and more, but the predominant production of electricity in the United States comes from fossil fuels. Fossil fuels are generated when organic material undergoes millions of years of heat and pressure in the Earthâ&#x20AC;&#x2122;s crust, forming multiple carbon-based geologic deposits. They are not easily obtainable, emit greenhouse gases when burned, and are a finite resource. Development of alternative energy sources, such as biofuels created from organisms, which derive their energy from sunlight and CO2 are increasing due to their potentially lower environmental and financial costs. Minimal resources are required to grow algae, and algae have the potential to produce high amounts of energy relative to their small size. If algal-based biofuels are successful in their production of energy and can serve as alternative energy sources, replacing fossil fuel burning, algae could help reduce greenhouse gases while also making energy cheaper and more accessible. sqonline.ucsd.edu

Applications of Algae Algae undergo photosynthesis, utilizing the sunâ&#x20AC;&#x2122;s energy and nutrients to make their living. They range in size from tiny microalgae to larger, multicellular forms like kelp. Algal biomass can then be used as a biofuel. Algae are diverse organisms which reproduce quickly in salt and waste water, and do not require arable land. By consuming CO2 to conduct photosynthesis and therefore release oxygen, they help reduce the impact that carbon dioxide has as a greenhouse gas. Algae can even help with purifying wastewater while also creating biomass and essentially serving as a source of biofuel.

Algae production would be beneficial for the medical industry, as well; some algae bioproducts include therapeutic proteins like the UV-protectant metallothionein, anti-cancer proteins, and even sub-unit vaccines. Other bioproducts include nutritional supplements that can be incorporated into animal feed and even used for human dietary needs. By exploring the benefits of algaeâ&#x20AC;&#x2122;s potential impact on the world, it is evident that taking advantage of the additional bioproducts algae create may bring about the environmental change that the world needs. SALTMAN QUARTERLY

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FIGURE 1: Algae have a lot to offer and have led to several innovations. Some include biofuel, medical alternatives, and biomaterials. There’s still much to be discovered, but the possibilities are endless.

Fashionable Algae Flip-Flops Chlamydomonas reinhardtii are single-cell green algae, and are one of the first genetically engineered algal species to be used for research purposes. C. reinhardtii has three modifiable genomes, one of which is the chloroplast genome. The chloroplast genome is a primary target for modification in biofuel research because it is easily transformed, can perform homologous recombination, and is crucial for protein production. Genetically engineering the chloroplast could help to maximize production of recombinant protein, to make algae more adaptable to environmental conditions, and even to modify how photosynthetic components work. The Mayfield lab, at the California Center for Algae Biotechnology at UC San Diego, is studying the potential for algae to derive biofuels and bioproducts like medication, as well as petroleum and plastic substitutes. Dr. Stephen Mayfield emphasized that algae play a bigger role than just providing biofuels; in fact, they can be used to create a biodegradable foam that can replace plastic and other petroleum products. There is a massive problem with the floating beds of trash within the ocean, and according to Dr. Mayfield, flip-flops make up a large part of the plastic found within these trash beds. 8

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Dr. Mayfield has worked on flip-flops using the foam material created from algae oil. It took several trials and years of research to generate the appropriate foam material. Certain hydrocarbon interactions could make the foam very brittle, so it was crucial to their research to understand how they could create the perfect sets of hydrocarbon interactions that could create sturdy but comfortable foam. Dr. Mayfield’s foam is biodegradable and more environmentally friendly than current petroleum-based materials. Because it degrades quickly, this foam could help in the reduction of growing garbage patches which negatively affect marine life. The biodegradation happens when microorganisms identify the polymers in these biodegradable materials as food and an energy source. It is easier for these microorganisms to degrade this specific material because the polymers found in it are also found in nature. The Mayfield Lab has also derived several bioproducts from

algae including one medication that helps individuals with irritable bowel syndrome. This medication is currently undergoing human trials and has shown improvements in users, according to Dr. Mayfield. Initially, when looking into research regarding algae, the first thought that arose was their potential as an alternative fuel source, but, as seen through Dr. Mayfield’s research, the various bioproducts derived from algae seemed to be making a larger impact. Dr. Mayfield believes that the recent surge of highly efficient electric vehicles may eventually replace fueldependent vehicles. With the future heading this way, biofuel is not necessarily the most important factor of algae, yet algae still prove to be versatile organisms as shown through the foams and medicines that have been developed from them. With algae playing a major role, the world may be headed for a greener future. sqonline.ucsd.edu

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With algae playing a major role, the world may be headed for a greener future.” References 1. The end of fossil fuels. (n.d.). Retrieved April 14, 2018, from https://www.ecotricity .com.uk/our-green-energy/energyindependence/the-end-of-fossil-fuels 2. Jose, S., & Archanaa, S. (1970, January 01). Environmental and Economic Sustainability of Algal Lipid Extractions: An Essential Approach for the Commercialization of Algal Biofuels. Retrieved April 14, 2018, from https://link.springer.com/ chapter/10.1007/978-3-319-51010-1_14 3. Shurin, J. B., Burkart, M. D., Mayfield, S. P., & Smith, V. H. (2016, October 4). Recent progress and future challenges in algal biofuel production. Retrieved April 14, 2018, from https://www.ncbi.nlm.nih.gov/pmc/ articles/PMC5054820/ sqonline.ucsd.edu

FIGURE 2: Flip-flops accumulate in large masses throughout the ocean. These flip-flops are obstructive to wildlife and are not easily degraded. In fact, flip-flops make up a large portion of what is in several floating trash beds.

4. Scranton, M. A., Ostrand, J. T., Fields, F. J., & Mayfield, S. P. (2015, February 18). Chlamydomonas as a model for biofuels and bio‐products production. Retrieved April 14, 2018, from http://onlinelibrary .wiley.com/doi/10.1111/tpj.12780/full 5. Gimpel, J. A., Specht, E. A., Georgianna, D. R., & Mayfield, S. P. (2013, May 16). Advances in microalgae engineering and synthetic biology applications for biofuel production. Retrieved April 14, 2018, from https://www.sciencedirect.com/science/ article/pii/S1367593113000616 6. Algae Basics. (n.d.). Retrieved April 14, 2018, from http://allaboutalgae.com/whatare-algae/ 7. Biofuels. (n.d.). Retrieved April 14, 2018, from http://www.alternative-energy-news .info/technology/biofuels/

8. Davison, A. (n.d.). Biomass Energy. Retrieved April 14, 2018, from http://www .altenergy.org/renewables/biomass.html 9. Kržan, A. (n.d.). Biodegradable polymers and plastics. Retrieved May, 2012, from www.icmpp.ro/sustainableplastics/files/ Biodegradable_plastics_and_polymers.pdf

WRITTEN BY

AISHWARYA VUPPALA Aishwarya is a Biochemistry and Cell Biology major from Earl Warren College. She will graduate in 2018.

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THE BRAIN

WRITTEN BY GRACE SAHYOUNI â&#x20AC;¢ ILLUSTRATED BY VARSHA RAJESH

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FEATURES The brain's reward circuit is located in the limbic system, which is an ancient brain structure. It functions to release chemical messengers in response to rewarding stimuli. The primary chemical involved in the reward circuit is dopamine, which helps reinforce behaviors that can be essential for survival. The function of this system has implications on the natural reward system of people who may engage in habitual behaviors that are harmful and can lead to addictions or conditions like diabetes.

kydiving involves jumping out of a plane 10,000 feet above ground simply for the thrill. Although this activity may only be for the risk-takers, why do humans engage in such behaviors? The answer lies in the pleasure center of the brain. This system is responsible for rewarding natural behaviors such as eating and is an integral component of a person's drive. The brain reward center helped primitive humans survive in harsh conditions by motivating them to seek food. In a modern world, the reward center acts as a mood booster in response to a variety of pleasurable activities such as drawing, cooking, or running. In contrast, addictive substances and activities such as drug abuse and gambling can hijack the brain’s reward system and reinforce harmful behavior. The limbic system is a primitive brain structure that is involved in emotional regulation and motivation among many other functions in all mammals. Within the limbic system resides the nucleus accumbens (NA) and the ventral tegmental area (VTA). Both structures are involved in the primary reward circuit in the brain called the mesolimbic dopamine pathway. Neurons of the VTA project to the NA along with other brain regions and release a neurotransmitter called dopamine. Therefore, activation of this pathway causes high dopamine levels to circulate in the limbic system, which leads to the feelings of euphoria associated with pleasure. sqonline.ucsd.edu

Circulating Triglycerides & the Reward Center Mohammed Shenesa, a graduate student in the Hnasko Lab in the neuroscience section of the Division of Biological Sciences, is researching the reward system in the context of post-meal neurophysiology. Shenesa is studying the effect of circulating triglycerides in the brain on circuitry activity. To do so, Shenasa performed rodent surgeries that included chronically catheterizing the carotid artery, the vessel that supplies the brain with blood, to directly infuse lipids to the brain. The purpose of this surgery was to deliver triglycerides into the brain in order to mimic the brain’s physiology after consumption of a high-fat and highsugar diet profile. Shenasa describes this method as “unique [because they] can infuse triglycerides to awake and freebehaving animals at very controlled rates so as to not alter peripheral blood chemistry.” The type of diet that the surgery emulates has been shown to highly reinforce the reward circuitry. Preliminary data reveals that circulating triglycerides upregulate markers of neural activity in the VTA, substantia nigra, cortex, and other brain regions compared to activity in saline infused control mice. These results show that circulating triglycerides activate the reward center, which explains the naturally pleasurable sensation of consuming high-fat content food. However, it is not simply the infusion of triglycerides that

is responsible for this pathway, but rather the processing of the triglycerides in the brain. The brain expresses an enzyme called lipoprotein lipase (LPL) which is responsible for hydrolyzing triglycerides into their components, glycerol and fatty acids. Knockdown studies of LPL attempt to study how LPL functions in the neural circuit. The results show that the nucleus accumbens displays the opposite behavioral effects, which suggest that the enzyme is an essential component in sensing triglyceride levels and mediating the effects they have on the reward circuit. Furthermore, a direct link between dopamine signaling and high-fat feeding and obesity has been discovered in human and rodent experiments. Research has shown that the amount of dopamine receptors in the brain is inversely correlated to body weight. This data indicates that obese individuals have less active dopaminergic neurons in the striatum compared to non-obese individuals. Therefore, the dopamine insensitivity people with obesity experience can explain their desensitized responses to food signals. These results suggest that diet and excess food may contribute to and also be a result of a disruption of the normal circuitry function. An alternative explanation is that people with obesity overwhelm their limbic systems with dopamine when they overeat. As a result, their limbic systems downregulate the number of dopamine receptors. The low dopamine signaling in individuals with obesity may be the result of overeating SALTMAN QUARTERLY

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and partly responsible for habitual behavioral responses such as overeating and not exercising, which exacerbates an already dysfunctional system. Although poor dietary and exercise habits play a role in the onset of diabetes and obesity, it is important to recognize that an individual’s choice to consume high-fat foods may be connected to a faulty reward circuit center in their brain. Society oftentimes blames the individual for their diabetes and obesity by attributing the cause to predominantly dispositional factors rather than situational. Triglycerides in the brain function similar to substances of abuse, and food preferences should be therefore recognized as positive reinforcers to an addict. Some of the ways in which high-fat diets act similar to substances of abuse include increasing expression of a transcription factor, delta Fos B in the striatum which has been shown to increase the motivation to seek rewarding behaviors. In addition, individuals with obesity or a drug addiction show a decreased activity of a type of dopamine receptor in the brain, which means less reward circuit activation. Both individuals also show a decrease in activity of the prefrontal cortex, a brain region that is responsible for inhibition and higher order cognitive function.

FIGURE 1: The mesolimbic dopamine pathway. Dopaminergic neurons project from the ventral tegmental area to various brain regions including the nucleus accumbens, amygdala, and prefrontal cortex.

abuse, contributing to the rapid escalation in prevalence of obesity and diabetes… frequently referred to as an epidemic.” He hopes to see some real-world applications in the future, such as clinical action that may include “inhibiting some key molecular mediator that facilitates harmful neuro-adaptations seen in addicted individuals.” This research has begun to uncover the complex network that links the reward circuitry and dopamine signaling, dietary triglycerides, and obesity.

Brain Injury & Risky Decision Making

Implications & Further Steps This research has implications on the natural reward centers of diabetic and obese patients who override this negative feedback and continue to eat regardless of the harmful consequences to their health. Shenasa expresses that “studying the interaction between circulating dietary triglycerides and the reward circuit ... can and has shed light on the potential for high-fat foods to act as substances of 12

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Although eating food is a necessary activity and a primary reinforcer, the reward center is also involved in secondary reinforcers that are not integral for survival and can even be dangerous. Some examples of harmful stimuli include varying levels of addiction to drugs, gambling, caffeine, etc. To further study this, Samreen Haque, an undergraduate student at the Delano-Wood Lab at UC San Diego is studying people who experience mild traumatic brain injury (mTBI) in order to quantify neurological

The understanding of this circuitry reveals the motives behind many of the natural and unnatural behaviors humans partake in their daily lives.” differences and to observe its effects on behavioral responses. Participants with mTBI were the subjects of the study because it is hypothesized that neurodegeneration in particular brain regions may be responsible for the negative behavior seen in an individual, post-injury. The subjects of the study included veterans who had a history of mTBI and the participants were matched with control individuals who were veterans with no history of mTBI. All sqonline.ucsd.edu

FEATURES

individual or a species to continue existing. Certain stimuli send positive or negative feedback signals to the system which cease or perpetuate the circuit’s activation. These stimuli can be overridden as in the case of the diabetic who continues to eat despite the rising levels of triglycerides that should promote cessation of consumption. The stimuli can also hijack the system and force someone into a loop of addiction and dependence in which they must seek higher and higher doses in order to achieve the same level of euphoria as previously experienced. The understanding of this circuitry reveals the motives behind many of the natural and unnatural behaviors humans partake in their daily lives. FIGURE 2: A neural circuit pathway with projections of dopaminergic regions synapsing on various brain regions. Triglycerides found in high fat high sugar diets upregulate the activation of the reward center, resulting in more dopamine release.

participants were subjected to MRI scans and then administered the Iowa Gambling Task (IGT) to assess real-life, rewardbased decision making. The IGT requires a participant to decide between small, long-term rewards that have little risk of loss versus large, immediate rewards that have a high risk of loss. Gamblers perform worse on this task because they often choose uncertain, immediate gratification, even though they experience great losses. In terms of the mTBI participants in the study, the results show that the mTBI group displayed lower volumes on the left nucleus accumbens volumes relative to the control group and this difference was statistically significant. The data is controlled for intracranial volume as well as PTSD symptoms, as these variables may have an influence on the dependent variable which is the volume of the left nucleus accumbens. The results also show that individuals with lower levels of left nucleus accumbens volumes demonstrated poorer sqonline.ucsd.edu

performance on the IGT. The nucleus accumbens, as discussed earlier, is a key player in the motivation and reward center of the brain. It also has a fundamental role in the dopaminergic signaling of the reward circuit and neurodegeneration of this brain region is associated with riskier behavior. This research may have implications on the neuroanatomy of an addict and how it contributes to the impaired decision making in individuals following an injury or neurotrauma, such as mTBI. In addition, further studies on the brain's morphological changes that occur when an individual engages in secondary reinforcing activities can provide insight into the link between reward circuitry, behavioral responses, and decision making strategies. The brain reward circuit pathway is integral to the motives people experience in their daily lives such as eating, drinking, and even interacting socially. The brain rewards these necessary motivations as well as other ones that are not integral for an

References 1. Neuroanatomy and Physiology of the “Brain Reward System” in Substance Abuse. (n.d.). Retrieved April 19, 2018, from http://ibg.colorado.edu/cadd1/a_drug/ essays/essay4.htm 2. Berland, C., Cansell, C., Hnasko, T., Magnan, C., & Luquet, S. (2016, March 18). Dietary triglycerides as signaling molecules that influence reward and motivation. Retrieved April 19, 2018, from https:// www.sciencedirect.com/science/article/pii/ S235215461630064X 3. Brevers, D., Bechara, A., Cleeremans, A., & Noel, X. (2013, September 05). Iowa Gambling Task (IGT): Twenty years after– gambling disorder and IGT. Retrieved April 19, 2018, from https://www.frontiersin.org/ articles/10.3389/fpsyg.2013.00665/full

WRITTEN BY

GRACE SAHYOUNI Grace is a Human Biology major from John Muir College. She will graduate in 2018.

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DISCOVERING NEW WRITTEN BY ARJUNE NIBBER â&#x20AC;¢ ILLUSTRATED BY GRACE LO

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FEATURES Recently, researchers at the Salk Institute of Biological Sciences and UC San Diego may have found a different way to identify neurons. By developing an epigenetic system of studying neurons these researchers have identified entirely new categories of neural cells, a finding that could be applied to all aspects of neuroscience. By analyzing small fractions of the brain, the researchers discovered 21 new subtypes of neural cells in the human brain. By expanding their research to more parts of the brain, the neuroscience community has the potential to solve the great mysteries of the human brain.

eural classification generally occurs today by characterizing neurons by physiological appearance, electrical signaling profile, or overall connectivity. Cellular and molecular markers can readily study these features, which is why the cells are categorized in this way. The procedures of cellular analyses presently used in studying the brain are powerful and efficient when it comes to single neurons, but they are inefficient in studying whole cell populations, a feat which, if accomplished, would lead to advances in neural classification. Researchers at UC San Diego and the Salk Institute of Biological Sciences may have found a new way to identify different subtypes of neurons through categorization by epigenetic analysis. Epigenetics is the study of chemical modifications in the genome that alter genomic expression without changing the genetic code itself; it is highly stable, and cell-type specific. Scientists at the UC San Diego Computational Neural Data and Dynamics Lab, as well as researchers led by Dr. Joseph Ecker and Dr. Margarita Behrens at the Salk Institute, hypothesized that by analyzing the epigenomic profile of a cell, specifically a DNA methylation signature unique to each cell type, they could identify neural subtypes in the mammalian brain. The study, presented in the paper “Single-cell methylomes identify neuronal subtypes and regulatory elements in mammalian cortex,” yielded 21 types of neurons in human brains, and 16 types in mouse brains. By expanding the pool of brain cell types, the study presents a sqonline.ucsd.edu

new approach in understanding the brain through identifying its building blocks.

From Neurons to Neural Circuits Neuroscientists undertaking studies of the brain usually initiate their projects by thoroughly researching the cellular phenomena of neurons. The rationale of doing so is that the vital connections of the diverse brain systems generally begin at the level of the cells; studying the microscopic structural and functional qualities of the nervous system will facilitate an understanding of how each of those cellular components cooperate to generate the intricate functions of neural circuits. An aspect of investigating this cooperation of neurons is scrutinizing their qualities in relation to one another; in what ways are they similar, different, and how do these conserved or diversified traits factor into the relationship the neurons may have with each other. It is therefore advantageous to establish a neural identity that is as specific as possible. Christopher Keown, a primary researcher and graduate student in the Computational Neural Data and Dynamics Lab, believes this specificity is “especially significant given the diversity of neurons in the cortex,” which is also demonstrated by this study. Developing more accurate labels for neurons would logically expedite the process of conducting neurobiological research and possibly improve results. After analyzing the cell, scientists can begin to turn their attention to reconstructing the larger, more complex elements of

the brain, such as neural circuits. As Dr. Hongkui Zeng of the Allen Institute of Brain Science explained in an interview with the San Diego Union-Tribune, “Once we have a parts list, we can then try to understand how the different parts—the cell types—are connected together, and how they work together to process information the brain receives.” It is for this reason that neuroscientists are invested in the advancement of more efficient methods of neural cell classification, and why the cell classification research performed in this study generated interest in the neuroscience community. This intrigue was not limited to neuroscience academia, but even extended to artificial intelligence experts like Dr. Zeng, as constructing computerized models that mimic human neural circuits is one of their primary goals. Thus, cellular neurobiology is a potential avenue through which the brain model could be updated with more accurate neural circuits, and several scientific disciplines would benefit from its advancement. The epigenetic neural research being performed at UC San Diego and the Salk Institute is producing findings that are contributing to this progress.

Epigenetics There are several methods available for analyzing qualities through which neurons are identified and segregated into categories such as electrophysiology, overall structure, and connectivity. However, each has associated limitations. For example, current cellular and molecular assays have proven SALTMAN QUARTERLY

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inefficient as they are difficult to scale to whole cell populations. mRNA transcript sequencing has successfully shown unique transcriptional profiles, but these change too rapidly in different environmental conditions for them to be useful in creating accurate profiles of neural cells. The resultant transcriptome, a profile of all the mRNA molecules synthesized by the cell, only yields information about highly expressed transcripts. Epigenetic analysis through DNA methylation, and subsequent development of a methylome, a complete profile of a single nucleus’ methylation pattern in its genome, compensates for some of these limitations. DNA methylation refers to the addition of methyl groups at various locations along a cell’s genome, resulting in regulated expression without changing the genetic code. DNA methylation is a stable genetic feature, meaning that it is readily studied and a methylome can be created. Methylation also clearly marks the regulatory elements on a genome and can be analyzed to determine what genetic information is relevant to that particular cell. Whereas transcriptional analysis analyzes regulatory information from a fraction of the genome, a methylome is able to ascertain regulatory information from greater than 97% of the genome. As each cell has a unique set of genetic code being expressed, each has a cell-specific methylome. It is this cell specificity that led the researchers to hypothesize that “epigenetic profiles using single-cell DNA methylomes could enable the identification of neuron subtypes in the mammalian brain.” By generating methylomes for thousands of neurons in brain tissues, researchers were able to compare the epigenetic signatures and reveal conserved portions. Conservation in the epigenetic signature implies that the neurons have the same regulatory features; therefore, analogous to how two structurally similar neurons would be grouped together, two neurons with a high degree of epigenetic conservation belong in the same subtype. This reasoning developed by the research team was even able to identify rare types of neurons that would ordinarily be missed with other methods, and overall yielded extremely promising results. 16

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FIGURE 1: There are two main modifications made to DNA that result in regulation. Adding a methyl group to a DNA base or adding an acetyl group to a histone results in conformational changes that expose or hide the genetic code from a ribosome.

The Experiment In postnatal synaptogenesis, neurons develop large quantities of DNA methylation at non-cytosine-guanine base pairing sites, and also experience substantial modification of cytosine-guanine site methylation. The consequence is a unique methylation pattern at gene regulatory elements, such as promoters and enhancers. The research team recorded methylomes for 3377 mice and 2784 human frontal cortical neurons through a modified bisulfite methylcytosine sequencing procedure. After developing this repository of methylomes, the researchers used computer algorithms to identify patterns in methylation. This was primarily done through measuring levels of methylation in non-overlapping regions of the genome, consisting of 100 kilobase pairs each; the distinctive levels of methylation in each region designated each neuron to a “cluster” of neurons with similar methylation patterns. To assign identities to the neural clusters, the researchers analyzed methylation levels at specific known molecular markers, including GABAergic neuron markers, cortical layer

markers, and inhibitory neuron subtype markers. For example, significant depletion of methylation at a subset of molecular markers (indicating increased expression of those genomic elements) was considered to assign a label to neurons with that pattern of depletion. This mechanism of classification was sufficient to assign most of the neurons studied in the experiment to a subtype. However it is worth noting that some neurons did not possess significant methylation patterns at classical markers, and thus were not able to be conclusively identified. The accuracy of the results was corroborated through comparison with mRNA sequencing data for the clusters; neurons of a specific cluster produced a similar transcriptome. The researchers concluded that they had identified 16 mouse and 21 human neural subpopulations, with interspecies conservation in some regulatory elements, and greater diversity in deep tissue neurons than those found in superficial layers of the cortex. In order to compare human and mouse regulation, the researchers examined methylation levels at homologous genes. Interspecies conservation of regulatory sqonline.ucsd.edu

FEATURES

is happening in the neural network. And these algorithms should be very useful for the further development of artificial intelligence.” Keown is already exploring applications of methylation patterns in studying neurological disorders, such as depression. In general, the research team anticipates that cell specific epigenomic profiles, as they were studied here, will have further applications in disease and drug exposure research, and possibly in understanding cognitive experience. Regardless of if these goals are realized or not, the research in this methylome study has brought us one step closer to completing the puzzle of the human brain. FIGURE 2: Humans share many cell types with other animals, such as mice, which are regularly used as models in experiments. By comparing epigenetic categories between the human and mouse sample, the researchers were able to verify the existence of a conserved epigenetic neural subtype.

elements in mouse and human frontal cortical genomes occurred at a higher amount in inhibitory than excitatory neurons, but homology was observed in some excitatory neural subtypes as well. Overall, human neurons exhibited a greater degree of methylation diversity when compared to the mouse dataset. Both of these findings are expected as consequences of evolutionary theory, as humans do share a common evolutionary ancestor with mice, but have evolved to possess much more complicated neurogenesis. The expanded diversity in deep tissue neurons compared to neurons found in more superficial layers of the mammalian cortex is also expected, based on events determined to occur in neurogenesis and synaptogenesis. Inhibitory and excitatory neurons possess distinct progenitor cells; these cells then differentiate into more diverse neurons that are added in an inside-out fashion; the higher diversity is centralized in deeper layers of the brain. This mechanism is conserved across mammalian cortical development, and so the pattern of increased regulatory diversity in deep tissue was seen in both mice and human samples. sqonline.ucsd.edu

References

Looking Forward The results of this research demonstrate that epigenetic analysis of the methylation pattern in neural cells is an efficient method for classifying neurons. It is a relatively novel experiment in which a new a high throughput categorization method was able to analyze regulation beyond just proteincoding regions of the genome. Epigenetic analyses of methylomes can simultaneously be used for marker gene prediction and regulatory element identification. After classification of the neurons through their regulatory genomic features, other analyses could be applied to determine cellular function similarities in the neural subtypes. According to Dr. Mukamel, one of the researchers leading the team, the point of the experiment was to produce a practical demonstration that the technology involved was viable, with the goal being to “create an expanded atlas of neurons’ conserved brain cell diversity.” Dr. Zeng believes this expanded atlas will allow scientists to produce the most accurate models of neural networks yet and that “we can develop algorithms that simulate this process that

1. Fikes, B. J. (2017, October 23). New kinds of brain cells discovered by Salk, UCSD scientists. Retrieved April 14, 2018, from http://www.sandiegouniontribune .com/business/biotech/sd-me-salk-brain20170810-story.html 2. Luo, C., Keown, C. L., Kurihara, L., Zhou, J., Li, J., Castanon, R., . . . Ecker, J. R. (2017, August 11). Single-cell methylomes identify neuronal subtypes and regulatory elements in mammalian cortex. Retrieved April 14, 2018, from http://science.sciencemag.org/ content/357/6351/600 3. Schwartzman, O., & Tanay, A. (2015, October 13). Single-cell epigenomics: Techniques and emerging applications. Retrieved April 14, 2018, from https://www. nature.com/articles/nrg3980 4. Trapnell, C. (2015). Defining cell types and states with single-cell genomics. Genome Research, 25(10), 1491–1498.

WRITTEN BY

ARJUNE NIBBER Arjune is a Physiology and Neuroscience major from John Muir College. He will graduate in 2019.

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TYPE 2 DIABETES: OBESITY IS NOT WRITTEN BY ANOKHI SAKLECHA â&#x20AC;¢ ILLUSTRATED BY VICTORIA HOZNEK

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FEATURES As a primary cause of worldwide mortality, type II diabetes has become an international health concern over the past several years. While the onset of diabetes is highly correlated with excess weight and obesity, the underlying molecular mechanisms of this link are little known. UC San Diego researchers Dr. Maria Araneta and Dr. Jerrold Olefsky have delved into this relationship to discover the true connecting factor between the two. Their research has set the foundation for earlier diagnostics of type II diabetes, as well as more personalized care and targeted therapies.

iabetes is the sixth-leading cause of death in the United States, according to the Centers for Disease Control and Prevention (CDC). It affects more than 370 million people around the globe, and kills 3.4 million per year. With the recent increase in health awareness, perhaps one would expect these numbers to be going down. However, diabetes has become a widespread public health concern, and it is only getting worse with time. As with any disease or disorder, the key to treatment and suppression is early detection. But, the problem with diabetes is that over a third of diabetes victims fail to display symptoms until their condition has progressed to levels of danger. And, while we are routinely assessing those at risk for diabetes, it is possible that we are missing a key piece to the puzzle. Until now, the term “at risk” has largely been used to describe obese or overweight individuals. However, for every four diabetics that are overweight, there is one that is not. And, it is this 20% that are often overlooked. So the question is, what should we really be looking for in risk factors? First, let's take a step back.

Diabetes: The Basics Diabetes is a chronic disorder, characterized by unusually high blood glucose levels. The disorder is caused by lost or reduced function of insulin, a key pancreatic hormone that regulates uptake of glucose from the blood. Consequently, those affected by diabetes are unable to respond to fluctuations in blood sugar levels, which can lead to both short and long-term health sqonline.ucsd.edu

issues. Diabetes is split up into two major classes, type I and type II. Type I, associated with juvenile onset, describes a complete loss of the beta cells that produce insulin. In other words, the body completely lacks the hormone responsible for regulating blood sugar. This class of diabetes accounts for approximately 10% of total diabetes cases and can be treated with insulin injections. Type II diabetes, the more common of the two, refers to decreased insulinsensitivity. While in most cases, the pancreas still produces insulin, over time, the body becomes resistant to it, resulting in increasingly abnormal blood glucose levels. Type II diabetes is correlated with obesity and weight gain, generally affecting older populations. As type II diabetics are not “insulin-dependent” from the start, they can often be treated with diet and lifestyle changes, as well as insulin and non-insulin medications. Although type I diabetes is often unpreventable, caused largely by autoimmune and genetic factors, research has shown that type II diabetes can be curbed through healthy eating habits, weight loss therapy, and increased exercise levels. According to the American Diabetes Association, 80% of type II diabetics are overweight or obese. Hence, there is a clear connection between weight and diabetes onset. In fact, until 2015, the American Diabetes Association declared the BMI screening cutoff for diabetes risk to be at 25.0 or above. However, as subsequent studies show, many type II diabetics remain under that lower limit. For example, Asian-American populations are one of the most at risk groups for type II diabetes. Yet, over 35% of Asian-American type II

diabetics have BMIs under this threshold of 25.0, signifying that they would likely go undiagnosed by the ADA’s guidelines. If BMI is so highly correlated with diabetes onset, how do we explain this discrepancy? It turns out that the answer may be in our interpretations of these BMIs.

The Plot Thickens Dr. Maria Araneta, a UC San Diego professor of epidemiology in the Department of Family Medicine and Public Health, conducts her research on the ethnic differences in diabetes, highlighting Asian populations and the aforementioned discrepancy. After years of analysis, she found that obesity itself does not result in diabetes; rather, it is the distribution and concentration of this excess weight, known as adipose, or fat, tissue. When this adipose tissue is concentrated near the abdominal internal organs, referred to as visceral adipose tissue (VAT), it can exponentially increase the risk for diabetes, independent of total BMI. Hence, regardless of overall weight, people with higher concentrations of adipose tissue near the abdominal internal organs may be more likely to develop diabetes. And, while this often includes obese and overweight individuals, it is not limited to these groups. But how exactly does adipose tissue relate to diabetes onset? This brings us to the work of Dr. Jerrold Olefksy, a UC San Diego professor of medicine in the Division of Endocrinology and Metabolism and an associate dean for scientific affairs. For the past several years, Dr. Olefsky has researched the molecular mechanisms of diabetes, focusing on the link between SALTMAN QUARTERLY

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FIGURE 1: This figure displays the molecular pathway of adipose tissue-induced insulin resistance. High concentrations of saturated free fatty acids results in the hyperactivation of ANT2, a mitochondrial membrane protein. This ANT2 activity subsequently causes a cell-wide state of hypoxia, releasing cytokine chemicals into the extracellular fluid. These cytokines then trigger an inflammatory response from the adipose tissue cells via macrophages. During this inflammatory response, the macrophages also secrete exosomes carrying miRNA fragments. At last, these miRNA fragments travel through the body, inactivating target mRNA sequences vital to insulin recognition.

adipose tissue and insulin resistance. At the core of his work is an inflammatory response, induced by high levels of adipose tissue. In essence, the inflammatory response sets off a signaling cascade that results in diabetes.

The Inflammatory Response It has been known for years that macrophageinduced adipose tissue inflammation is associated with both obesity and diabetes. Macrophages are immune cells that play a key role in the initiation and maintenance of body-wide inflammation. In 2007, Olefsky and his team discovered that this inflammatory response of adipose tissue, triggered by an accumulation of macrophages, is a primary causing factor of insulin resistance in type II diabetics. By inhibiting the inflammatory pathway in mice, insulin resistance was avoided despite a high-fat diet. The team concluded that inflammation, and not obesity itself, leads to diabetic symptoms. However, both the causation and aftereffect of this inflammatory response were still undetermined. His team commenced studies on mouse adipocytes 20

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to elucidate the root of the previously observed inflammation. Over time, they were able to track it down to a single culprit protein: adenine nucleotide translocate 2 (ANT2). ANT2 is a mitochondrial membrane protein that acts as a metabolic proton pump in adipocytes. In areas of high adipose tissue concentrations, there are typically elevated levels of saturated free fatty acids. These fatty acids, in turn, hyperactivate ANT2, increasing its oxygen consumption. By hoarding overall oxygensupplies, ANT2 leaves the rest of the cell in a state of hypoxia, or decreased oxygen. The hypoxia then stimulates a release of cytokine chemicals, which induce the inflammatory response that can trigger insulin resistance. But how exactly does this inflammatory response engender an insensitivity to insulin? This was Olefsky’s most recent focus.

The “Missing Link” In September of 2017, his team published a study on the “missing link to insulin resistance”: exosomes. Exosomes are extracellular vesicles released by cells, generally involved in cell-to-cell

communication. Through fluorescent labeling, Olefsky discovered that the macrophages involved in adipose tissue inflammation were secreting exosomes carrying microRNA (miRNA) fragments. These exosomes were observed traveling from adipocytes, through the blood, and into liver and muscle tissue. In an effort to more closely examine these cellular components, Olefsky’s team isolated the exosomes from obese mice with insulin resistance. They then inserted the “unhealthy” exosomes into normal, non-diabetic mice. Within a short period of time, these previously healthy mice began exhibiting symptoms of insulin resistance. Additionally, treatment of obese, diabetic mice with “healthy” exosomes resulted in a complete elimination of insulin resistance. Thus, this experiment supports the importance of macrophage-derived exosomes in the cause of type II diabetes. In terms of cellular processes, typically, once exosomes arrive at their target tissue, they employ their miRNAs to fulfill specific intracellular functions. Often, the miRNA binds to and inactivates a particular mRNA segment. While additional studies must be conducted on the exact mechanisms of this sqonline.ucsd.edu

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After years of analysis, [Araneta] found that obesity itself does not result in diabetes; rather, it’s the distribution and concentration of this excess weight, known as adipose, or fat, tissue.”

Explaining Ethnic Differences

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4. Carnethon, M. R., De Chavez, P. J., Biggs, M. L., Lewis, C. E., Pankow, J. S., Bertoni, A. G., ... Dyer, A. R. (2012). Association of Weight Status With Mortality in Adults With Incident Diabetes. JAMA, 308(6), 581–590. 5. Chivers, Tom. Diabetes: The facts and figures. The Telegraph, 10 Oct. 2014.

The Bigger Implications

exosome class, the team has pinpointed one “suspect” miRNA: microRNA 155. It is predicted that this miRNA is involved in the inactivation of proteins critical to insulin recognition.

There are broad-scale implications of Olefsky's research. Not only can they lead to earlier detection and more personalized treatment plans, but they also indicate that it is the inflammatory response of adipose tissue, and not excess weight itself, that causes type II diabetes—precisely what Araneta goes on to claim in her own research. Through extensive comparisons in diabetes incidence, BMI, and adipose tissue levels among a myriad of races and ethnicities, Araneta also concluded that excess weight should not be the primary indicator of diabetes risk. During her research, Araneta found that Asian populations—especially those of Filipino descent—have one of the highest risks for type II diabetes onset. However, as a whole, their BMIs are significantly lower than other ethnicities. Through further analysis and study compilations, Araneta

3. Araneta, Maria Happy. Diabetes and Asian Americans: The Need to “Screen at 23”. The Association of Asian Pacific Community Health Organizations, 9 Nov. 2017. Speech.

found that many of these Asian-American at-risk populations have a higher percentage of body fat when compared to white people of the same BMI. This observation especially rings true with regards to VAT in Asian-American women. In a qualitative comparison of a 160-pound (BMI = 25) African-American woman, and a 115-pound (BMI = 20) Filipina-American woman, the VAT areas were calculated to be 25.4 cm3 and 84.0 cm3, respectively, exhibiting a dramatic difference in fat composition.

Taking Araneta’s findings with Olefsky’s, it can be inferred that the elevated adipose tissue concentrations in Asian subgroups can explain the heightened diabetes risk in these populations, despite the normalrange BMIs. Both Olefsky's and Araneta’s research findings have the potential to revolutionize detection, diagnosis, and treatment techniques for type II diabetes in the future. By targeting specific signaling pathways, measuring adipose tissue levels, and understanding the molecular basis of the disease, perhaps we can better identify at-risk patients at their most premature stages, setting precedent for optimized monitoring and care.

References 1. Araneta, M. R., & Barrett-Connor, E. (2005). Ethnic Differences in Visceral Adipose Tissue and Type 2 Diabetes: Filipino, African-American, and White Women. Obesity Research, 13(8), 1458–1465. 2. Araneta, M. R., Kanaya, A. M., Hsu, W. C., Chang, H. K., Grandinetti, A., Boyko, E. J.,... Fujimoto, W. Y. (2015). Optimum BMI Cut Points to Screen Asian Americans for Type 2 Diabetes. Diabetes Care, 38(5), 814–820.

6. Centers for Disease Control and Prevention. Diabetes Report Card 2017. Atlanta, GA: Centers for Disease Control and Prevention, US Dept of Health and Human Services; 2018 7. Lee, Y., Kim, J., Osborne, O., Oh, D., Sasik, R., Schenk, S., ... Olefsky, J. (2014). Increased Adipocyte O2 Consumption Triggers HIF-1α, Causing Inflammation and Insulin Resistance in Obesity. Cell, 157(6), 1339–1352. 8. Mcnelis, J., & Olefsky, J. (2014). Macrophages, Immunity, and Metabolic Disease. Immunity, 41(1), 36–48. 9. Ying, W., Riopel, M., Bandyopadhyay, G., Dong, Y., Birmingham, A., Seo, J. B., ... Olefsky, J. M. (2017). Adipose Tissue Macrophage-Derived Exosomal miRNAs Can Modulate In Vivo and In Vitro Insulin Sensitivity. Cell, 171(2).

WRITTEN BY

ANOKHI SAKLECHA Anokhi is a Biochemistry and Cell Biology major from John Muir College. She will graduate in 2018.

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TAKING A LOOK AT WRITTEN BY SKYLAR RAINS â&#x20AC;¢ ILLUSTRATED BY LAUREL BOWLING

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FEATURES As our knowledge and ability to synthesize new chemicals develops, unintended consequences have risen. Human-made chemicals are entering the environment at alarming rates and do not readily degrade. This results in an unprecedented accumulation of chemicals in the environment. Ecosystems like the ocean are being contaminated by these chemicals, which is posing a threat to both humans and marine life. Researchers at UC San Diego and the Scripps Institute for Human Health are working to understand the effects these chemicals have on organisms in order to combat the threat they pose to environmental health.

ay by day the oceans become more polluted. Ocean toxicologists seek to answer the question: what are the effects of this growing pollution? Toxicology is the study of the adverse effects of chemical, physical, or biological agents on living organisms and the ecosystem, including the prevention and remediation of these effects. Researchers at the Scripps Institution of Oceanography (SIO) are diving into this issue head first, seeking to discover just how many human-made chemicals reside in our ocean animals, and how those chemicals affect their physiology. Some human-made chemicals, like dichlorodiphenyltrichloroethane (DDT), have been shown to damage the environment. DDT is an insecticide that was created during World War II and used throughout the second half of the 20th century until it was shown to have many adverse environmental effects. As a result, DDT was banned in the United States in 1972; shortly after that, the majority of the world stopped using it. Currently, DDT is only used for combating malaria in some African countries. It was thought that after its ban, DDT would disappear from the environment. However, DDT has been classified with a group of toxins called persistent organic pollutants (POPs), that do not readily degrade, persist in the environment, and accumulate in the tissues of animals. Thus, almost 50 years after its ban, DDT still exists in the environment and has made its way into the oceans. sqonline.ucsd.edu

DDT in Marine Mammals Researchers at the Scripps Center for Oceans and Human Health (SCOHH) have identified DDT-related compounds accumulating in southern California bottlenose dolphins. This buildup of DDT and its metabolites in living organisms occurs when chemicals bypass the plasma membrane of cells and are stored in fats and proteins of organisms. These chemicals increase or “bioaccumulate,” as one moves up the food chain as organisms within each successive trophic level concentrate everincreasing amounts of toxins within their tissues. The researchers at SCOHH collected blubber samples from eight sexually mature male bottlenose dolphins from nearshore and deeper off-shore regions of the Southern California Bight (SCB). Parts of the SCB are considered a Superfund site, a location the Environmental Protection Agency has designated as contaminated by hazardous waste. As a result, it is a candidate for cleanup due to the potential risks it poses to human health and the environment. SCB got this designation because of past contamination with DDT from the Montrose Chemical Corporation of California. The company dumped thousands of tons of DDT and waste products into a nearby sewer system. The dolphins analyzed in this study were found fatally stranded on beaches from 1995 to 2010. Their blubber was analyzed via comprehensive gas chromatography coupled with time-

of-flight mass spectrometry. Researchers looked for DDT-related compounds (known compounds and potential DDT-related transformation products or metabolites) and Dicofol, a pesticide currently used and synthesized from DDT. The researchers at SCOHH confirmed the presence of 45 DDT-related compounds in the samples of dolphin blubber. This includes the six major isomers of DDT, breakdown products, and related compounds. The majority of the compounds found are not typically monitored in the environment. This discovery is a breakthrough because the study identified DDT transformation products beyond those that were previously known to occur and normally monitored. This provides evidence that the extent to which chemicals can be present in marine life is more complicated than previously thought. Bioaccumulation is a very problematic issue. Compounds like DDT, fire retardants, and mercury enter the ocean from sewage outfalls and the atmosphere via volatization. The chemicals volatilize into the atmosphere, spread out across the globe, and then enter the ocean. Once in the water, a variety of factors like adsorption, dissolution, volatility, and photostability can alter the chemicals’ interactions and behaviors within the environment. These processes are explained by chemodynamics, which determines the separation of chemicals into different environmental compartments. Compartments are defined parts of SALTMAN QUARTERLY

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FIGURE 1: An illustration of the gradient of total POP concentrations found in yellowfin tuna caught in various locations throughout the globe. Tuna caught in the Northwest Pacific Ocean (NWPO) were found to have the lowest total POP concentrations, whereas tuna caught in the Northeast Atlantic Ocean (NEAO) had the highest. The tuna caught in the Southwest Pacific Ocean (SWPO), Indian Ocean (IO), Gulf of Mexico (GOM), and Northeast Pacific Ocean (NEPO) were found to have levels between the two extremes, as displayed through the color gradient.

the environment, like fish, sand, or the atmosphere. Chemicals are more prevalent in some compartments more than others. This becomes dangerous because chemicals like DDT and other pollutants end up in fish and other living organisms when they ingest primary producers that are polluted with the toxic chemicals. These POPs are stored in the fish tissues, then passed into higher trophic-level consumers such as the dolphins in this study. Another study conducted by the Kurle lab at UC San Diego’s EBE Section of the Division of Biological Sciences also demonstrated that these POPs in the SCB make their way into California sea lions and into highly endangered California condors that eat contaminated sea lion carcasses. POPs are toxic and adversely affect human and animal health and consequently harm the environment. What makes POPs especially dangerous is that they can spread throughout the world via atmospheric transport by wind and water. These POPs persist in the environment for long periods of time and make their way up the food chain through bioaccumulation. This issue is so serious that the United Nations joined forces to draft the Stockholm Convention, a treaty to reduce or eliminate the production and release of twelve key POPs. DDT and polychlorinated biphenyls (PCBs), a known carcinogen previously used in production 24

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of dielectrics and coolant fluids, and also a contributor to the SCB’s classification as an EPA Superfund site are two of the twelve chemicals included in the treaty.

Persistent Organic Pollutants in Fish Another lab at UC San Diego is studying the effects of chemicals on ocean environments. Under the guidance of Dr. Amro Hamdoun, the Hamdoun Lab at SIO studies POPs with respect to their effects on humans who consume fish. These chemicals are taken up by fish in a manner similar to how DDT is. The study examined the geographic differences in POP levels of yellowfin tuna. This research is essential because the distribution of POPs across the oceans remains largely unknown and is important for guiding consumers away from eating contaminated fish. Also, understanding the differences in residue may lead to a future where extremely contaminated fish can be eliminated from the food supply. Nearly three billion people rely on fish as a major food source, so preventing consumption of POP contaminated fish is essential in order to reduce the risk of human exposure to POPs. The Hamdoun lab’s study sampled sections of dorsal muscle from 117 yellowfin tuna from twelve separate locations across the globe. Samples were collected from

places such as the Atlantic Ocean, Gulf of Mexico, Indian Ocean, and the South and North China Seas. From the sample, POP levels were examined using combined liquid or gas chromatography and mass spectrometry. The analysis screened for different pollutant classes: including 209 PCBs, 29 types of organochlorine pesticides (OCPs), and nine polybrominated diphenyl ethers (PBDEs). Fish from certain sites were not screened for all of these POPs. From this data, total POP levels were compiled. The researchers defined “total POP levels” as the sum of all OCPs, PBDEs, and PCBs measured in fish from the eight sites where all these chemicals were tested. Testing found that POP levels varied significantly among sites. The three sites with the lowest total POP concentrations were the Northwest Pacific Ocean (0.41 ± 0.16), the Southwest Pacific Ocean (0.54 ± 0.21), and the Indian Ocean (0.69 ± 0.30). The three locations with the most total POP concentrations were the Gulf of Mexico (10.05 ± 6.2), the Northeast Pacific Ocean (12.29 ± 6.8), and the Northeast Atlantic Ocean (14.93 ± 5.4). The study found that the fish caught from sites offshore of North America and Europe had far higher POP concentrations than the fish from sites located in the waters of Asia and Oceania. In each site, the fish caught had differences in the individual pollutant classes that sqonline.ucsd.edu

FEATURES

Although these linger, now is not the time to accept defeat. We must actively seek out ways to reduce the amount of POPs in our atmosphere and oceans before it is too late.”

comprised total POP levels. For example, in the Gulf of Mexico fish had higher concentration of PCBs, whereas fish caught in the Northeast Pacific Ocean had higher concentrations of OCPs, on average. The researchers attribute these geographic differences in tuna POP levels to “historical deposition, atmospheric transport effects and/ or local circulation patterns.” In short, there are many factors that contribute to the variability of pollution levels in regions of the ocean and consequently in fish. In an interview, Hamdoun stated that it is becoming increasingly important to be aware of where fish meant for consumption is caught. Hamdoun elaborated that further research should be conducted on developing new technology to lower the cost of analysis of contaminant levels in fish so that it can be possible to sample fish throughout the ocean on a larger scale. He recommended that we apply our understanding of the biology of chemicals we have gained from pharmaceutical research, such as the processes by which chemicals cross membranes and degrade in the body, and apply it to industrial chemicals. If this were done, we could make industrial chemicals sqonline.ucsd.edu

FIGURE 2: Featured is the short beaked common dolphin, Delphinus delphis. The dolphin is only one of many animals to be heavily affected by toxic bioaccumulation spanning the Earth's oceans.

References

which are functional and do not end up in food, breastmilk and persist in the environment.

What Does the Future Hold? Overall, there is no question that humanmade chemicals like POPs are present in marine life. More research needs to be performed to fully understand the effects of these chemicals on both animals and humans. Researchers at UC San Diego are tirelessly working to understand this issue to improve the health of both our oceans and our population. What can we do as scientists and concerned members of humanity? We can seek to lessen the effects of bioaccumulation, strengthen the testing done on compounds to assess for possible environmental consequences, and encourage public policy to impose harsher punishments on companies that refuse to comply with environmental regulations. Although the effects of bioaccumulation linger, now is not the time to accept defeat, and we must actively seek out ways to reduce the amounts of POPs in our atmosphere and oceans before it is too late.

1. Mackintosh, S. A., Dodder, N. G., Shaul, N. J., Aluwihare, L. I., Maruya, K. A., Chivers, S. J., . . . Hoh, E. (2016). Newly Identified DDT-Related Compounds Accumulating in Southern California Bottlenose Dolphins. Environmental Science & Technology, 50(22), 12129–12137. 2. Nicklisch, S. C., Bonito, L. T., Sandin, S., & Hamdoun, A. (2017, June 26). Geographic Differences in Persistent Organic Pollutant Levels of Yellowfin Tuna. Retrieved from https://www.ncbi.nlm.nih.gov/ pubmed/28686554

WRITTEN BY

SKYLAR RAINS Skylar is a Physiology and Neuroscience major from Eleanor Roosevelt College. She will graduate in 2018.

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RESEARCH 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.

A monarch butterfly, Danaus plexippus, drinks nectar from a butterfly weed, Asclepias tuberosa. Photo by Tomomi Yoshida SALTMAN QUARTERLY

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RESEARCH

RESTING STATE BRAIN DYNAMICS SUPPORTING CREATIVITY Subathra Raj

Thurgood Marshall College Cognitive Science Major with a Neuroscience Specialization Class of 2019

Advisor: Ying Choon Wu, Ph. D

UC San Diego, Swartz Center for Computational Neuroscience Creativity is often seen as the expression of fluid thought and defined as the ability to generate original, useful contributions to human endeavors in the form of new ideas, representations, material objects, and so forth. This study examines how different types of virtual search tasks can impact creative thinking, as well as neurodynamics conducive to creative thought. In particular, we examine how a task that involves open-ended exploration modulates resting state electroencephalographic (EEG) alpha power (8–12 Hz) relative to a task that simply involves following a series of instructions. Our results indicate that alpha power variability is linked to task-related changes in performance on the Alternative Uses Task (AUT), which is a psychometric measure of creative abilities allowing for an operational measurement of creativity. This study advances our understanding of human creativity from a neurocognitive perspective.

INTRODUCTION Psychologists have linked scope of attention to aspects of information processing that support insight and creative thought1,2. Broad or diffuse attention is characterized by less efficient inhibition or filtering of task-irrelevant information, leading to the greater likelihood that disparate concepts come to be connected. This contrasts with narrow or focused attention, where there is less frequent connection of differing concepts. Individuals distinguished by higher levels of diffuse attention3 or lower latent inhibition abilities1 have been associated with superior immediate and longterm creative achievement, respectively. Further, visual search tasks have led to improved scores on measures of creative thought when targets and distractors are scattered over a large versus small display field, eliciting broad or narrow attentional mode, respectively4. Electroencephalography (EEG) is a method that is used to record the electrical activity of the brain through non-invasive electrodes applied to the scalp. Thus far, several studies have indicated a relationship between the topography and magnitude of resting state EEG power, and the likelihood of experiencing insight5,6,7. Kounios et al5 demonstrate that during rest, spectral activities in the alpha frequency band (8–12 Hz) tend to be diminished over the right hemisphere in people who experience insight more often. Alpha power represents alpha waves, one of the numerous types of brain waves recorded sqonline.ucsd.edu

through EEG, and is most prominent during times of wakeful relaxation8. Because suppression of alpha power is linked to cortical excitation9,10, this outcome may reflect more engagement of visual cortex on the part of high insight problem solvers. Our project investigated whether creative thinking abilities can be at least transiently enhanced by participating in virtual reality based tasks. These tasks are designed to stimulate either a broad or narrow focus on the participant’s virtual environment, such that changes in resting state EEG dynamics that might accompany modulations of creative abilities could be analyzed. In the task that stimulates broad attention, the participant was prompted to discover a creative solution to the problem at hand, whereas in the task stimulating narrow attention they followed a series of instructions. By understanding more about brain states that support creativity, it may be possible to develop neurofeedback training protocols to enhance creative abilities or predict optimal periods for creative work on the basis of resting state EEG. Solving the discovery-based task is expected to lead to changes in resting state alpha activities relative to the instruction-based task.

METHODS AND MATERIALS A. Participants. The Institutional Review Board of UC San Diego approved this experiment protocol. Twenty-three volunteers were recompensed for their participation at a rate of $15 per hour. All were neurologically healthy university students who gave informed consent.

B. Materials. TA 14-channel wireless Emotiv Epoch headset was

used to collect EEG data. The game development platform, Unity, was used for the virtual reality tasks. Unity was chosen over other platforms due to its capacity for creating high-quality 3D games through which to present the scenarios of broad and narrow attention for both the “creative game” and “control game” conditions, respectively. Both game scenarios involve participants getting into the house, but they were prompted to do so differently in each condition. In the “control game,” participants were asked to follow directions on a series of notes that led them to a key used to unlock a door. In the “creative game,” they were asked to discover a way to break into the house. The instrument used to evaluate creative thinking was the Alternative Uses Task (AUT) (Torrance 1966). In brief, the AUT is a widely used test of divergent thinking that requires participants to enumerate as many novel uses for a conventional item (e.g. a paper clip) as possible within a fixed time. Fluency scores were determined by recording how many unique uses each participant produced for a given item, and were assessed for AUT sessions administered both before and after each virtual reality task. Differing fluency scores after each VR task would indicate a change in levels of creativity as a result of completion of the control or creative task, where a higher fluency score indicates a higher level of creativity.

C. Procedure. Each subject participated in two experimental

sessions, with one session involving the control, instruction-based game and the other involving the creative, discovery-based game. Two scenarios were created using Unity—each tasking the player with finding a way to enter a locked house after having lost the key. In the narrow attention version, the player followed a trail of notes with SALTMAN QUARTERLY

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Following Instructions Discovery

Figure 1. Scalp locations of O1 and O2 channels

control

creative

Pre

10.3 (7.9)

9.6 (4)

Post

10.1 (4.4)

10.4 (4.5)

Table 1. Mean fluency scores (mean number of words produced per three minute test session) before and after the control and creative problem solving tasks.

instructions where to look for a hidden key. In the broad version, it was necessary to discover alternative entry points and devise methods for accessing them using serendipitous tools in the environment. The order of these sessions was counterbalanced such that the instructionand discovery-based games were presented in alternating orders across individuals, with intervals between sessions ranging from 3 to 31 days. Participants received oral and written instructions for each task. A 3-minute resting state pre-game baseline EEG was recorded at the start of each session, followed by the first AUT session. The participants then played either the instruction- or discovery-based computer game for 15 minutes, and a 3-minute resting state postgame baseline EEG was recorded following the game, and followed by the second AUT session. The four words used for the AUT were “paperclip,” “brick,” “button,” and “newspaper.” The order of these words were counterbalanced by incorporating a random number generator into the script of the experimental program in order to present a randomly selected word during each of four trials for each subject without repetition of any of the AUT words. Over the course of their two sessions, each participant was presented all four of the words amongst the AUT sessions preceding and following both the “control” and “creative” games.

D. Data Acquisition. EEG data were recorded over 14 scalp

locations using the wireless Emotiv Epoch headset with a sampling rate of 128 Hz. AUT responses were recorded using a voice recorder before being manually transcribed for each session.

E. Behavioral Data Analysis. A fluency score was obtained for

each participant’s AUT session by counting the number of responses for each session. Participants whose fluency scores improved after the creative game were separated from those whose fluency scores decreased for analysis of their EEG power spectra.

F. EEG Analysis. 1) Preprocessing: EEG signals were analyzed using MATLAB (The

Mathworks, Inc.) and the open source toolbox, EEGLAB (Swartz Center for Computational Neuroscience, University of California San Diego). EEG time series data were inspected visually to exclude poor-quality channels. Next, data from baseline rest periods were extracted from the full dataset and divided into one-second segments. 28

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-5

Decrease in inFluency FluencyScores Scores Decrease

Increase in in Fluency FluencyScores Scores

Figure 2. Differences obtained by subtracting mean alpha power on pre-game baseline from post-game baseline over the O1 electrode for each participant. Positive values reflect greater power on post-game versus pre-game baselines; negative values, the reciprocal.

Segments of data heavily contaminated by movement and other nonbrain artifacts were identified and excluded via EEGLAB algorithms sensitive to statistically improbable values. Thirteen subjects were eliminated from this analysis because it was not possible to obtain clean data for both the Emotiv O1 and O2 channels located above the occipital lobe area. 2) Pre- and Post-Game Baselines: Out of the 14 Emotiv Epoch channels, channels O1 and O2 were selected for further analysis of the pre- and post-game baseline EEG (Figure 1) because these channels presented the lowest levels of EEG background noise and were clean for the majority of the participants. Estimates of power spectral density were computed from baseline rest EEG (3 to 50 Hz) and graphed for each subject. Mean power spectra for alpha level frequencies (α: 8–12 Hz)were computed by averaging power estimates within this frequency range.

RESULTS A. AUT Fluency Scores. After the discovery-based problem solving

task, four participants generated more alternative uses relative to their pre-game baseline, whereas six participants generated fewer. Mean fluency scores, defined as the mean number of words produced per three minute session, from these two groups were analyzed separately (Table 1a, 1b). The group whose fluency score decreased or increased following the “creative game” will be referred to as the “decrease in fluency scores” versus the “increase in fluency scores” group, respectively. The “decrease in fluency scores” participants also showed a decrease in fluency following the instruction-based task. The “increase in fluency scores” participants also showed an increase in fluency following the instruction based task. However, the fluency scores decreased or increased by a larger magnitude following the “creative game” compared to following the “control game” for both groups.

B. Resting-state Dynamics. Figure 2 and Figure 3 plot grand

averages of resting-state spectral power derived from left and right occipital EEG (O1 and O2, respectively) from the ten participants with clean O1 and O2 channel data. Over the left occipital channel, alpha power gained in magnitude after the discovery-based task for all participants; however the magnitude of this gain was greater for sqonline.ucsd.edu

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Following Instructions Discovery

4 2

0 -2 -4

Decrease in Fluency FluencyScores Scores Decrease in Increase in Fluency Increase FluencyScores Scores

Discovery Discovery

-5 FollowingInstructions Instructions Following

-6 Figure 3. Differences obtained by subtracting mean alpha power on pre-game baseline from post-game baseline over the O2 electrode for each participant. Positive values reflect greater power on post-game versus pre-game baselines; negative values, the reciprocal.

Figure 4. Mean alpha power on post-game baseline over the O1 electrode for each participant. Positive values reflect greater power on post-game baselines; negative values, the reciprocal.

the increasing fluency scores group (Figure 2). This effect appears confined to the left hemisphere (Compare Figure 3 with Figure 2).

DISCUSSION AND CONCLUSIONS This study yielded three noteworthy findings. First, discovery-based problem solving led to increased resting-state alpha activity levels centered over the left occipital region of the scalp, whereas minimal pre-post differences for either group were detected over the right occipital homologue. This finding suggests an important role for the left-hemisphere in supporting aspects of creativity. In our study, resting state alpha power of the increasing fluency group tended to be higher after the participants were tasked with discovering a game solution versus simply following directions. On the other hand, pre- versus post-game levels of resting state alpha were comparable irrespective of task for the decreasing fluency group. This pattern of outcomes is consistent with other research suggesting that alpha activities tend to increase during creative thought, perhaps reflecting more internally versus externally focused attention12. Importantly, though, the overall levels of resting-state alpha power were higher for the decreasing fluency group relative to those whose fluency scores actually benefited from the discovery-based task. In other words, the upward shift in alpha power appears more important than overall magnitude. It is noteworthy that the pattern of outcomes described here differs substantially from outcomes described in related studies, such as that of Kounios et al5, who reported reduced rather than enhanced alpha power and greater right rather than left hemisphere effects in participants who tended to solve puzzles with insight. One explanation for these disparities relative to the present study centers on the differences in experimental tasks. In Kounios5, participants were asked simply to solve a series of anagram puzzles. In the present work, the task involved multiple possible solutions and an openended search space. Importantly, while the right hemisphere has traditionally been implicated in insight13 and semantic processing14 that could support creativity, it appears that right hemisphere engagement is not always crucial during creative problem solving. These findings supplement our understanding of human creativity and its corresponding electrical activity levels present in the brain. sqonline.ucsd.edu

Using findings from this and similar studies, neurofeedback training protocols involving alpha wave stimulation can be developed to enhance creative thinking, and ideal periods for creative work can be predicted, in keeping with existing techniques15. This study also exemplifies the utility of 3D gaming as a tool to stimulate problem-solving. Next, we plan to perform the experiment with an EEG cap capable of recording 128 channels in order to obtain both more abundant and cleaner data in comparison to the wireless Emotiv Epoch. We also plan to score the AUT for originality, flexibility, and elaboration to supplement the fluency scores in order to determine how these other aspects of creative thinking are linked to resting state EEG. References 1.

Carson SH, Peterson JB, Higgins DM (2003) Decreased latent inhibition is associated with increased creative achievement in high-functioning individuals. Journal of Personality and Social Psychology 85: 499. 2. Rowe G, Hirsh JB, Anderson AK (2007) Positive affect increases the breadth of attentional selection. Proceedings of the National Academy of Sciences 104: 383–388. 3. Kasof J (1997) Creativity and breadth of attention. Creativity Research Journal 10: 303–315. 4. Friedman RS, Fishbach A, Förster J, Werth L (2003) Attentional priming effects on creativity. Creativity Research Journal 15: 277–286. 5. Kounios J, Fleck JI, Green DL, Payne L, Stevenson JL, et al. (2008) The origins of insight in resting-state brain activity. Neuropsychologia 46: 281–291. 6. Kounios J, Frymiare JL, Bowden EM, Fleck JI, Subramaniam K, et al. (2006) The prepared mind: Neural activity prior to problem presentation predicts subsequent solution by sudden insight. Psychological Science 17: 882–890. 7. Wu YC, Jung M, Lock D, Chao E, Jung TP (2014) Discovering optimal brain states for problem solving with EEG. 6th International IEEE/EMBC Conference on Neural Engineering. San Diego, California. 8. Torrance EP (1966) Torrance Tests of Creative Thinking. Bensenville, IL: Scholatic Testing Service. 9. Mednick SA (1962) The associative basis of the creative process. Psychological Review 69: 220–232. 10. Taft R, Rossiter JR (1966) The Remote Associates Test: Divergent or convergent thinking? Psychological Reports 1966: 1313–1314. 11. Chun-ling L, Jung M, Wu YC, She HC, Jung TP (2015) Neural correlates of mathematical problem solviing. International Journal of Neural Systems Vol. 25 no. 2 2015: 1550004-11550004-17.

Acknowledgments This research was supported by the Chancellor’s Research Excellence Scholarship (CRES).

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DIFFERENCES IN HETEROSPECIFIC POLLEN DEPOSITION ACROSS POLLINATION SYNDROMES IN MONTEVERDE, COSTA RICA Kara Powell1 , AndrĂŠs Camacho2, Emilia Triana2

UC San Diego, Revelle College, Ecology, Behavior, and Evolution The Monteverde Institute, Monteverde, Puntarenas, Costa Rica

1 2

Pollination is an essential ecosystem service that is crucial for plant reproduction. The efficiency of pollination can be reduced due to the transfer of heterospecific pollen, defined as pollen from different species. Flower characteristics vary in order to attract certain types of pollinators. This phenomenon is classified in groups of flower morphology characteristics known as pollination syndromes. Little research exists that describes heterospecific pollen transfer (HPT) within these pollination syndromes. I investigated pollen deposition across pollination syndromes in Monteverde, Costa Rica in two different sites: San Gerardo and Curi Cancha. I mounted stigmas from each location onto microscope slides in order to count the amounts of both conspecific and heterospecific pollen. I then classified each species into a certain pollination syndrome using floral characteristics outlined by the USDA. I divided the plant species in our data into two categories, generalist and specialist, based on their strategy for attracting pollinators. I found that there was no significant difference in the amount of conspecific and heterospecific pollen deposition between generalist and specialist plant species. Heterospecific pollen deposition is not as affected by pollination syndrome specificity as I predicted.

INTRODUCTION Pollination allows plants to achieve sexual reproduction, increasing their ability to adapt to changing environments. Without pollinators, plants would be forced to create clones of themselves in order to reproduce, which would reduce the amount of genetic variability in a given population. Genetic variability is an essential part of population success because it provides a chance for survival when the population is faced with novel diseases, disasters, or human interferences1. Many flowers have morphological characteristics that are specialized to attract certain types of pollinators2. The occurrence of specialization for certain types of pollinators led to the establishment of pollination syndromes, which are groups of floral characteristics that are meant to attract a certain type of pollinator. 30

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Examples of pollination syndromes include bee-pollinated flowers, hummingbird-pollinated flowers, and moth-pollinated flowers. Some of these pollination syndromes are more specific than others, since their morphological characteristics prevent certain pollinators from accessing the pollen. Examples of specialist pollinators include hummingbirds, since their long, tubular beaks allow them to reach the nectar reward at the end of long, tubular flowers3. An example of a plant pollinated by hummingbirds in this study is Razisea spicata, which is pollinated by long-billed hummingbirds. However, pollinators can also be generalists, visiting many different species of flowering plants that do not have restrictive floral morphology. Generalist flowers are those that are visited by generalist pollinators, and they are more likely to receive visits from a wider array of pollinators than specialist flowers1. Due to the diversity of flowers visited by generalist pollinators, they are more likely to deposit heterospecific pollen, or pollen from a species other than the flower being visited. Heterospecific pollen can reduce or inhibit reproduction success4. In the same way that there is limited space on the stigma, there is also limited space on the bodies of pollinators. Pollinators that visit multiple species will carry multiple types of pollen, reducing the amount of pollen for any given species that the pollinator has visited. This reduces the potential for male reproductive success for all species involved5. Additionally, heterospecific pollen on the stigma can form blockages that prevent pollen tubes from generating, altering female reproductive success 6. Another study that measured the effect of heterospecific pollen on the stigmas of flowers found in the understory of the Costa Rican cloud forest found that less pollen tubes were generated in the presence of heterospecific pollen7. Due to interspecific pollen competition, a higher prevalence of heterospecific pollen has been shown to reduce seed output, thus reducing plant fitness8. In a study conducted in an alpine meadow in Colorado in which two hummingbird-pollinated speciesâ&#x20AC;&#x2122; flowering periods overlap for some time, seed set is reduced during the period of overlap 9. Within natural systems, there might be natural selection pressure to evolve differing flower morphologies in order to reduce the amount of heterospecific pollen transfer (HPT). The first objective of this study is to compare the differing levels of heterospecific pollen present in flowers that belong to certain pollination syndromes. I speculated that more specialized syndromes, such as hummingbird-pollinated flowers, would receive less heterospecific pollen than syndromes that appeal to more generalist pollinators (as in bee-pollinated flowers).

Heterospecific Pollen Deposition in Razisea spicata. In an

attempt to reduce the transfer of heterospecific pollen, plants within a community evolved morphological characteristics meant to attract a certain type of pollinator and prevent others from pollinating.10 This phenomenon is termed the Sexual Architecture Hypothesis. This hypothesis can also refer to different sized floral structures involved in pollination, such as different length styles and filaments. sqonline.ucsd.edu

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Styles are the structures that support filaments, which in turn support the pollen-producing anthers. R. spicata has similar length filaments across individuals, but the lengths of the styles differ. Previous studies have suggested that stigma position is an important floral trait in determining the amount and species of pollen received,since flowers with styles of the same length have morphologies that position the stigmas in different locations to be received by the pollinator 11. In a study comparing the deposition of heterospecific pollen between patches of R. spicata of different densities, researchers found that heterospecific pollen deposition was sporadic in flowers pollinated by long-billed hummingbirds12. The second objective of this study aimed to determine if the sporadic nature of heterospecific pollen deposition was due to different style lengths. My prediction was that the amount of heterospecific pollen deposition would depend on the style length between the anthers and the stigma, with certain lengths having higher amounts of heterospecific pollen than others.

MATERIALS AND METHODS The study was conducted in Monteverde, Costa Rica, collecting only from ecological reserves in order to prevent using species that were planted for use as ornamental foliage. I collected flower samples over

a two-week period from 13 November 2017 to 27 November 2017. I visited two sites, the San Gerardo Research Station (at the Children’s Eternal Rainforest) and Curi Cancha Reserve. (San Gerardo is a Lower Montane Rain Forest at 1550–1850 m elevation, Curi Cancha is a Lower Montane Wet Forest at 1450–1600 m elevation.13) In each site I walked multiple trails and collected the flowers that were visible from the trail and not dangerous to access (usually within two meters from the ground). I usually collected all flowers that were available, but if they were abundant I would limit myself to approximately four flowers. I collected flowers from the ground as long as the parent plant could be identified. I covered 10.7 kilometers of trail in San Gerardo and 6.2 kilometers of trail in Curi Cancha. Flowers were collected and identified using A Field Guide to Plants of Costa Rica14 and A Guide to Tropical Plants of Costa Rica 15. After collecting the flowers and taking photos and samples of the plant specimens, I mounted the anthers and stigmas onto a microscope. By doing this, I created a preserved anther library in order to be able to identify the types of pollen present on each stigma as either conspecific or heterospecific. For individuals of the species R. spicata, I also recorded the length of the style. Upon returning from the field, I counted the number of conspecific grains and heterospecific grains present on each stigma using a light microscope.

Appendix 4: Pollinator Syndrome Identification Chart—USDA15

Trait

Ant

Bat

Bee

Beetle

Bird

Butterfly

Fly

Moth

Wind

Color

inconspicuous

white, green, purple

bright white, yellow, blue, UV

white, green

scarlet, orange, red, white

bright red or purple

pale, dark brown, or purple pink, white

pale red, purple, pink, white

pale green, brown or colorless

none

present

none

present

none

strong, mustry emitted at night

fresh, mild, pleasant

none to strongly fruity or foul

none

faint, fresh

putrid

strong, sweet, emitted and night

none

abundant, somewhat hidden

usually present

sometimes present

ample, hidden

usually absent

ample, hidden

none

ample

limited, often sticky, scented

ample

limited

abundant, small, smooth

bowlshaped, closed during the day

shallow with landing platform, tubular

cluster of small flowers or large, bowlshaped, solitary

large funnel-like, strong perch support

narrow tube with spur, wide landing pad

shallow funnel-like or complex with trap

regular, tubular without lip

regular and small

Nectar Guides Odor

none/faint

Nectar

Pollen

Flower Shape

small, low-growing close to stem

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I divided the plant species that I collected into four different pollination syndromes (bee, beetle, bird, and butterfly). I classified them using the information available in the literature for previously studied species that already had a known pollination syndrome and then I classified the remaining unstudied species using the USDA criteria16. Once the plant species were divided in this way, I categorized the pollination syndromes into two groups: specialist and generalist. Specialist flowers include bird-pollinated flowers and butterfly-pollinated flowers, while generalist flowers include beepollinated flowers and beetle-pollinated flowers.

RESULTS General Results. In total, I collected 581 stigmas, 251 from San

Gerardo and 330 from Curi Cancha. I collected 20 unique species from San Gerardo and nine species from Curi Cancha. I collected from 167 individual plants from both sites. Most of the specialist flowers in our dataset are bright red and tubular, thus, pollinated by hummingbirds. R. spicata was the most common flower available to collect at the time of our study, which is reflected in the amount of stigmas and individuals from this species in our dataset. Cococicelum pubescens and Fabaceae were only present in Curi Cancha, while Blachea, Calathea, Commelinaceae, Melastomataceae, Miconia, Monochaetum vulcanicum, Solanum, and Phytolacca

were only present in San Gerardo. The distribution of stigmas and individuals collected in both sites can be found in Figure 1. The amount and type of pollen grains found varied greatly between species. The highest amounts of conspecific pollen deposition occurs in Centropogon, Columnea consanguinea, other Gesneriaceae, Poaceae, and Malvaviscus palmanus. Heterospecific pollen deposition in San Gerardo was concentrated in Centropogon, Columnea consanguinea, and other species of Columnea, which are specialist flowers. Centropogon received approximately 11 times more conspecific pollen than heterospecific pollen. Conspecific pollen transfer was approximately 11 times more common than heterospecific pollen transfer. The majority of species received little to no pollen at all (Figure 2).

Pollen Deposition in Specialist and Generalist Flowers. Figure

3 shows that flowers classified as generalist received relatively the same amount of conspecific and heterospecific pollen as those that were classified as specialist. There was high variation in the number of pollen grains counted for each stigma, which led to large standard deviation values.

Pollen Deposition in Razisea spicata. In total, I collected 56

stigmas from R. spicata. I divided the stigmas into categories based on the length in centimeters that the style protruded past the

300 250 Number Collected

200 150 100 50 0

s s s e e e e e a a a a a a a a a a x n dr cea che the ogo sen ne ine caly cea cea mat anu cea oni icum loce cea icat uvo nom oni n a e a a a a a c u m a a n a r p l i R t l l a Bl Ca trop puv olu ang cho Fab ner ate alm ma M lcan hito Po a s ter he S o ia z s cu s p w C ns on o P u n e As t s t e m Ap v i n G oa z C elu cu co a c las m ca Ra lg avis Me ic tu es ea oni a c e d n a Id alv m co m ha Tr lu Dry nc M Co o o C M Number of Individuals Collected

Number of Stigmas Collected

Figure 1. Total number of stigmas collected and number of individuals sampled, from both sites.

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anthers. Figure 4a shows the number of stigmas collected from each length category. There were four categories total: equal length with anthers, 0.5 cm past anthers, 1 cm past anthers, and 2 cm past anthers. Longer styles were relatively rare, with most styles being placed in the “equal” or “1 cm” categories. Figure 4b shows the number of pollen grains deposited in each length category. The stigmas in the “equal length” category received the most conspecific pollen. There was a decreasing

amount of pollen deposition as the distance between the stigma and the anthers increases. The stigmas in the two centimeter category received very little conspecific pollen. The only category that received heterospecific pollen was the “equal length” category, although the amount of pollen remained small. Figure 4b shows the number of pollen grains deposited in each length category. The stigmas in the “equal length” category received the most conspecific pollen. There was a decreasing

Number of Pollen Grains

1000 900 800 700 600 500 400 300 200 100 0

a ter As

m lu ice

c Co

s ve pu

s en

a ne

m lu Co

a ne

s on c a

i gu an

c ba Fa

c vis a alv M

p us

us an alm

ac Po

zis

ta ica p as

s vo Ru

Number of Pollen Grains

1400 1200 1000 800 600 400 200 0

s e e e e a a a a a a a a a a x n dr cea che the ogo ne ine caly cea mat anu cea oni icum loce cea icat nom oni n a a a a a c u m a a n a l Bl Cal trop olu ang cho ner ater alm mat Mi lcan hito Po a sp Sol a za ter he w C ns P n As ti on Ges a cu us p sto Ap vu zis Ce an c ela co ia c a o m c s i g a s R v al M on etu de ne Id alva ra m r ym ha c T u l M D on Co M Conspecific

Heterospecific

Figure 2. Sum of pollen grains deposited for all individuals of each species collected in a) Curi Cancha b) San Gerardo. Each graph displays only the species that were present in that particular site. Error bars show standard deviation.

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Figure 3. A comparison of type (conspecific or heterospecific) and amount (number of pollen grains present) of pollen deposition between generalist and specialist plant species in both sites. Error bars show standard deviation.

DISCUSSION Prevalence of Heterospecific Pollen Deposition. Our results found less heterospecific pollen transfer than I originally expected. This could be due to a number of factors. It could be flowering phenology: the tropical climate of this region provides opportunities year-round for flowering, since resources are available throughout the year. In order to reduce competition for pollinators, plants may diverge their flowering phenologies throughout the year, rather than concentrating it in a single season17 . The diversity of pollinators also plays a role in reducing the amount of heterospecific pollen transfer. Monteverde is located within a biodiversity hotspot18. Thus, the diversity of pollinators in the area is overwhelming; even within the hummingbirds there are two separate pollinator guilds. Longbilled hummingbirds and short-billed hummingbirds have different foraging behaviors and thus carry different species of pollen19 . This is an example of specialization, since these two guilds each focus on certain species instead of competing for the same nectar sources. Thus, the high diversity of pollinators can lead to more specialized interactions, reducing the chance for heterospecific pollen transfer. Differences in Heterospecific Pollen Transfer Between Species. Heterospecific pollen deposition in San Gerardo was concentrated in hummingbird- pollinated species with similar floral morphologies: Columnea consanguinea, Columnea sp., and Centropogon. Columnea is a genus in the family Gesneriaceae, giving this family the highest amount of pollen transfer in our dataset. This could mean that Gesneriaceae is more efficient at attracting pollinators, or that the pollinators that are the most suited to pollinate this family are more active during this time of year. Columnea consanguinea has been witnessed to have hummingbird pollinators 20 . It is safe to assume that related plants are visited 34

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by hummingbirds as well due to similar flower morphologies. Hummingbirds likely visited each of these types of flowers within a single foraging period in order to transfer this amount of heterospecific pollen. In Curi Cancha, the flowers that received the most heterospecific pollen were previously classified as generalists. Poaceae and Ruvos both have small, white flowers that are attractive to bees. One possible explanation for this result is that the flowers collected from Ruvos and Poaceae were concentrated in a relatively small forest clearing in the center of the Reserve. They were interspersed among each other, providing an opportunity for pollinators to transfer pollen between the two species. Heterospecific Pollen Transfer in Generalist and Specialist Pollination Syndromes. The difference between the two syndrome types in the amount of heterospecific pollen received is not statistically significant at a 95% confidence interval (p=0.67). I predicted that generalist plant species would receive more heterospecific pollen deposition than specialist plant species, due to the pollinator accessibility of the morphologies of generalist flowers. The results display more specialist HPT and less generalist HPT than expected. A possible explanation for why specialist flowers received more heterospecific pollen than was expected is their distribution within the forest. Many of the specialized flowers in this study were dispersed intermittently, with individuals being separated by long distances (with the exception of R. spicata, which was found in intermittent clusters). Since specialist flowers are more spread out in their distribution, a specialist pollinator likely visits many flowers of a different species as they travel through the forest. Specialization has been thought to evolve in the circumstance of low focal plant density in order to reduce heterospecific pollen transfer21. However, our study found that the amount of heterospecific pollen was not significantly different between specialist and generalist flowers. Perhaps without specialization, flowers with intermittent distributions would have a much higher rate of heterospecific pollen transfer. Specialization may reduce HPT to relatively the same level experienced by generalist flowers. sqonline.ucsd.edu

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Conspecific

Heterospecific

Number of Pollen Grains Deposited

40 35 30 25 20 15 10 5 0

Figure 4. a) The distribution of style lengths of the stigmas collected from both sites. 0.5

equal

Length of Style Above Anther (cm)

Flowers that were classified as generalist may not have been visited by as many generalist pollinators as originally expected. Even if these flowers were visited by generalist pollinators, there may have been unique opportunities for the pollinators to forage on the same species for extended periods of time. Most of the generalist flowers in this study were found in large clusters where a pollinator could visit multiple individuals of the same species, eliminating the possibility of heterospecific pollen transfer. Open areas of forest tended to harbor large populations of generalist flowers, usually clustered by species. Due to the way flowers were distributed in the sites that were visited, it is possible that floral distribution may have an more of an effect on heterospecific pollen deposition than originally thought. Pollinator foraging behavior is another possible factor that may have influenced our results. In a study comparing the foraging behaviors between a generalist pollinator, the bumblebee 22, and a specialist pollinator, the butterfly, each type of pollinator had different foraging strategies. Bumblebee flights are relatively short, with individuals preferring to visit the next closest flower to the one previously visited. In contrast, butterflies are more likely to skip flowers and fly longer distances in between floral visits23. Due to this behavior, the generalist pollinator may be reducing the amount of heterospecific pollen transfer if the flowers are clustered by species, as they were in this study. Behavior differences between generalist and specialist pollinators were factors that we did not consider when making our original prediction. Taking these explanations into consideration, we must accept that there are more factors influencing heterospecific pollen transfer than just the level of specialization of floral morphology. Pollen Deposition in Razisea spicata. While conducting this study, I became interested in the heterospecific pollen deposition within the species R. spicata due to the differing style lengths across individuals. I found that the amount of pollen deposition decreases as the length of the style increases. This could be due to deposition of pollen from the anthers of the same flower, defined as self-pollination. As a breeze passes through the forest or an animal brushes against the plant, pollen can be shaken off the anthers, and sqonline.ucsd.edu

it is more likely to be deposited on the stigma if the stigma is closer. R. spicata receives large amounts of self pollen24. However, it must be taken into account that not all flowers in this sample experienced deposition solely with pollen originating from the same plant. Since the “equal length” category received some heterospecific pollen, there is evidence that pollinators are transferring pollen between individuals. Thus, there is a possibility that the conspecific pollen in the “equal length” category contains some grains that were transferred through a pollinator.

CONCLUSION Overall, the amount of heterospecific pollen that I found was low across most pollination syndromes. I expected to find more heterospecific pollen from my previous experience and other pollination research10,14. However, it is important to note that these other studies were conducted in temperate zones where flowering is usually a seasonal occurrence. This concentrates pollen transfer into a shorter time period, increasing the chances for heterospecific pollen transfer. In this situation, HPT may be a more influential factor in plant reproductive success than in the tropics. Since there is less seasonal variation in weather in tropical forests, plants are able to produce flowers throughout the entire year. This reduces the amount of overlap in flowering periods where HPT is more likely to occur. There is debate in the field of pollination biology as to whether or not the specialization of floral syndromes is effective in limiting the types of pollinators that visit the flowers17. Other factors, other than the morphological characteristics that separate flowers into different pollination syndromes, may have an effect on pollinator attraction. For example, flower size is a characteristic independent of pollination syndrome that alters the attractiveness of a flower to a pollinator25. Pollinators tend to prefer larger flowers rather than smaller ones 26. This controversy must be taken into consideration when trying to explain why the levels of heterospecific pollen differ between pollination syndromes. There was little heterospecific pollen deposition in R. spicata. I originally thought that the style lengths in this species likely SALTMAN QUARTERLY

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Number of Stugmas Collected

0.5

equal

Length of Style Above Anther (cm)

Figure 4. b) The number of pollen grains deposited in each style length category.

differed in order to reduce the amount of heterospecific pollen transfer. After learning that heterospecific pollen transfer is a rare occurrence, I now speculate that it is an attempt to reduce selfpollination. The stigmas that are separated from the anthers by more than one centimeter had a relatively low chance of self-pollen deposition compared to stigmas that were placed directly next to the source of pollen. Further research is needed, perhaps investigating seed set in the different morphotypes, in order to confirm that this is why the different style lengths exist. References

1. Waser, Nickolas, and Ollerton, Jeff. Plant-Pollinator Interactions: From Specialization to Generalization. Chicago, IL: The University of Chicago Press, 2006. 2. Fenster, C.B. et al. Pollination syndromes and floral specialization. Annual Review of Ecology, Evolution, and Systematics. 35, 375–403. (2004). 3. Fenster, C.B. Selection on Floral Morphology by Hummingbirds. Biotropica. 23, 98-101. (1991). 4. McLernon, S.M. et al. Heterospecific pollen transfer between sympatric species in a mid successional old-field community. American Journal of Botany. 83, 1168-1174. (1996). 5. Wesselingh, R.A., Burgers, H.C., Den Nijs, H.C. Bumblebee pollination of understory shrub species in a tropical montane forest in Costa Rica. Journal of Tropical Ecology. 16, 657–672. (2000). 6. Murcia, C., Feinsinger, P. Interspecific pollen loss by hummingbirds visiting flower mixtures: effects of floral architecture. Ecology. 77, 550–560. (1996). 7. Waites, A.R., Agren, J. Pollinator visitation, stigmatic pollen loads and among-population variation in seed set in Lythrum salicaria. Journal of Ecology. 92, 512–526. (2004). 8. Kohn J.R., Waser N.M. The effect of Delphinium nelsonii pollen on seed set in Ipomopsis aggregata, a competitor for hummingbird pollination. American Journal of Botany. 72, 1144-1148. (1985). 9. Waser, N.M. “Competition for pollination and floral character differences among sympatric plant species: a review of evidence.” In Handbook of Experimental Pollination Biology, edited by C. E. Jones and R. J. Little, 277-293. New York, NY: Van Nostrand Reinhold, 1983. 10. Qiang F., Shuang-Quan H. A directed network analysis of heterospecific pollen transfer in a biodiverse community. Ecology. 94, 1176-1185. (2013). 11. Feinsinger, P. et al. Floral neighborhood and pollination success in four hummingbird‐ pollinated cloud forest plant species. Ecology. 67, 449-464. (1986). 12. Holdridge L.R. Life Zone Ecology. San Jose, Costa Rica: Tropical Science Center, 1967. 13. Garguillo, M., Magnuson, B., Kimball, L. A Field Guide to the Plants of Costa Rica. New York, NY: Oxford University Press, 2008. 14. Zuchowski, W. A Guide to Tropical Plants of Costa Rica. Miami, Fl: Zona Tropical Publication, 2006.

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15. USDA. “Animal pollination.” USDA Forest Service. United States Department of Agriculture. Accessed December 12, 2017. http://www.fs.fed.us/wildflowers/ pollinators/animals/inde x.shtml. 16. Devaux, C., Lande, R. Displacement of flowering phenologies among plant species by competition for generalist pollinators. Journal of Evolutionary Biology. 22, 1460-1470. (2009). 17. CEPF. “Explore the biodiversity hotspots.” Critical Ecosystem Partnership Fund. Accessed February 15, 2018. https://www.cepf.net/our-work/biodiversity-hotspots 18. Murray, K.G. Evaluation of character displacement among plants in two tropical pollination guilds. Ecology. 68, 1283–1293. (1987). 19. Rodríguez, S., Almarales, A, Fenster, C.B. Evaluation of pollination syndromes in Antillean Gesneriaceae: evidence for bat, hummingbird, and generalized flowers. Journal of Ecology. 97, 348–359. (2009). 20. Sargent, R.D., Otto S.P. The role of local species abundance in the evolution of pollinator attraction in flowering plants. American Naturalist. 167, 67–80. (2006). 21. Papaj, Lewis, Alcinda. Insect Learning. Boston, MA: Springer, 1993. 22. Schmitt, J. Pollinator foraging behavior and gene dispersal in Senecio (Compositae). Evolution. 34, 934–943. (1980). 23. Linhart, Y.B. et al. Forager behavior, pollen dispersal, and inbreeding in two species of hummingbird-pollinated plants. Evolution. 41, 679–682. (1987). 24. Hegland S.J., Totland. Relationships between species’ floral traits and pollinator visitation in a temperate grassland. Oecologia. 145, 586–594. (2005). 25. Conner J.K., Rush, S. Effects of flower size and number on pollinator visitation to wild radish, Raphanus raphanistrum. Oecologia. 105, 509–516 (1996).

Acknowledgments I would like to give acknowledgment to the EAP instructors (Frank Joyce, Federico Chinchilla, Sofía Flores and Félix Salazar), especially our advisors, Andrés Camacho and Emilia Triana, and Eladio Cruz for assistance in flower identification. Special thanks to the staff at the Estación Biológica and at the reserves, and to the Rodríguez-Horner family for housing me during our data collection. I was also very lucky to have a positive working environment and support network thanks to my fellow students, they inspired and motivated me throughout the duration of the program. Funding for this project was provided by the UC Education Abroad Program and the Monteverde Institute, which I am very grateful for.

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RESEARCH

EFFECTS OF ANXIETY, DEPRESSION, AND FATIGUE ON DECISION MAKING ABILITIES IN HUMANS Michaela Juels1 Carly Shevinsky1 Pamela Reinagel1

Divison of Biology, UC San Diego, La Jolla, CA 92093

Many mental illnesses alter reward circuits in the brain, which could adversely affect decision making. For example, in both anxiety and depression, individuals are particularly risk-aversive and more cautious in their decisions1. While many studies have focused on the effects of mental illness on strategic and cognitive tasks, little research has gone into how mental illness affects decision making. The present manuscript focuses on “snap decisions”, in which a person must respond to visual information in a fraction of a second. In the pilot study described here, human subjects (N=13) were assessed for anxiety, depression, and fatigue using Patient Reported Outcome Measurement Information System (PROMIS), scored for tiredness and stress on a selfreporting scale, and tested for snap decision-making using a visual motion discrimination task. Correlations were evaluated between the five psychological measures (three PROMIS scores and two self-reported measures) and five performance metrics from the decision-making task. We found preliminary evidence for a correlation between depression and two performance metrics: perceptual threshold (the weakest motion signal a person can reliably detect) and lapse (probability of mistakes on easy trials). Perceptual threshold was negatively correlated with depression (r= -0.63), suggesting heightened perception. Lapse was also negatively correlated with depression (r= -0.53), suggesting higher vigilance. These findings were unexpected, since depression is generally associated with impaired decision making and cognition. Due to small number of subjects and the large number of comparisons made, statistical significance was not reached. The result of this exploratory study identified the two best candidate interactions for focused hypothesis-testing in a second larger cohort.

processing sensory information in a fraction of a second. Some types of work, such as firefighter2 police3, or military4, require making rapid decisions frequently and with serious consequences. Yet these same professions are emotionally taxing and can lead to symptoms associated with anxiety, depression, or fatigue. Therefore it is important to know whether such symptoms could impair snap decision making. This knowledge could encourage mental wellness programs to recognize, treat or prevent anxiety, depression or fatigue in these professions. This research is also vital to college populations, due to the high prevalence of anxiety and depression among college students 5. According to the American Psychological Association, surveys found that 41.6% of college students’ deal with anxiety and 36.4% with depression as of 2013.6 Another survey conducted across 109 different college campuses, found that at least once in the past 12 months 60.8% of students felt “overwhelming anxiety” and 39.1% “felt so depressed it was difficult to function”7. Additionally, 87% of college students experienced at least mild fatigue 8.If there is a connection between these mental health concerns and snap decision making it could have direct consequences for test taking and other areas of academic performance, in addition to their well-being. Snap decision making has been studied in humans and monkeys using a visual motion discrimination task9,10. They tested the ability of subjects to judge the direction of visual motion signals at varying motion strengths. This task measures two things about decision making: subjects must decide how long to view the stimulus before responding; and then decide which direction the motion was going. From these data they created a psychometric curve that could analyze lapse (accuracy at high motion strength), threshold (motion strength that resulted in 75% accuracy), and reaction time. Diagnostic tools are already being used in a clinical setting to better understand patients’ mental health status. For example, PROMIS (Patient Reported Outcome Measurement Information System) is a set of standardized health measures produced by the NIH that can be used by researchers and clinicians to promote better treatment. Relevant to this study it includes standard scales for anxiety, depression and fatigue. This research expands on the diagnostic tools that can be used to assess these mental health conditions; specifically, in regards to decision making abilities. The present manuscript explores if certain psychological effects could alter snap decisions. Specifically, this study hypothesizes that depression, anxiety or fatigue, as measured by clinical questionnaires, may affect decision making capabilities in humans as measured by performance on a snap decision task. This research could lead to better understanding of how these illnesses affect daily functioning.

MATERIALS AND METHODS INTRODUCTION Snap decision making, or making a decision instantaneously, is an important part of human life. From quickly changing lanes on a freeway to catching a falling object, these decisions require sqonline.ucsd.edu

Study Population and Procedures. Subjects were recruited from the University of California, San Diego campus via flyers. Recruiting from a college population is beneficial because of the prevalence of depression and anxiety. Anxiety is the most common of psychiatric disorders among college students, with depression as SALTMAN QUARTERLY

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Data Collection and Analysis. Data were collected from subjects

using a pre-testing questionnaire which asks general questions such as gender and handedness, and a section with PROMIS Assessments11 regarding their psychological state. Specifically, subjects completed the PROMIS Fatigue, Short Form 4a; Emotional Distress—Anxiety, Short Form 4a; and Emotional Distress—Depression, Short Form 4a12. PROMIS assessments were scored according to publisher instructions to obtain T-scores,which are normalized to the distribution of raw scores among American adults. Subjects were then tested on the snap decision making task. During the task, the subjects were asked to make decisions about the direction (left or right) of a visual motion signal (white dots) in hundreds of trials (Figure 1A). The motion coherence (difficulty of task) varied randomly from trial to trial. Data saved by the task software were analyzed using Matlab to collect each subject’s reaction times, number of trials completed, percent trials over three seconds, and accuracy. By varying the coherence of the task, one can construct a psychometric curve using the Palamedes Matlab Toolbox13. These types of curves provide two useful pieces

100% coherence

50% coherence

of information regarding their performance: lapse and threshold (Figure 1B). Threshold is the weakest motion signal for which the subject can correctly identify its direction 75% of the time. Lapse is the percentage of trials the subject gives the wrong response when all the dots on the screen move in the same direction (coherence is 100%), which is the easiest stimulus condition to discriminate. Decision-Making Performance Metrics. To reduce the risk of false positive results, a statistical plan was formed in advance to restrict analysis to performance metrics that varied across subjects and were relatively uncorrelated with each other. To accomplish this, preliminary data from another experiment (same motion discrimination task) were analyzed (N=18 subjects; data not shown). Five performance metrics were found to vary across subjects and be relatively uncorrelated with each other: (1) Reaction Time, the median reaction time to respond over all the trials in a 2 hour session; (2) Trials, the total number of trials completed in a 2 hour session; (3) Threshold, the coherence (motion strength) at which a subject had 75% accuracy; (4) Lapse, the error rate at high motion strength; (5) Late Responses, the percentage of trials in which the subject took longer than 3 seconds to decide. A sixth metric, overall accuracy, was also considered, but it was highly correlated with Threshold (r=-0.96, P=1.3x10-16). Of these two correlated measures, Threshold was chosen because Accuracy could depend on whether a subject happened by chance to get more hard or easy trials in that session, while Threshold would not be

Lapse

% Correct

a close second. Another study found that 87% of college students experienced at least mild fatigue. Therefore, any findings from this experiment can be helpful to college students regarding their mental states and decision making performance. Subjects were financially compensated for their participation, including a performance-based bonus to motivate them to perform the task as well as they could. For this cohort, the funds available to compensate subjects were sufficient to test 13 subjects. All procedures were conducted with the approval and oversight of the UCSD Institutional Review Board.

Threshold

log% coherence

Figure 1. Visual Snap Decision Test. a) Schematic representation of motion coherence. Hundreds of small white drifting dots are displayed against a black background on a computer monitor. 100% coherence is defined as all of the dots moving the same direction, while 50% coherence is when half of the dots are moving in the same direction and the other half are moving in random directions. b) Schematic representation of a psychometric curve. Sigmoidal curve of coherence (motion strength) plotted against percent correct (accuracy). Lapse is how close to 100% correct a subject is when the coherence is high (direction of stimuli is obvious). Threshold is the coherence at which the subject has 75% correct.

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RESEARCH affected by that. Although Threshold and Late Responses were also significantly correlated (P=-0.03) this correlation was weak (r=- 0.40), and therefore both measures were retained. In this study the five selected performance metrics were measured for all N=13 subjects. Distributions of these metrics (including the N=18 previous and N=13 new subjects) were analyzed for normality by Lilliefors Test to determine whether to use Pearson correlation (for Gaussian variables) or Spearman correlation (non-Gaussian variables) when comparing to psychological measures. Psychological Measures. There were ten questions that had been used with previous subjects (N=18) as well as the subjects reported here (N=13); and three PROMIS questionnaires used only in this study (N=13). Analysis for this study focused on the following five items: (1) Tired: self-reported tiredness on a scale of 1–10. (2) Stress: self-reported stress level on a scale of 1–10; (3) Fatigue: level of fatigue based on the PROMIS Fatigue scale 4a (4) Anxiety: level of anxiety based on the PROMIS Anxiety scale 4a (5) Depression: level of depression based on the PROMIS Depression scale 4a. For the questions that had been used previously the distribution of responses was analyzed for all N=18 previous and N=13 new subjects. For the PROMIS scores the distribution was analyzed for

N=13 subjects. All these measures varied across our sample (data not shown). These distributions were checked for normality by Lilliefors Test to determine whether to use Pearson or Spearman correlation when testing for correlations amongst measures or correlations with performance metrics. One advantage of using the PROMIS questionnaires is that they are statistically validated. The scores are T-scores, which are normalized to a mean of 50 and standard deviation of 10, based on data from a large sample of the general population of adults (age 18+) in the United States. Statistics in our small sample appear to be representative of the reference population (not shown). Scores above 55 probably represent clinically diagnosable conditions. In our sample, one person scored >55 for fatigue, two scored >55 for depression and four scored >55 for anxiety (one individual scored >55 on all three scales). Psychological help referral sheets were provided to any subject whose scores indicated a possible clinical concern.

RESULTS This study tested N=13 subjects with PROMIS assessments (Fatigue, Anxiety and Depression), self-reported tiredness, self-reported stress, and a snap decision-making task.

Correlation(r)

tired

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fatigue

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depression tired

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depression

P value

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tired 0.04

stress 0.03

fatigue 0.02

anxiety 0.01

depression tired

stress

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Figure 2. Correlations among pre-testing questionnaire questions. Data from N=13 subjects are shown. In the top panel, color indicates the correlation between one questionnaire question (row) and every other question (column), for the five questions of greatest interest to this study: self-reported tiredness, self-reported stress, PROMIS fatigue score, PROMIS anxiety score, PROMIS depression score. Correlation of each question with itself is by definition r=1. In bottom panel, color indicates the p-values associated with each correlation. Correlations between two Gaussian variables used Pearson correlation; if either variable was Non-Gaussian then Spearman’s correlation was used. Values P>=0.05 are shown as the color of P=0.05. By Bonferroni correction for N=10 comparisons, P<0.005 is required for significance.

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Figure 3. Correlation between mental health and performance metrics. Each graph plots each subjectâ&#x20AC;&#x2122;s score on the indicated mental health scale vs. a performance measure from the snap decision task. Data from N=13 subjects are shown. Correlations between two Gaussian variables used Pearson correlation; if either variable was Non-Gaussian then Spearmanâ&#x20AC;&#x2122;s correlation was used. a. Self-reported tiredness vs. threshold. b. Self-reported tiredness vs. lapse. c. Self-reported tiredness vs. median reaction time (secs). d. Self-reported stress vs. threshold. e. Self-reported stress vs. lapse. f. Self-reported stress vs. median reaction time (secs). g. PROMIS fatigue score vs. threshold. h. PROMIS fatigue score vs. lapse. i. PROMIS fatigue score vs. median reaction time (secs). j. PROMIS anxiety score vs. threshold. k. PROMIS anxiety score vs. lapse. l. PROMIS anxiety score vs. median reaction time (secs). m. PROMIS depression score vs. threshold. n. PROMIS depression score vs. lapse. o. PROMIS depression score vs. median reaction time (secs). None of the correlations were significant to P<0.002, which is the significance threshold for N=25 comparisons by Bonferroni correction. Relationships between Psychological Measures. Self-reported stress was correlated with both the PROMIS anxiety score (r=0.66, p=0.01) and PROMIS depression score (r=0.64, p=0.02). Selfreported tiredness was correlated with PROMIS depression 40

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score (r=0.58, p=0.04). All other correlations were weak and/ or nonsignificant. After correction for multiple comparisons (N=10 pairwise comparisons) none of the correlations among psychological measures were significant in this small population, sqonline.ucsd.edu

RESEARCH

so these candidates would need to be confirmed in a larger confirmatory study. Relationships between Performance and Mental State. When comparing the PROMIS and other questionnaire questions to the five pre-selected performance metrics (Figure 3), the strongest and most significant effect was that PROMIS depression score was negatively correlated with threshold in the decision task (Figure 3m; r= -0.63; P=0.02). This would mean that depressed subjects were able to discriminate weaker motion signals more accurately. Depression was also negatively correlated with lapse (Figure 3n; r= -0.53; P=0.06). If this bears out in future, studies it would mean they were less likely to make a mistake on a really easy trial.

DISCUSSION Other effects that did not reach significance in this small sample, but still merit further study in a bigger population were: subjects who said they were tired had faster median reaction times (Figure 3c; r= 0.32, P=0.29), as did subjects who were depressed (Figure 3o; r=-0.35, P=0.24). Subjects who were anxious did fewer trials overall (not shown; r= -0.47, P=0.11). Like depressed subjects, perceptual thresholds were lower for those who were tired (Figure 3a; r= -0.33 P=0.28) or stressed (Figure 3d; r= -0.44, P=0.13). Also like depressed subjects, those who were fatigued had lower lapse (Figure 3h; r = -0.33, P=0.28), indicating higher vigilance. Finally subjects who were anxious had more trials with responses later than 3 seconds (not shown; r=0.47, P=0.11). Because we made 25 comparisons (5 performance measures x 5 psychological measures) none of these effects met the multiple comparison threshold for significance (P<0.002, Bonferroni correction). Casting a wide net enabled us to identify the strongest candidates for a follow-up confirmatory study, however. Depression was correlated with improved task performance, in regards to threshold and lapse. This was unexpected, given that depression has often been associated as resulting in decreased cognitive ability. For example, depression has been linked to cognitive impairments in attention, short-term memory and executive function14. One study found that in a series of cognitive tests, depressed individuals showed impairment in cognitive inhibition, or the ability to tune out irrelevant stimuli. They showed slower response times and more errors compared to controls, demonstrating problems with working memory function15. Increased perceptual performance of depressed individuals could be explained by hypervigilance, which could result from depressive realism. Depressive realism is a cognitive model that describes the negative informational processing in depression. Healthy individuals demonstrate a “self-serving” bias, in which they feel responsible for positive events and not responsible for negative sqonline.ucsd.edu

ones. The opposite trend is found true in depression; internal attribution of negative events and external attribution to positive events. Although this was previously a psychological hypothesis, neuroimaging studies have demonstrated the neural correlates associated with this cognitive model16. If depressed individuals were more focused on negative events, they would be more cautious in their decisions and perhaps make fewer careless errors. Those with depression show different responses to emotional stimuli in an affective go/no-go task compared to healthy controls17. The affective go/no-go tasks use “happy” or “sad” words, in order to compare the processing of emotional and neutral words between depressed and healthy individuals. Subjects with depression showed elevated neural activity to sad targets in emotional processing regions of the brain, as well as differential response in a region involved in behavioral inhibition.17 Although our task did not involve emotional stimulation, from these studies it is evident that depressed individuals may use their brains differently in cognitive tasks. Other strong correlations were found among the mental health assessments. Stress correlated with anxiety (r=0.66) and depression (r=0.64). Self-reported tiredness correlated with depression (r=0.58). These are reasonable findings, given that stress is a well-known side effect of anxiety and a risk factor for depression18. Additionally, lack of energy is a symptom used to diagnosedepression in the Diagnostic and Statistical Manual of Mental Disorders (DSM-V). These correlations suggest that perhaps psychological measures could be combined into compositescores, such as using Principal Component Analysis, to identify diagnostic clusters for analysis of decisionmaking. There were many other findings in this study that merit further investigation:anxiety and number of trials with responses longer than 3 seconds (r=0.47), anxiety and number of trials (r= -0.47), and depression and lapse (r= -0.53). Further research is needed because of the small sample size and unequal sampling (very few reported higher than average PROMIS scores). We need to confirm if the correlations found in this pilot study are statistically significant in a second independent sample of subjects. To address both of these problems, a future study will test more subjects on the same decision-making tasks, specifically recruiting for depressed and anxious individuals, as well as happy and “stress free” individuals. The follow-up study will use the longer 8-question PROMIS scales to allow greater sensitivity and validity of measures19. By focusing only on two psychological measures (PROMIS Depression and Anxiety) and only two performance measures (threshold and lapse) our confirmatory study can have much higher statistical power. In addition, statistical power will be increased by using Benjamini-Hochberg procedure for multiple comparison correction. Even though the results of this study were not statistically significant, the strong correlations are useful as preliminary data for guiding future research. SALTMAN QUARTERLY

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Although this research focuses on snap decisions, it would be important to also look at more deliberative decision processes. Deliberative decision processes are not instantaneous, but require critical and conscious thought. Deliberative decisions in the mental health realm are mainly concerned with choosing treatment and therapies. Patients who have greater jurisdiction in their treatment outcomes have a multitude of benefits such as: improvement of symptoms, greater adherence and higher patient satisfaction20. Shared decision making becomes tricky however, when dealing with mentally ill patients. Therefore, a diagnostic tool to assess the decision-making abilities of a patient would be extremely useful to determine how much discretion should be left up to the patient. Ultimately, understanding the effects of these mental states is important because of the widespread occurrence of these illnesses throughout the world and on college campuses. It is still not fully understood how these psychological states affect the brain, or how they affect decision making. Better understanding of these effects is crucial for better understanding and treatment and because of the frequency and importance snap decisions have in everyday life. References 1. Murphy, F. C. , J. S. Rubinsztein, A. Michael, R. D. Rogers, T. W. Robbins, E. S. Paykel, and B. J. Sahakian. "Decision-making Cognition in Mania and Depression," Psychological Medicine 31, no. 04, 679–93 (2001): doi: 10.1017/s0033291701003804. 2. Carey, M.G., Al-Zaiti, S.S., Dean, G.E., Sessanna, L. and Finnell, D.S. "Sleep Problems, Depression, Substance Use, Social Bonding, and Quality of Life in Professional Firefighters," Journal of Occupational and Environmental Medicine / American College of Occupational and Environmental Medicine, 928–933. (August 2011): doi: 10.1097 JOM.0b013e318225898f 3. Wickramasinghe, N.D., Wijesinghe, P.R., Dharmaratne, S.D., and Agampodi, S.B. "The Prevalence and Associated Factors of Depression in Policing: A Cross Sectional Study in Sri Lanka," SpringerPlus, 5(1):1776 (2016): doi: 10.1186/s40064–016-3474–9 4. Gadermann, A.M., Engel, C.C., Naifeh, J.A., Nock, M.K., Petukhova, M., Santiago, P.N., Wu, B., Zaslavsky, A.M., and Kessler, R.C. "Prevalence of DSM-IV Major Depression Among U.S. Military Personnel: Meta-Analysis and Simulation," Military Medicine 177, no. 8S (2012): doi: 10.7205/milmed-d-12-00103. 5. Blanco, C., Okuda, M., Wright, C., Hasin, D.S., Grant, B.F., Liu, S. M., and Olfson, M. "Mental Health of College Students and Their Non-college-attending Peers: Results from the National Epidemiologic Study on Alcohol and Related Conditions.," Archives of General Psychiatry (December 2008):, doi: 10.1001/archpsyc.65.12.1429 6. "College Students’ Mental Health Is a Growing Concern, Survey Finds," Monitor on Psychology (June 2013): accessed March 29, 2018, http://www.apa.org monitor/2013/06/college-students.aspx. 7. "American College Health Association-National College Health Assessment Spring 2007Reference Group Data Report (Abridged)," Journal of American College Health 56, no.5 (2008): 477–88. doi:10.3200/jach.56.5.469–480. 8. Nyer, M., Mischoulon, D., Alpert, J.E., Holt, D.J., Brill, C.D., Yeung, A., Pedrelli, P., Baer, L., Dording, C., Huz, I., Fisher, L., Fava, M., and Farabaugh, A. S. “College students with depressive symptoms with and without fatigue: Differences in functioning, suicidality, anxiety, and depressive severity,” Annuals of Clinical Psychiatry : Official Journal of the American Academy of Clinical Psychiatrists, 100-108 (May 2015): accessed March 30, 2018, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4539614/. 9. Huk, A.C. "Neural Activity in Macaque Parietal Cortex Reflects Temporal Integration of

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Visual Motion Signals during Perceptual Decision Making," Journal of Neuroscience 25, no. 45 (2005): doi:10.1523/jneurosci.4684–04.2005. 10. Palmer, J., Huk A.C., and Shadlen, M.N. "The Effect of Stimulus Strength on the Speed and Accuracy of a Perceptual Decision," Journal of Vision 5, no. 5 (2005): doi:10.1167/5.5.1. 11. Ader, D.N. "Developing the Patient-Reported Outcomes Measurement Information System (PROMIS)," Medical Care 45, no. Suppl 1 (2007): doi:10.1097/01 mlr.0000260537.45076.74. 12. Web accessed 3/31/18: http://www.healthmeasures.net/explore-measurementsystems promis/obtain-administer-measures. 13. Prins, N. Palamedes: Matlab routines for analyzing psychophysical data (2009). http:/ www.palamedestoolbox.org 14. Marazziti, D., Consoli. G., Picchetti, M., Carlini, M., and Faravelli, L. "Cognitive Impairment in Major Depression," European Journal of Pharmacology 626, no. 1, 649–654, (2010): doi:10.1016/j.ejphar.2009.08.046. 15. Gohier, B., Ferracci, L., Surguladze, S.A., Lawrence, E., Hage, W.E., Kefi, M.Z., Allain, P.,Garre, J., and Gall, D. "Cognitive Inhibition and Working Memory in Unipolar Depression," Journal of Affective Disorders, 116, no. 1–2, 100-105, (2009): doi:10.1016/j jad.2008.10.028. 16. Seidel, E. M., Satterthwaite, T.D., Eickhoff, S.B., Schneider, F., Gur, R.C., Wolf, D.H., Habel, U., and Derntl, B. Neural correlates of depressive realism—an fMRI study on causal attribution in depression. Journal of Affective Disorders, 268–276, (May 2012): doi:10.1016/j.jad.2012.01.041 17. Chamberlain, S.R and Sahakian, B.J. "The Neuropsychology of Mood Disorders," Current Psychiatry Reports 8, no. 6, 458-463, (2006): doi:10.1007/s11920-006-0051-x. 18. Leonard, B.E. "Stress, Norepinephrine and Depression," Acta Neuropsychiatrica 14, no. 04, 26 (Suppl): S11–S16 (2002): doi:10.1034/j.1601–5215.2002.140403.x. 19. Health Measures, PROMIS Depression Scoring Manual. 20. Malm, U., Ivarsson, B., Allebeck, P., and Falloon, I.R. "Integrated Care in Schizophrenia: a 2-year Randomized Controlled Study of Two Community-based Treatment Programs," Acta Psychiatrica Scandinavica 107, no. 6, 107(6):415–23, (2003): doi:10.1034/j.1600–0447.2003.00085.x.

Acknowledgments MJ – designed experiment, conducted experiments, analyzed data, wrote manuscript CS – designed human decision making task, collected preliminary data used to inform statistical plan PR – supervised and advised on experiments, analysis, and manuscript. The authors thank other members of the Reinagel lab for valuable discussion. James Proudfoot at the UC San Diego Altman Clinical and Translational Research Institute (ACTRI) Biostatistics Unit, for help with study design and statistical analysis. Funds Ledell Family Scholarship for Science and Engineering (to MJ) UCSD General Campus Research Grant RO034B-REINAGEL "Neurobiology of Sensory Decision-Making"- supported the subject compensation fees.

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A DRIVER OF CANCER METASTASIS: VASCULOGENIC MIMICRY Rishi N. Modi Sural K. Ranamukhaarachchi Vasculogenic mimicry (VM) describes the vessel-like networks created by aggressive cancer cells to perfuse a tumor with blood. The phenomenon has become accepted as a mechanism that supports cancer growth and progression to metastasis. In order to further understand VM, genotypic and phenotypic analyses have been conducted in vitro and in vivo. This research has drawn strong ties between VM formation and cancer patient prognosis, underscoring the critical need to continue to explore the mechanistic underpinnings of vasculogenic mimicry and its relationship to metastatic cancer.

INTRODUCTION Tumors require consistent blood flow for growth and metastasis1. The source of this blood flow had long been credited to tumorassociated angiogenesis—the branching of cardiovascular vessels into a tumor.1. However, the discovery of a separate mechanism has brought about a paradigm shift in the understanding of tumor microcirculation. First categorized in aggressive uveal melanoma cells, de novo vascular channels were observed to be the main source of sustenance during initial tumor growth,1 and to significantly perpetuate metastasis thereafter2. This fluid-conducting network of tumor cells has been coined as “vasculogenic mimicry”. The term vasculogenic differentiates these networks from angiogenic vessels, which develop from pre-existing blood vessels. While the channels are similar to blood vessels in that they conduct fluid, the term mimicry clarifies that, unlike vessels,

they are not actually lined with endothelial cells3. Over the past two decades, cancer researchers have aimed to detail the mechanisms behind and clinical relevance of vasculogenic mimicry.

OVERVIEW OF VASCULOGENIC MIMICRY Vasculogenic mimicry was initially classified by Maniotis et al. in 1999 as an interconnected pattern of channel-like structures formed within a mesh of highly aggressive and metastatic cancer cells (Figure 1)4. At the time, the presence of this morphology was characterized by a positive periodic acid-Schiff (PAS) stain, and a negative CD31 stain5. These stains confirmed that the observed channels were lined with extracellular matrix (ECM) proteins instead of typical endothelium. The classification of these PAS(+)/CD31(-) patterns as vasculogenic mimicry was met with controversy, as contemporary researchers claimed that the structures were fibrovascular septa, which are simply arrays of collagen6. The patterns were eventually confirmed to be VM, as it was shown that fibrovascular septa were not only uncommon, but also lacked prognostic significance in uveal tumors, unlike VM3. VM has since been linked to a notable decrease in patient survival rate across a variety of cancers, including melanomas, carcinomas, sarcomas, gliomas, glioblastomas, and astrocytomas7,8. Subsequent studies have shown the correlation between VM formation and metastatic potential. Initial in vitro work to identify this connection involved studies of melanoma cells on top of 2D Matrigel matrices. Only the cells isolated from existing VM networks (aggressive cells with high metastatic potential) were found to reform VM patterns4. Recently, work done in the lab of Dr. Fraley in the department of Bioengineering at UC San Diego has built on this correlation by showing that certain 3D ECM conditions can induce VM network formation in a variety of aggressive cancer cell types (Figure 2)9.

TRANSCRIPTIONAL BASIS OF VASCULOGENIC MIMICRY A deeper understanding can be gained from analyzing the genetic profile of vasculogenic mimicry. Multiple studies have highlighted the differences between VM and angiogenic vessels. Both morphologies show upregulation of genes such as vessel growth factors; however, basic angiogenic factors are not only absent in VM, but have no effect on VM growth when artificially introduced10.

Figure 1. Schematic model of the epithelial-mesenchymal transition (EMT) by which cancer cells acquire enhanced migration properties. Cells typically displaying an epithelial phenotype de-differentiate into a pluripotent state, before shifting to a mesenchymal phase and adopting a migratory phenotype. The VM phenotype can be formed as cancer cells align during this process

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Extracellular Matrix

VM Vessels

Tumor Cells

Figure 2. Schematic model of a cross-section of a tumor with vasculogenic mimicry. Tumor cells are shown in organized chain clusters, surrounded by ECM protein layers. A three-dimensional rendering would show that the vasculogenic mimicry â&#x20AC;&#x153;vesselsâ&#x20AC;? are a result of the gaps within a meshwork of tumor cell chain aggregates.

Figure 3. Schematic model of the epithelial-mesenchymal transition (EMT) by which cancer cells acquire enhanced migration properties. Cells typically displaying an epithelial phenotype de-differentiate into a pluripotent state, before shifting to a mesenchymal phase and adopting a migratory phenotype. The VM phenotype can be formed as cancer cells align during this process.

The VM phenotype significantly shows an upregulation of ECM protein production; quantitative polymerase chain reaction analysis has shown up to a 97-fold enrichment of ECM specific genes in aggressive cells3. Also significantly upregulated in VM structures are matrix metalloproteinases (MMPs). VM networks use these MMPs to actively manipulate their microenvironment; this finding implies that aggressive cancer cells modify the matrix around them in a way that perpetuates their metastasis10. These genetic insights serve to delineate VM from the ostensibly similar angiogenic, endotheliumassociated vessels. Further gene analysis has revealed similarities between VM and other biological mechanisms. One such process is cancer cell epithelial-mesenchymal transition (EMT) (Figure 3). Epithelial cancer cells de-differentiate and gain mesenchymal properties as they proliferate and convert from benign to metastatic cells. Four of the main transcription factors modulating this process (Twist, ZEB1, Sna1, and Sna12) are involved in dominant signal pathways during cancer metastasis as well1. Another mechanism with genetic similarity to VM is stem cell signaling. Many genes upregulated during vasculogenic mimicry are highly active in stem cell signaling pathways, where they regulate pluripotency and differentiation11. A prominent example is the Nodal signaling pathway, which is critical in cancer cell metastasis and tumorigenicity13, 14. Nodal mRNA is notably present in VM

structures in melanoma and breast cancer cells, and in the presence of Nodal neutralizing antibodies, VM showed reduced engagement in 3D15. Research into vasculogenic mimicry has been expanding beyond in vitro work and is gaining significant traction as an indicator of clinical prognosis. A retrospective clinical study of over 3000 patients confirmed that the presence of VM structures in patient biopsies accurately predicted a poor survival outcome12. In a study conducted by Fraley et. al., a transcriptional module of 70 genes linked to VM was predictive of patient survival and conserved across nine separate tumor types (Figure 4)9. These findings bolster the significance of VM as a disparate physiological structure and present several avenues for future research into the connections between VM and cancer.

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DISCUSSION AND FUTURE STEPS Introductory research into the significance of vasculogenic mimicry, from a bench top and clinical perspective, has been explored over the past two decades. The pathway links between VM and other mechanisms serve as the preliminary direction from which a better understanding of vasculogenic mimicry is being derived. Continued work will contribute to targeted therapeutic strategies and companion diagnostics in order to improve cancer survival sqonline.ucsd.edu

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THBS1

In 70 gene module

EDN1

In 70 gene module

Normal Tissue

Breast Carcinoma

In 70 gene module

JAG1

Figure 4. Sections of primary breast carcinoma tissue. Tumor cells displaying VM are stained for three of the most upregulated genes in Velez et al.â&#x20AC;&#x2122;s 70-gene module.

rates and provide a promising future for both cancer treatment and stem cell research. References 1. Liu, Q. et al. The relationship between vasculogenic mimicry and epithelialmesenchymal transitions. Journal of cellular and molecular medicine 20, 1761â&#x20AC;&#x201C;1769. 2. Folberg, R., Hendrix, M. J. & Maniotis, A. J. Vasculogenic mimicry and tumor angiogenesis. The American journal of pathology 156, 361-381. 3. Lin, A. Y. et al. Distinguishing fibrovascular septa from vasculogenic mimicry patterns. Archives of pathology & laboratory medicine 129, 884â&#x20AC;&#x201C;892. 4. Maniotis, A. J. et al. Vascular channel formation by human melanoma cells in vivo and in vitro: vasculogenic mimicry. The American journal of pathology 155, 739-752. 5. Qiao, L. et al. Advanced research on vasculogenic mimicry in cancer. Journal of cellular and molecular medicine 19, 315-326. 6. Foss, A. J. et al. Reassessment of the PAS patterns in uveal melanoma. The British journal of ophthalmology 81, 240-246; discussion 247â&#x20AC;&#x201C;248 (1997). 7. Takeda, K., Kawai, M., Sakuta, M. & Takemura, T. [Paralytic ileus and atonic bladder in a case of mitochondrial encephalomyopathyâ&#x20AC;&#x201D;electrophysiological, chemical and pathological study with evidence of the peripheral nerve involvement]. Rinsho shinkeigaku = Clinical neurology 29, 643â&#x20AC;&#x201C;646 (1989). 8. van der Schaft, D. W. et al. Tumor cell plasticity in Ewing sarcoma, an alternative circulatory system stimulated by hypoxia. Cancer research 65, 11520â&#x20AC;&#x201C;11528, doi:10.1158/0008-5472.canâ&#x20AC;&#x201C;05â&#x20AC;&#x201C;246810.1158/0008-5472.CANâ&#x20AC;&#x201C;05â&#x20AC;&#x201C;2468. (2005).â&#x20AC;Š 9 Fraley, D. O. V. et al. 3D collagen architecture induces a conserved migratory and transcriptional response linked to vasculogenic mimicry. Nature Communications 8, 1651.

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10. Paulis, Y. W., Soetekouw, P. M., Verheul, H. M., Tjan-Heijnen, V. C. & Griffioen, A. W. Signaling pathways in vasculogenic mimicry. Biochimica et biophysica acta 1806, 18â&#x20AC;&#x201C;28, doi:10.1016/j.bbcan.2010.01.00110.1016/j.bbcan.2010.01.001. Epub 2010 Jan 14. (2010). 11. Seftor, R. E. et al. Tumor cell vasculogenic mimicry: from controversy to therapeutic promise. The American Journal of Pathology 181, 1115-â&#x20AC;&#x201C;125. 12. Yang, J. P. et al. Tumor vasculogenic mimicry predicts poor prognosis in cancer patients: a meta-analysis. Angiogenesis 19, 191â&#x20AC;&#x201C;200. 13. Topczewska, J. M. et al. Embryonic and tumorigenic pathways converge via Nodal signaling: role in melanoma aggressiveness. Nature Medicine 12, 925-932. 14. Strizzi, L., Hardy, K. M., Kirschmann, D. A., Ahrlund-Richter, L. & Hendrix, M. J. Nodal Expression and Detection in Cancer: Experience and Challenges. Cancer Research 72, 1915â&#x20AC;&#x201C;1920. 15. Strizzi, L. et al. Nodal as a biomarker for melanoma progression and a new therapeutic target for clinical intervention. Expert Rev Dermatol 4, 67â&#x20AC;&#x201C;78.

Acknowledgments Rishi N. Modi: UC San Diego, Thurgood Marshall College, Bioengineeringâ&#x20AC;&#x201D;Biotechnology, 2018 Sural K. Ranamukhaarachchi: UC San Diego, John Muir College, Physiology & Neuroscience, 2018

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IDENTIFICATION AND CHARACTERIZATION OF PACSIN-1 AS AN LRP1 CELL SIGNALING CANDIDATE IN PERIPHERAL NERVE INJURY

Damaged axon

Direction of degeneration

PACSIN-1 released from axoplasm

Schwann cell

LRP-1

1) Cell Survival 2) Migration

Figure 1. Schematic of PACSIN-1 interaction with Schwann cells. This figure illustrates the proposed mechanism of action of PACSIN-1 in the injured nerve. Following injury the axon degrades, releasing its contents (PACSIN-1) into the extracellular space. PACSIN-1 diffuses across the matrix before binding to Schwann cell LRP1, subsequently activating cell survival signaling and migration through ERK1/2 and Akt.

BACKGROUND The peripheral nervous system is capable of marked regeneration following injury and recently, Schwann cells have been implicated as major respondents to injury. Low-density lipoprotein receptor-related protein 1 (LRP1) is an endocytic cell-signaling receptor responsible for binding over 80 ligands1. Following peripheral nerve injury, there is a massive upregulation of the production and recruitment to the Schwann cell membrane of LRP12. Signaling through LRP1 is pro-survival, and multiple papers from the Campana lab show that LRP1 is responsible for a majority of the response known as the Schwann cell repair response. Activation of LRP1 increases survival rates, migration, and proliferation of Schwann cells in the injured nerve3. Deletion of LRP1 specifically in Schwann cells results in an ablation of this response, thus leading to the current conclusion that LRP1 is an orchestrator of the Schwann cell repair response4. Current projects include studying the diverse ligands that bind to the LRP1 receptor. Through tandem mass spectrometry (MS/ MS), several novel ligands were identified in both the injured and uninjured nerve as high-affinity binders. From these ligands a strong candidate for binding in the injured nerve was chosen, Protein Kinase C and Casein Kinase Substrate in Neurons 1 (PACSIN-1). Notably, PACSIN-1 normally functions as a synaptic receptor responsible for restructuring of the membrane, endocytosis, and transportation of vesicles. All studies performed were under the condition that PACSIN-1

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is introduced extracellularly, showing that an intracellular protein can function as cell-surface ligand in peripheral nerve injury. The importance of this statement is not to be underappreciated, as neurons in the damaged nerve degrade and release their contents into the surroundings and Schwann cells are near axons. To confirm the results of MS/MS, pull-down assays were performed that showed that PACSIN-1 binds to the CCR2 and CCR4 extracellular domains of LRP1, which are known to elicit ligandinduced cell signaling responses. Next, western blots were performed to demonstrate the capability of PACSIN-1 to activate pro-survival and migratory signaling pathways extracellular signal-regulated kinases and protein kinase B (ERK1/2, Akt) in primary cultured Schwann cells. To illustrate the concept that PACSIN-1 binds and signals in an LRP1-dependent manner, LRP1 was genetically silenced using electroporation. Preliminary data suggests pro-survival and promigratory signaling in response to PACSIN-1 is ablated. Recently, LRP1 has been shown to be a co-receptor for N-methyl D-aspartate receptor (NMDA-R) and ablation of the functionality of NMDAR results in a loss of LRP1-dependent migratory capabilities of Schwann cells in the injured peripheral nerve5. Utilizing a chemotactic gradient, we showed that PACSIN-1 induces migration in Schwann cells, confirming functional consequences cell signaling. Current studies utilizing an NMDAR antagonist are being performed to confirm whether the LRP1-NMDAR interplay is responsible for the migration of Schwann cells in response to PACSIN-1. To re-iterate, this is the first time an axonal intracellular protein has been found to activate LRP1dependent cell signaling in response to peripheral nerve injury. REFERENCES 1. Strickland, D.K., Gonias, S.L., Argraves, W.S. Diverse roles for the LDL receptor family. Trends in Endocrinol. Metab., USA 13, 66–74 (2002). 2. Campana et al. The Low-Density Lipoprotein Receptor-Related Protein Is a ProSurvival Receptor in Schwann Cells: Possible Implications in Peripheral Nerve Injury. Journal of Neuroscience, USA 26, 11197–11207 (2006) 3. Campana et al. The Hemopexin Domain of Matrix Metalloproteinase-9 Activates Cell Signaling and Promotes Migration of Schwann Cells by Binding to Low-Density Lipoprotein Receptor-Related Protein. Journal of Neuroscience, USA 28, 11571–11582 (2008) 4. Mantuano, E., Jo, M., Gonias, S.L., Campana, W.M. Low Density Lipoprotein Receptorrelated Protein (LRP1) Regulated Rac1 and RhoA Reciprocally to Control Schwann Cell Adhesion and Migration. Journal of Biological Chemistry, USA 285, 14259–14266. 5. Mantuano, E., Lam, M.S., Shibayama, M., Campana, W.M., Gonias, S.L. The NMDA receptor functions independently and as an LRP1 co-receptor to promote Schwann cell survival and migration. Journal of Cell Science, USA 128, 3478–3488

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BREVIA

AN INVESTIGATION OF THEROPITHECUS GELADA AS A MODERN ANALOG FOR EXTINCT HOMINID PARANTHROPUS BOISEI Isabel Hermsmeyer1, Peter Fashing2, Nga Nguyen3, Margaret Schoeninger4 Ecology, Behavior, and Evolution PI: Margaret Schoeninger, Ph.D. BACKGROUND

When studying extinct species, we often look at comparable living species to make inferences about characteristics that are not preserved in the fossil record, such as behaviors or soft tissue. In the case of Paranthropus boisei, a human relative that lived ~2.5–1.3 million years ago, recent isotopic data suggest they were graminivorous (consumes grasses and sedges)1. Researchers have suggested that the only living graminivorous primate2, Theropithecus gelada, might make a useful analog.3 Here we evaluate the suggestion by comparing four dietary indicators. These indicators included body size, which suggests their likely energy requirements4, dental morphology which suggests diet type5, dental microwear which are microscopic marks left behind by food consumed6, and lastly, we analyzed stable isotopes, focusing on the stables isotopes of carbon, C12/C13. These isotopic signatures can be found through biological material (e.g. hair, teeth) and tell us if they consumed C3 or C4 plants. C3 are comprised mainly of leafy plants that most primates consume, whereas C4 plants are comprised mainly of grasses7, which are lower in nutritional quality and an abnormal primate diet choice, making the C4 signature of extinct primate Paranthropus boisei all the more curious.

METHODS

Figure 1: Charted carbon isotope values for T. gelada and P . boisei 1 respective to C3 and C4 ranges.

was -1.3 ± 0.9‰, a clear C4 signature1 (Fig. 1). Our T. gelada data alternatively show an average of -21.7 ± 3.4‰, a clear C3 signature (Fig. 1).

DISCUSSION

Microwear is the only dietary indicator with similarities. Similar striations but different directionality suggests they consumed foods with similar mechanical properties but in alternative ways. Body size was dissimilar, where P. boisei was over two times the size of T. gelada, suggesting they had very different energy intake requirements. Dental morphology was different, suggesting their food was different or eaten in different ways. Isotopic signature was also different, where despite T. gelada being graminivorous, they are still C3 consumers like most primates. Alternatively, P. boisei is a uniquely majority C4 consumer. This is likely because T. gelada live in higher elevations3, where plants are more likely to be C3. Altogether, these differences suggest using T. gelada as an analog for P. boisei has substantial limitations. Future directions include analyzing other dietary indicators, like dental musculature and body proportion.

For body size, craniodental morphology, and dental microwear, we compiled and compared previously published data on each of the respective species. For stable isotopes,4 T. gelada hair samples REFERENCES were collected by Fashing and Nguyen from naturally deceased 1. Cerling, T. E., et al. 2011. Diet of Paranthropus Boisei in the Early Pleistocene of East Africa. Proceedings of the National Academy of Sciences108.23: 9337–341. individuals at Guassa, Ethiopia (3438 m asl)3 and sent to UC San 2. Jablonski, N.G. 1993. Evolution of the Masticatory Apparatus in Theropithecus. Diego’s Paleodiet lab for processing. These new T. gelada data were Theropithecus: The Rise and Fall of a Primate Genus (n.d.):299-330. compared to previously published P. boisei isotopic data which 3. Fashing, P.J. Nga, N. Vivek, V.V. Jeffrey, T.K. 2014. Gelada Feeding Ecology in an 1 looked at 24 teeth from 22 individuals . Intact Ecosystem at Guassa, Ethiopia: Variability over Time and Implications for Theropith and Hominin Dietary Evolution. American Journal of Physical All hair samples were cleaned then homogenized. Samples were Anthropology 155.1: 1–16. analyzed in an automated fashion on a Thermo-Finnigan Delta 4. Strier, K. 2016. Primate Behavioral Ecology. Routledge; 5. XP Plus, Conflow and Costect EA in the Analytical Laboratory at 5. Wood, B. Lieberman, D.E. 2001. Craniodental variation in Paranthropus boisei: a Scripps Institute for Oceanography (SIO). (For complete methods, developmental and functional perspective. American Journal of Physical contact ihermsme@ucsd.edu). Anthropology 116:13–25. 6. Ungar, P.S. Grine, F.E. Teaford, M.F. 2008. Dental microwear and diet of the Plio

RESULTS

Body size (sexes averaged) of P. boisei was approximately 129.5 cm and 42 kg,5 and T. gelada at 62.5 cm and 14.75 kg8. Dental morphology for P. boisei shows small canines with large, flat molars9 while T. gelada has large canines and high-crowned molars2. Dental microwear for P. boisei showed fine striations, lacked pitting, and moderate directionality6. T. gelada ’s microwear consisted of fine striations, occasional large pits, and high directionality10. For stable isotopes, the average δ13C value for Cerling’s P. boisei samples sqonline.ucsd.edu

Pleistocene hominin Paranthropus boisei. PLoS One 3:e2044 7. Schoeninger, M.J. 2014. Stable Isotope Analyses and the Evolution of Human Diets. Annu. Rev. Anthropol. Annual Review of Anthropology 43.1: 413–30. 8. Jolly, C.J. 2007. Baboons, mandrills, and mangabeys: afro-papionin socioecology in a phylogenetic perspective. In: Campbell CJ, Fuentes A, MacKinnon KC, Panger M, Bearder SK, editors. Primates in Perspective. New York: Oxford U Pr. p 240–51. 9. Wood, B.A. 2007. Paranthropus boisei: Fifty Years of Evidence and Analysis. Yearbook of Physical Anthropology 50: 106-132 10. Teaford, M.F. 1993. Dental microwear and diet in extant and extinct Theropithecus: preliminary analyses. In: Jablonski NG, editor. Theropithecus: The Rise and Fall of a Primate Genus. Cambridge, UK: Cambridge University Press. p 331–349.

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SENIOR

HONORS THESES 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 2018.

The botanical building and lily pond in Balboa Park in San Diego over look a field of orange and yellow coneflowers, echinacea purpurea hybrid. Photo by Tomomi Yoshida 48

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THE DIVISION OF BIOLOGICAL SCIENCES SENIOR HONORS THESES PROGRAM

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. Each student in the program has a faculty mentor who provides guidance throughout the year. During the spring quarter of each year, students in the program participate in a research showcase which gives them the opportunity to discuss their research with faculty and their fellow students. These are the abstracts of all the exceptional research projects conducted by undergraduates in the program during the 2017â&#x20AC;&#x201C;18 academic year.

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YALIN DENG Revelle College Physiology & Neuroscience and Linguistics Major PI: Fred Gage Ph.D. Salk Institute for Biological Studies

SEAN GUY Sixth College Molecular Biology Major

Studying the Role of Protocadherin Genes in Regulation of Human Neuronal Morphology In Vitro Protocadherins (PCDH) are a subgroup of the cadherin superfamily of homophilic cell- adhesion proteins. It was found that PCDH alpha genes promote survival and migration of neurons, axonal targeting, and synaptogenesis in mice brains (Yagi, 2013). Hence, we sought to examine how regulation of PCDH genes found in our screen would regulate neuronal morphology. Human neurons can be generated in vitro with pluripotent stem cell culture, and we examined whether knockdown of PCDH genes regulated neuronal morphology. We observed significant alterations in neuronal morphology following knockdown of PCDH genes in human pluripotent-stem- cell-derived neurons in vitro.

Measuring the Variation in Local Mutation Rate Along the Yeast Genome

Mutations are the ultimate source of genetic and phenotypic diversity, which in turn drives adaptive evolution. Mutation rates vary PI: Sergey Kryazhimskiy, across the genome in many organisms, but our understanding Ph.D. UC San Diego Division of the magnitude, causes, and evolutionary consequences of of Biological Sciences, this variation is poor. Here, we are developing an IlluminaSection of Ecology, sequencing-based fluctuation test for measuring genome-wide variation in local mutation rates with 10 kb resolution. To this Behavior & Evolution end, we generated a collection of 563 strains of baker’s yeast with the URA3 marker integrated into different genomic locations. In a preliminary screen of this collection, we identified 50 strains in which the local mutation rate resulting in URA3 loss-of-function is elevated by two orders of magnitude relative to the genome-wide baseline. Relating mutation rates to local factors, such as replication timing and chromatin structure, will provide insights into structural and functional influences on mutation rates in the eukaryotic genome.

CAROLINA GONZALEZ BRAVO

Uncovering the Secret Life of Genes

The structural organization of cells is often regarded as consisting primarily of membrane-enclosed organelles. Notwithstanding, there are a number of cellular organelles that do not exhibit a delimiting membrane. These structures are still capable of assembling, maintaining their structural integrity, and compartmentalizing through the driving force of intrinsically PI: Galia Debelouchina Ph.D. disordered regions (IDR). While a number of intracellular UC San Diego, Division of structures have been identified as having this form of phase Chemistry & Biochemistry, separation, many questions regarding their function and properties Division of Biochemistry remain. Studying phase separation can help us better understand the mechanisms that underlie a number of protein aggregation and neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). An in vitro model of HP1α in relation to chromatin will be replicated with the purpose of investigating the liquid-phase nature that has been exhibited by this chromo domain containing protein. This project seeks to address the HP1α conformation that is generated during this heterochromatin-mediated gene silencing mechanism and subsequently address some of the environmental conditions of phase-separated heterochromatin.

Earl Warren College Biochemistry & Cell Biology Major Chemistry Minor

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THESES ZHIJAN LI Sixth College Biochemistry & Cell Biology, Molecular Synthesis Major

Noncatalytic Domains of TOE1 are Important for its 3’- End snRNAs Trimming Activity

Research from our lab has shown that TOE1, an unconventional deadenylase, trims the 3’ end of snRNAs to promote their maturation during snRNA biogenesis. The catalytic domain of TOE1, which is conserved across mRNA deadenylases, is PI: Jens Lykke-Andersen, functionally important for snRNA trimming; however, other Ph.D. UC San Diego, Division domains of TOE1, which may be important for TOE1 target of Biological Sciences, recognition remain uncharacterized. To test the importance of TOE1 domains in trimming, I created stable cell lines expressing Section of Molecular Biology TOE1 mutants with specific domain deletions. Gene specific 3’-end sequencing of total RNA reveals that deletion of regions results in higher fractions of 3’-end extended snRNAs. This suggests that these domains are relevant to snRNA trimming. Ongoing efforts focus on using the mutants to study the molecular mechanism of snRNA trimming by TOE1. Elucidating these molecular processes will lead to a better understanding of snRNA biogenesis and how deadenylases may act on unconventional substrates.

MANDY LAI Revelle College Neuroscience and Physiology Major

Target Specificity and Spatial Organization of Auditory Cortex Projection Neurons

Sensory information processed in the primary auditory cortex (A1) is directed to a variety of spatially and functionally distinct targets, including contralateral A1, inferior colliculus, PI: Jeffry Isaacson Ph.D. striatum, and thalamus. These areas play distinct roles in UC San Diego Division of sound-guided behavior and cognition. However, it is unclear Biological Sciences, whether the organization of A1 projections supports transmission of redundant auditory information to all target regions or Department of Neuroscience transmission of specific aspects of auditory information to different target regions. Understanding the nature of these projections may shed light on brain disorders, such as autism and schizophrenia. To address this question, we used the retrograde tracer, cholera toxin B (CTB), conjugated to different fluorescent markers to visualize and characterize the spatial organization of A1 projections. Our results suggest that individual A1 principal cells rarely target more than one downstream region, and are thus organized to transmit unique channels of auditory information to functionally distinct areas of the brain.

TAEYEON JESSICA KIM Sixth College Biochemistry & Cell Biology Major, Social Issues in Healthcare Minor

Deregulation of miRNA Profiles in MSA May Alter Oligodendrocyte Maturation Pathway and Contribute to α-syn Accumulation

Multiple Systems Atrophy (MSA) is a fatal neurodegenerative disorder affecting around 100,000 people in the U.S.; however, there is still little known about the molecular basis of PI: Paula Desplats Ph.D. the disorder and thus no disease-modifying therapy available. UC San Diego School of MSA is characterized by the accumulation of α-synuclein (α-syn) in oligodendrocyte cells and an increased proportion of immature Medicine, Departments of oligodendrocytes in the brain. Mature oligos, in contrast to immature Neurosciences & Pathology oligos, clear α-syn aggregates more efficiently. Deregulation of miRNA profiles in MSA may play a role in altering oligo maturation pathways, contributing to α- syn accumulation and demyelination. We established a protocol to differentiate oligodendrocytes from adult rat hippocampal neuronal progenitor cells and used lentiviral vectors to overexpress α-syn. We identified several miRNAs deregulated at different stages of oligo maturation associated with accumulation of α-syn. Changes in miRNA levels and gene expression related to MSA were also measured in human MSA postmortem brains to identify miRNA-mRNA inverse correlations.

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JOSE MENDOZA Revelle College Physiology & Neuroscience Major Chemistry and French Literature Minor

Investigating the Role of Endogenous Opioids in Learning and Motivation

Endogenous opioid neuropeptides are distributed throughout reward and motivation circuits in the brain. To determine how endogenous opioids contribute to goal-directed actions, we systemically administered naloxone (NLX) to mice prior to performing a goal-oriented operant conditioning PI: Matthew Banghart, Ph.D., task. Intriguingly, we found that the NLX-treated mice made UC San Diego Division of significantly less lever presses than their saline-treated littermates. Biological Sciences, Open field and rotarod experiments suggested that gross locomotion and coordination, respectively, remained intact. Free feeding and Section on Neurobiology drinking tests showed that food- and water-restricted mice receiving NLX consumed slightly less food and water, respectively, compared to controls, indicating that NLX treatment did not cause satiety. These experiments suggest that in the context of a goal-directed task, although endogenous opioids do not regulate the movement required to perform the task, they may regulate general motivation irrespective of the nature of the reward. Alternatively, they may be involved in learning actionoutcome associations, a possibility that is currently under investigation.

JOSEPH MILLER Thurgood Marshall College Biochemistry & Cell Biology Major Psychology Minor

Impact of PDLIM1 Expression on Vascular Development of the Retina

The blood-retinal barrier (BRB) is a unique set of properties possessed by the endothelial cells of the retinal vasculature that limits vascular permeability by maintaining a precise biochemical environment for proper retinal function. Many PI: Richard Daneman, Ph.D. retinal diseases are characterized by BRB disruption, but UC San Diego School of questions remain regarding the molecular changes that drive Medicine, Departments of BRB dysfunction and repair. To address this question, we used the oxygen-induced retinopathy (OIR) mouse model of retinopathy of Pharmacology & Neurosciences prematurity and performed RNA sequencing on isolated endothelial cells. We found that PDLIM1 was strongly upregulated in endothelial cells in OIR, and this result was confirmed via immunofluorescent staining of hypoxic retinas. Furthermore, PDLIM1 transfection inhibits the Wnt signaling pathway in HEK-293 cells. As vascular development in the retina is dependent upon Wnt signaling, we postulate that PDLIM1 inhibits abnormal neovascularization in disease. Our final aim is to determine how PDLIM1 affects BRB function and neovascularization in the context of retinal disease.

XAVIER ORAIN Revelle College Molecular Biology Major

Defective Mechanism of Nerve Growth Factor Secretion in the Context of HSAN Type V

Hereditary Sensory Autonomic Neuropathy Type V, a rare but severe peripheral sensory neuropathy, is associated with a PI: Chengbiao Wu, PhD. missense mutation (R100W) in nerve growth factor (NGF). We UC San Diego School hypothesize that the point mutation NGFR100W induces a deficit of Medicine, Department in secretion of mutant mature NGF that contributes to sensory of Neuroscience neuropathy in the NGFR100W knockin mouse model. Our objective is to test our hypothesis using mouse embryonic fibroblasts (MEFs) cultured from wild-type, heterozygote and homozygote genotypes. We used ELISA to measure the level of NGF in the media. Our data shows NGF secretion in culture media is significantly reduced in NGFR100W cells in a gene-dosage-dependent manner. These results suggest that reduced mature NGF secretion from target tissues, either due to a reduction in cellular synthesis of NGFR100W protein or retarded secretion of NGFR100W, results in NGF deficiency that leads to peripheral sensory neuropathy in HSAN V.

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THESES MARCUS TURNER Revelle College General Biology Major Social Issues in Healthcare Minor

Circuit and Behavioral Investigation via Optogenetic Manipulation in Target-CellType-Defined Corticostriatial Pathways

Parkinson’s disease is characterized by impaired motor movement caused by malfunctions in basal ganglia circuitry. Corticostriatal (CSTR) projection pathways connect the PI: Takaki Komiyama, Ph.D. motor cortex and basal ganglia. However, the pathways’ UC San Diego, Division of function during movement is unknown. By implementing a Biological Sciences, motor skill task in which head-fixed mice walk on a motorized Section of Neurobiology rotation ladder, we aim to examine the circuit and behavioral effects by activating specific pathways using optogenetics. Using a rabiesvirus-mediated tracing technique, we express channelrhodopsin (ChR2), a light-gated cation channel, in CSTR pathways targeting direct pathway medium spiny neurons (dMSN), indirect pathway medium spiny neurons (iMSN), or cholinergic interneurons. We postulate that activating these pathways will not elicit movement, but instead modulate movement such that mice will speed up with dMSN-projecting neuron activation, halt with iMSN-projecting neuron activation, and not be affected by cholinergic interneuron activation.

YUYA ZHAO John Muir College Biochemistry & Cell Biology Major

Loss of RIAM in B cells impairs T-independent Immune Responses

Rap1-GTP-interacting adapter molecule (RIAM) regulates integrin activation, a cellular adhesion process that is important for multiple stages of immune responses. Previous PI: Joseph Cantor, PhD., studies have shown that germline RIAM knockout mice UC San Diego School of have defects in T-dependent humoral immunity, while T-cellMedicine, Department specific RIAM knockout mice display poor T cell activation and resultant effector functions. In this study, we utilized Cre-Lox of Medicine recombination to investigate the role(s) of B-cell-intrinsic RIAM in B-cell-mediated immune responses in mice. RIAM is necessary for B cell trafficking and residence in the marginal zone of the spleen, as naive spleen B cell subset analysis showed increased follicular B cells and decreased marginal zone B cells. Loss of RIAM in B cells did not cause observable defects in the T-dependent humoral responses, but humoral responses to both TI-1 and TI-2 antigens were impaired. These data reveal that RIAM expression in B cells is required for T-independent immune responses.

ANH-DAO TONG John Muir College Biochemistry & Cell Biology Major, Social Issues in Healthcare Minor

A Maize CCAAT Box Binding Transcription Factor that Regulates Defense Against Fusarium venenatum

Elucidating the maize defense response against pathogens is important for increasing yield of one of the most consumed crops consumed worldwide. In this project, homozygous PI: Steven P. Briggs, Ph.D., UC San Diego, Division transposon insertional mutants for ca2p6, a CCAAT box binding of Biological Sciences, transcription factor, were obtained. In a 32 hour elicitation Section of Cell and experiment, the stems of five-week old maize plants were slit and Developmental Biology inoculated with heat-killed Fusarium venenatum. These mutants produced more total phytoalexins, secondary metabolites associated with biotic stress, than their wild-type counterparts did, suggesting that ca2p6 plays an inhibitory role in the defense response. There were 72 significantly overexpressed or underexpressed proteins (p &lt; 0.01, &gt; 1.5-fold) between treated mutant and wild-type plants. One significantly underexpressed protein in the treated mutants was an enzyme involved in synthesis of jasmonate, a plant hormone critical for plant defense. Further analysis and research is needed to elucidate exactly how ca2p6 affects the maize defense response.

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BULOUERE PRINCESS WODU Eleanor Roosevelt College Biochemistry & Cell Biology Major

MechanoBiology of Articular Cartilage Impact Damage

Articular cartilage is subjected to impact in a variety of surgical settings. Impact that transmits excessive energy associated with deformation leads to chondrocyte death, yet cartilage PI: Robert L. Sah, M.D., ScD., can withstand and transmit high levels of pure hydrostatic UC San Diego Jacobs pressure. We tested the hypothesis that the mechanobiological School of Engineering, mechanism of damage during impact involves excessive strain Departments of Bioengineering due to lateral expansion and impactor-surface incongruity. The & Orthopaedic Surgery effects of drop-tower loading of articular cartilage was compared Bioengineering for (1) non-loaded controls (CTRL), (2) traditional unconfined compression (UCC), (3) loading with a â&#x20AC;&#x153;confined compression capâ&#x20AC;? (CCC) to prevent lateral expansion, and (4) loading with a hydrogel-containing CCC (CCC+H) to minimize surface incongruity. Chondrocyte viability, assessed by fluorescence microscopy, was reduced from CTRL by UCC but preserved by CCC and CCC+H. These results identify mechanobiological causes of cartilage damage during impaction utilizing a protective cap that has the potential for surgical use.

VISHAL VENKATRAMAN Revelle College Physiology & Neuroscience Major

Measuring Rat Impulsivity and Cognition Using Delay Discounting and Visual Attentional Engagement Tasks

The aim of this research was to investigate the effects of impulsivity on rat decision- making in different behavioral contexts. The project studied the effects of methylphenidate on behavioral measures of brain function. The open-field exploration test measured gross locomotion and risk tolerance, and the delay discounting task measured the ability to forego the opportunity for a small, immediate water reward in favor of a larger, but delayed water reward. In the visual attention task, rats discriminated between visual stimuli of varying attentional demand. We hypothesized that methylphenidate would increase tolerance of reward delay and reduce errors in the visual attention task. The effects of methylphenidate on the delay discounting task was inconclusive due to development of location-based biases. Methylphenidate did not have any significant effect on performance in the visual attention task. PI: Pamela Reinagel, Ph.D., UC San Diego, Division of Biological Sciences, Section of Neurobiology

PASSENT YOUSSEF Earl Warren College Biochemistry & Cell Biology Major

Oxidative Stress is a Cause of Reduced Fitness in Copepod Hybrids

According to the Dobzhansky-Muller model of hybrid incompatibility, matings between geographically isolated conspecific populations sometimes resultss in some degree of genetic inPI: Ronald Burton, Ph.D., compatibility. In this speciation process, hybrids show reduced Scripps Institutution fitness resulting from interactions between differentiated genes. of Oceanography, Such fitness breakdown happens after the F1 hybrid generation, when incompatibilities accumulate and become unmasked. Marine Biology Studies have shown that a direct relationship between genetic Research Division incompatibility and reduced fitness of hybrids exists. Because the highest rates of mutations occur in the mitochondrial genome (mtDNA), incompatibilities may evolve between mtDNA and the nuclear genome, affecting mitochondrial performance in hybrids. Here, we compared reactive oxygen species (ROS) levels in interpopulation hybrids to parental populations in copepod Tigriopus californicus, using a fluorescent assay. Comparisons between three hybrid lines and their parents showed one case of significant elevation of hybrid ROS production. This result is consistent with the hypothesis that hybridization can result in incompatibilities between mtDNA and the nuclear genome.

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THESES KHUSH KHARIDIA John Muir College Human Biology Major, Psychology Minor

Inhibition of D1R-Expressing Neurons in the Dorsal Striatum Promotes Meth Addiction-Like Behavior

Evidence from previous studies indicates that methamphetamine (meth) self-administration upregulates dopamine D1 receptors (D1Rs) in the dorsal striatum. It is therefore hypothesized that D1-receptor-expressing, medium-sized spiny neurons in the dorsal striatum may PI: Chitra Mandyam Ph.D. contribute to reinforcing effects of meth and produce dependence-like behavior. Here we UC San Diego School of seek to determine if inhibiting D1R expressing neurons in the dorsal striatum alters meth Medicine, Department self-administration. A viral vector-mediated approach was used to overexpress the inhibitory (Gi coupled-hM4Di) designer receptors, exclusively activated by designer drugs (DREADDs) of Anesthesiology engineered to only respond to exogenous ligand clozapine-N-oxide (CNO). Preliminary findings from behavior data reveal that CNO treatment increased responding for meth compared to vehicle saline in a within-subject treatment paradigm. Postmortem tissue analysis reveal that CNO treatment reduced neuronal activation in the dorsal striatum. This suggests that normal functioning and activity of D1R-expressing neurons in the dorsal striatum is necessary for reducing meth addiction-like behavior.

JACQUELYN TRUONG Revelle College Human Biology and Molecular Synthesis Majors PI: James Kadonaga, Ph.D. UC San Diego Department of Biological Sciences, Section of Molecular Biology Research Division

ANIKA ULLAH Revelle College Human Biology Major, Computing in Visual Arts Minor

Recruitment of TRF2 to TATA-less Promoter Sites via Transcription Factor Interactions Drosophila TRF2 is a TBP (TATA-binding protein)-related RNA polymerase II transcription factor that is used in place of TBP at TATA-less gene promoters containing either the TCT or the DPE promoter element. Unlike TBP, TRF2 does not bind DNA and is likely recruited to the promoter through protein-protein interactions that mostly remain unknown. Potential sites of interaction with transcription factors TFIIB, the Taf1 subunit of TFIID, Mot1, NC2, and TFIIA have been identified based on regions of homology of TRF2 to TBP. It has also been shown that transcription factors M1BP and DREF bind to TRF2. The 37 amino acid residues exposed on the surface of TRF2 have been mutated in clusters of three residues. The ability of these mutant TRF2 proteins, wtTRF2, and TBP to bind the transcription factors mentioned above will be compared to determine the mechanism of TRF2 recruitment to TATA-less promoters.

The After-Antibiotics Study: Investigating the Implications of Antibiotic Treatment for Urinary Tract Infection on the Urinary Microbiome and Metabolome in Young Women

Until recently, the urinary tract infection (UTI), a disease affecting 1 in 3 women globally by the age of 24 [1], was thought to be caused by the invasion of a single foreign pathogenic bacterial species into a sterile urinary tract [2]. However, contemporary PI: Rob Knight, Ph.D., advances have revealed that urine is not sterile: like other environments of the human body, the urogenital tract has its own distinct community of bacteria (microbiota) that UC San Diego School of Medicine, play a role in maintaining a healthy urinary tract [3].Current UTI treatment does not take UC San Diego Jacobs School into account the effect of antibiotics on protective microbes in the bladder [4] and their of Engineering, Department of contribution to antibiotic resistance [5]. This research project characterizes the collateral effects Pediatrics Research Division of antibiotic and non-antibiotic UTI treatments on the urinary microbiome and metabolome pre- and post-antibiotic treatment through shotgun sequencing, metabolomics, and multi-omics visualization. This work is designed to advance the prevention of UTIs in college-aged women.

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• Bottom row (left to right): Michael Endow, Sharada Saraf, Kwang-Tao Chou, Jason Chien, Siddhant Ambulkar, Jaidev Bapat, Yogitha Chareddy, Cade Oost, Pranav Iddamsetty, Madalyn De Viso • 2nd (left to right): Elizabeth Bottenberg, Michelle Pablo, Alexandra Vargas, Lisa Chik, Tushara Govind, Dominique Sy, Samreen Haque, Kristina Lapira, Olivia Gerald • 3rd row (left to right): Jordan Raus, Alicia Pacheco, Crystal Chan, Madeleine Yu, Lauren Brumage, Maddy Rae, Zarina Gallardo, Maya Gopalakrishnan, Nathan Troup, Yanmeng Shi, Lee Diego Lacasa • Top row (left to right): Sean Guy, Catherine Frusetta, Serena Tan, Sneha Ganguly, Arya Natarajan, Hanna Richkind, Julia Goebel, Liam Huber, Grace Lo, Edward Abarado

HEAD ADVISERS

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

FACULTY ADVISORY BOARD

ALUMNI ADVISORY BOARD

Timothy Baker, Ph.D Suckjoon Jun, Ph.D James Wilhelm, Ph.D Carolyn Kurle, Ph.D

Rahul Nachnani

Elvira Tour, Ph.D Chih-ying Su, Ph.D James Cooke, Ph.D

Special Thanks: Patera Design

STAFF EDITOR-IN-CHIEF Cade Oost

REVIEW BOARD MANAGER Jason Chien

EXECUTIVE EDITOR Madalyn De Viso

RESEARCH DESIGN EDITOR Yanmeng Shi

EDITOR AT LARGE Siddhant Ambulkar

FEATURES DESIGN EDITOR Arya Natarajan

FINANCE MANAGER Lisa Chik

SPECIAL SECTIONS DESIGN EDITOR Lee Diego Lacasa

HEAD PRODUCTION EDITOR Tushara Govind

WEBMASTER Pranav Iddamsetty

FEATURES EDITOR Rithvik Shankar

ONLINE EDITOR Sharada Saraf

RESEARCH EDITORS Kwang-Tao Chou Yogitha Chareddy

ONLINE REPORTERS Liam Huber Madeleine Yu Madison Rae Lauren Brumage Maya Gopalakrishnan

HEAD TECHNICAL EDITOR Jaidev Bapat TECHNICAL EDITORS Chinmay Kalluraya Alicia Ho Rebecca Hu Andrea Pedneault Jordan Raus

REVIEW BOARD

Nathan Troup Kyle Okamuro Anita Dev Angel Sorri-Battaroff Sean Guy Luke Okamuro Kishan Desai

PUBLICITY CHAIR Katherine Smith

Danny Styrpejko Kim Lol Vidit Goyal Saksham Gupta Darren Lam Julia Goebel Cassidy Lam

PUBLICITY COMMITTEE Elizabeth Bottenberg Vanessa Chen Belinda Liu Crystal Chhan Hanna Richkind Talisa Rodarte COMMUNITY OUTREACH CHAIR Kristina Lapira COMMUNITY OUTREACH COMMITTEE Samantha Madala Roberto Mercado Tiffany Huynh Saksham Gupta Belle Hilton Olivia Gerald BLOG MANAGER Alexandra Vargas BLOGGERS Rachel Sebastian Theresa Bui Henri Skinner Nikhil Jampana

Sneha Ganguly Serena Tan Kaitlyn Quach Guangzhao Liu Talisa Rodarte Sanjana Balagere Shibangi Pal

STAFF WRITERS Skylar Rains Grace Sahyouni Aishwarya Vuppala Anokhi Saklecha Arjune Nibber HEAD ILLUSTRATOR/ PHOTOGRAPHER Laurel Bowling PHOTOGRAPHERS Hope Hofffman-Larson Fredy Garcia Michael Endow Henry Shafron Daniel Melnick ILLUSTRATORS Qiuwan Liu Connie Mach Grace Lo April Damon Varsha Rajesh Vicky Hoznek

Amber Chung Aaron Trando Alicia Pacheco Torrey Rhyne

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