Sq vol11

Volume 11 2013-2014 sq.ucsd.edu

Undergraduate Research Journal Division of Biological Sciences

LETTER FROM THE EDITOR

In your hands you hold the 11th installment of the Saltman Quarterly Undergraduate Research Journal. The Saltman Quarterly journal is dedicated to the memory of the late Paul Saltman. We have written each volume with his vision in mind and this volume is no different. However this volume is not just dedicated to Paul Saltman. It is also dedicated to you; the student, the researcher. It is dedicated to your curious spirit, your willingness to take the time to read these pages and to continuously expand

ON THE COVER

your education. Our mission is to disseminate knowledge to our unique readership. I say unique because even though you are one in 6,000 biologists at UC San Diego, your interests will peak at different pages in this journal.

You might read our feature on axonal growth or a research manuscript on soil microbial diversity. No matter what your background, this volume contains something for any person with a passion for the science of life. The reason why we are so broad is because of how broad our staff is. The volume is a collaboration between undergraduates, graduates, professors, and doctors. We accomplish our mission in the creation and distribution of our journal and in a broader sense, by dedicating our time to fostering these relationships between student and teacher, our efforts are committed to the vision of Dr. Saltman.

This year SQ went through a metamorphosis. We came in as a group of determined students with very specific goals in mind and came out as

a team of professionals with booming confidence and much to say. And I am not talking figuratively; our changes are very tangible and visible throughout our brand new quarterly publications, weekly online articles and daily community blogs. You can read through any of these publications and see that we indeed have much to say. We speak on behalf of the UC San Diego biology community and our voice can be visualized through these pages. Your curiosity is what drives us and just as your eagerness for knowledge will never cease, our dedication to expressing your voice will never be exhausted.

Read at will, discover on.

Kevin Perez Editor-in-Chief 2013-14

Generously underwritten by

the Saltman family and supported by This young Crotalus scutulatus, also known as the Mojave rattlesnake, was found in the backyard of a home in San Bernardino County. The rattlesnake in this photo is in a defensive stance characterized by the s-shape posture on its neck and upper body in which it coils like a spring to give a quick bite when harassed. Despite its negative reputation, rattlesnakes in general will not bite unless provoked. Not only do rattlesnakes help control the mice population, but their venom can also be used to derive life-saving medicine.

Photo taken by Kyung Hyun Lee

The cover photo of a roaring lion was taken by Taylor Sanderson at the San Diego Zoo on April 8th, 2013.

The views expressed in this publication are solely those of Saltman Quarterly, its principal members and the authors of the content of this publication. While the publisher of this publication is a registered student organization at UC San Diego, the content, opinions, statements, and views expressed in this or any other publication published and/or distributed by Saltman Quarterly are not endorsed by and do not represent the views, opinions, policies, or positions of the ASUCSD, GSAUCSD, UC San Diego, the University of California and the Regents or their officers, employees, or agents. The publisher of this publication bears and assumes the full responsibility and liability for the content of this publication.

TABLE OF CONTENTS Insectivorous Birds Visit Resting Monkey Troops to Monitor Associated Food Source

by David Dannecker and Mary Jade Farruggia

PAUL SALTMAN: MENTOR

by Arjun Patel

FEATURES

9 13

5

Microbial Islands: Soil Microbe Diversity in Canopy Soil Patches

by Josh Kenchel Foraging and Feeding Preferences of Co-occurring Carollia Bat Species on the Shared Resource of Piper fruit

by Ariana Ananda, Maria F. Obando Quesada, and Susan Whitehead

BRIDGING THE GAP

by Rahul Lodhavia

MAKING THE PAIN FLY AWAY

by Vaishali Talwar

17

AT THE ROOTS OF SALT STRESS

by Lily Huang

23

THE SHRINKING SHORE

by Humphrey Lin

33 37 43

47

BREVIAS

51

SENIOR HONORS THESES

65

STAFF & ACKNOWLEDGEMENTS

RESEARCH

Ability of the Cane Toad (Chaunus marinus) to Associate Artificial Lights with Food

by Mary Jade Farruggia

29

PAUL SALTMAN :

MENTOR

1928

Paul Saltman is born in Los Angeles, CA 1949

B.S. in Chemistry from Caltech 1953

Ph.D. in Biochemistry from Caltech 1967

Joins UC San Diego as Provost of Revelle College 1972

1987

When we attend college, we are doing more than just furthering our education. In the

time that we spend here, we form important relationships with students and faculty and even become a part of a larger community outside the university. One of the great pioneers in establishing this kind of far-reaching community was the late Dr. Paul Saltman. Dr. Saltman was a renowned professor in biochemistry and a leader at UC San Diego who changed what it meant to be a student at this university.

Dr. Saltman first came to UC San Diego as a professor in the Department of Biology in 1967 and soon was appointed as Provost of Revelle College. He was tasked with decreasing student unrest over the Vietnam War. Instead of trying to crush student opposition with force or strict rules, Dr. Saltman opened his office door to students, as he would for the rest of his time at UC San Diego. Students could walk into his office and voice their concerns, talk about class, or just chat about anything.

Describing a student’s visit to his office, Dr. Saltman said, “I hate barriers. People use desks to keep people away. So I sat down beside her in a small chair...” This downto-earth approach to communicating with students worked well. Dr. Saltman believed that the key to fixing the conflict between the administration and students was to form a relationship built on mutual respect. This system really changed the way students saw their faculty and began to create a closer knit bond between the two groups.

Becomes Vice Chancellor for Academic Affairs at UC San Diego Publishes The California Nutrition Book Receives Career Distinguished Teaching Award

1994

1999

Paul D. Saltman Endowed Chair in Science Education is established at UC San Diego

Dr. Saltman also strongly believed to answer those questions.” He focused Much like Dr. Saltman, we want to do in applied knowledge in education on giving teachers more exposure to this through scientific communication and made sure to incorporate this science by having them learn from and education. We believe that having into curriculum changes at UC San university professors so that they could a more scientifically knowledgeable Diego. He once said, “I think that in not only be more knowledgeable but student body will improve the utility of this new curriculum that we are trying really understand why it was important our community. We want our readers to develop at the University of to feel passionately about what California at San Diego, we are they learn from our journal and She came and sat on the edge of to tell someone else about what trying to take the laboratory of the city, and no longer just the a chair in my office. I have a typical they read and make that person laboratory in the classroom on passionate about it as well. desk cluttered with homework the campus, and say, ‘Are you Thinking like a true biochemist, interested in society? Put your and a big chair. But I hate barriers. Dr. Saltman once said, “I really money where your mouth is. People use desks to keep people think the real reason that I’m here Get out in society and make is because I want to somehow away.

So I sat down beside her in a these changes.’” He encouraged communicate to you the joys students to be involved in their small chair... of teaching... so that perhaps I community and use what they Dr. Paul Saltman can seduce you in your way to learned to make a difference. be involved in the joys of the UC San Diego’s outreach efforts to know more about science. He felt teaching experience… these moments were increased further when Dr. that if teachers were more confident and that we have together will be catalytic Saltman began the Program for Teacher passionate about the science that they moments.” Dr. Saltman is still here with Enhancement in Science and Technology. taught, then students would develop an us in this journal, and we hope that his This National Science Foundationenthusiasm for science as well. Through message will inspire you to go out into funded program was started to improve his strong desire to educate and really the community and create more of these teachers’ understanding of science “turn someone on” with teaching, Dr. catalytic moments. within Los Angeles and San Diego grade Saltman increased the integration of schools. Dr. Saltman was shocked by the people into the university community.

WRITTEN BY ARJUN PATEL teachers’ lack of scientific knowledge This year, when the editors and Arjun is a Physiology and Neuroscience and had stated, “When a kid asks ‘Why writers of Saltman Quarterly were major from Eleanor Roosevelt College.

He will graduate in 2014. is the grass green? Why is the blood red? contemplating the purpose of this Why is the sky blue?’ you’d better know journal, we realized that what we really a lot of physics, chemistry and biology want to do is build a community as well.

“

”

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Photo taken by Elena Sojourner at the San Francisco Aquarium of the Bay.

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

FEATURES

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Spinal cord injury affects several hundred thousand people in the United States. It brings about damage to neurons, inducing neuronal death and axon degradation leading to physical dysfunction. Researchers at UC San Diego are currently looking into possible therapeutic agents into mitigating and even reversing the deleterious consequences of spinal cord injury. Dr. Zou and his colleagues are studying spinal cord injury by applying their knowledge of developmental neurobiology to the adult nervous system, bridging the gap between the two fields.

Bridging the Gap Rahul Lodhavia staff writer

Illustration by Elizabeth Cai 9

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The Big Connection

Back to the Basics

In the year 2012, an average of 270,000 individuals Before they could tackle the issue of solving spinal cord suffered spinal cord injuries. Every year, 250,000 more injury, the Zou lab first decided to study the development traumatic spinal cord injuries occur. It can happen of the nervous system and the growth of neurons and axons to anyone, with 83 percent of injuries occurring due during the stages of mammalian development in order to to events in daily life. Due to irreversible spinal cord gain a greater understanding of axonal growth. With this injuries, thousands of people have lost the ability to knowledge, they would be able to provide insight into perform normal body functions. For example, 53 the similar molecular mechanisms that occur following percent of all spinal cord injuries result in paraplegia, spinal cord injury. Dr. Zou used a mouse model to study the loss of motor and sensory function of the torso the nervous system during embryonic development and lower limbs, while 47 because of the genetic, percent result in tetraplegia, behavioral, and biological “ The Zou lab is one of the first similarities mice share with complete paralysis of the neck and below. Currently, no labs to make the connection humans. During embryonic concrete solution exists to fix molecules called between neuronal growth during development, damaged and dead neurons, morphogens direct and govern embryonic development and the the development of tissue and leaving injured individuals without a cure. This is because molecular mechanisms following various specialized cell types when neurons in the spinal cord within tissue. These morphogens adult spinal cord injury.” are damaged or injured, they die diffuse through the tissues of and can never grow back. an embryo during prenatal The spinal cord serves as the information highway development. Concentration gradients within cells are between the brain and the rest of the body. It takes in used to drive the differentiation of unspecialized cells stimuli from other parts of the body and sends those into specialized, specific cell types. Through reserach, the stimuli to the brain for processing. The brain then Zou lab was able to realize the importance of a family of relays information down the spinal cord which directs morphogens called the Wnt family. the function of different parts of the body. If the Dr. Zou and his colleagues found that during connection between the brain and the rest of the body neuronal development and axon growth in embryonic were to be severed due to an injury, the body would not development, the Wnt proteins are expressed in high be able to fully function. Because spinal cord injury is concentrations. These Wnt proteins bind to their irreversible, many suffer permanent loss of function in complementary receptor called Frizzled receptors, which many parts of their body. However, Dr. Zou and his are also induced during development. The binding of Wnt proteins to Frizzled receptors create a cascade of colleagues have been studying elements of the spinal secondary signals which contribute to the development cord both before and after injury in hopes of studying and growth of neurons and their axons. They found that and understanding this phenomenon. The Zou lab is in the presence of the Wnt4 signaling molecule, the axon one of the first to show that axon guidance molecules experienced growth and sprouting. To test the extent of important in development play an important role after the role the Wnt-Frizzled interaction has in neuronal spinal cord. With the great advancements in their growth, they introduced secreted Frizzled-related research, the future holds exciting news.

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FEATURES other cell types in the nervous system. Currently they are working to study the effects of the Wnt signaling pathway in myelination and glial cells. Myelination of neurons is extremely important in neuronal function as it allows for neuronal connections while glial cells provide neuronal support. Knowing the function of the reinduced molecules in both cell types is critical in developing a successful therapy to spinal cord injury.

Dr. Zou’s research in connecting developmental neurobiology with adult spinal cord neuron function and injury has set the foundation for many other researchers.

His work in revealing the function of the Wnt in spinal cord development has provided much needed insight in axon growth inhibition and stimulation. At the Peter MacCallum Cancer Centre in Australia, researchers using Dr. Zou’s work have generated antibodies that block the Wnt-Ryk signaling pathway. It has the potential to therapeutically target Ryk activity in spinal cord and peripheral nerve injury. Furthermore, these antibodies can be used as treatment in Wnt-dependent cancers by blocking the Wnt-Ryk signaling pathway. Ovarian cancer, which exhibits the overexpression of Ryk, can be one such targetable cancer. This is just one of the many implications that cutting edge research like that done by the Zou lab has for the future of studying spinal cord injury and other diseases.

The Zou lab hopes to continue to make substantial progress in uncovering more complex molecular mechanisms related to spinal cord development and injury. Though there is still more science to be uncovered and understood, Dr. Zou welcomes future progress with optimism. As he said, “There is much work to be done, but the future holds exciting news.”

Wnt-Ryk binding and promote neuronal sprouting and axon regeneration. Through their experiments, they were also able to determine that the Wnt-Frizzled pathway is the pathway mainly responsible for axon and neuronal growth.

Their findings could potentially lead to further studies and increased knowledge in the development of a solution to spinal cord injury and other related neuropathologies.

More recent research done by the Zou lab showed that a peripheral conditioning injury induced the expression of another Wnt receptor, related to Ryk. They found that after grafting compatible bone marrow stromal cells that secreted Wnt inhibitors, Wnt repulsion was blocked and regeneration increased by 60 percent. From their experiments, the Zou lab determined that the combination of Wnt signaling manipulation and conditioning lesion– mimicking growth enhancement may produce greater regeneration in injured spinal cord neurons.

To Be Continued Figure 1 Following spinal cord injury, reinduced Wnt-Ryk signaling inhibits the regeneration of sensory axons in adults.

proteins (sFRPs) that work to block the interaction of Wnts with the Frizzled receptor. After introducing these proteins, researchers found that the normal positive effect of Wnt4 was reduced significantly after just an hour. From this experiment, they concluded that Wnt proteins play an extremely important role in the growth of neurons during embryonic development and function best when they are able to bind to their complementary Frizzled receptor. Additionally the Wnt proteins act as guidance molecules and facilitate development, playing a key role in the development of the embryonic spinal cord neurons.

Bridging the Gap

Following their research in embryonic development, the Zou lab studied the active role Wnt proteins take following injury to the spinal cord. Though Wnt proteins are found in high concentration during embryonic development, they exist at very low concentrations in adult vertebrates.

However as found by the Zou lab, Wnt protein levels are found to be high in adult vertebratesfollowing injury to the spinal cord. Following manual lesions of the spinal cord in mice, researchers noticed the presence of Wnt proteins in higher concentration than what was normal. They found that three main Wnt proteins are re-induced: Wnt1, Wnt4, and Wnt5a. Additionally another Wnt receptor called Ryk, is also reinduced alongside the Frizzled receptor after injury. However, the Wnt protein has a higher affinity for Ryk and therefore binds with Ryk after injury. The signaling pathway generated by the Wnt-Ryk binding has a repulsive effect causing the retraction of axons and subsequent neuronal degeneration. The Zou lab found that by injecting Ryk function-blocking antibodies they were able to prevent 11

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Although great progress has been made by the Zou lab, the challenge to fully solve spinal cord injury is profound because of the complex nature of the biology being studied. Following injury, many molecules are reinduced that have many other functions. Their functions are relatively unknown but the Zou lab hopes to explore the function of these molecules and the other functions of the Wnt proteins. The Wnt-Ryk signalling pathway gives a potential target area of study, but it is not fully clear whether or not it is the only repulsive pathway.

The other molecules secreted following spinal cord injury could also have an inhibitory role. Furthermore, past research has shown that myelin and glial scar release inhibitory molecules that prevent the regeneration of neurons following spinal cord injury. However as Dr. Zou mentions in his 2013 Review, there are limitations involved in studying reinduced molecules in glial scar cells. It is known that glial scar cells provides inhibitory cues for axon regeneration. However, glial scarring is beneficial in providing a barrier against inflammation and protecting the area from secondary injury. Different approaches may be required to mitigate these effects, including varying experimental design by changing lesion sites and animal species. These other inhibitory molecules and classes of axons must be further studied and understood before research can move on to develop a conclusive treatment. “Before going directly into proposing a therapeutic agent, we must first understand the science behind spinal cord injury and the mechanism of Wnt signaling,” Dr. Zou explained. “Our knowledge of the full function of Wnt is not fully complete and we must look more into it.”

The Exciting Future

The Zou lab hopes to fully understand the Wnt family of morphogens. Researchers within the Zou lab are working hard to fully understand the role that these Wnt proteins play following spinal cord injury in neurons as well as sqonline.ucsd.edu

References

1. Halford MM, Macheda ML, Parish CL, Takano EA, Fox S, Layton D, Nice E, Stacker SA. “A Fully Inhibitory Monoclonal Antibody to the Wnt receptor RYK.” PubMed. 2013 Sep 18;8(9):e75447. Web. 2. Hollis ER 2nd, Zou Y. “Reinduced Wnt signaling limits regenerative potential of sensory axons in the spinal cord following conditioning lesion”. Proc Natl Acad Sci U S A. 2012 Sep 4;109(36):14663-8. Print.

3. Liu Y, Wang X, Lu CC, Kerman R, Steward O, Xu XM, Zou Y. “Repulsive Wnt signaling inhibits axon regeneration after CNS injury” J Neurosci. 2008 Aug13;28(33):8376-82. Print.

4. Lyuksyutova et al. “Anterior-Posterior Guidance of Commissural Axons by Wnt-Frizzled Signaling.” Science. 12 December 2003: 302 (5652), 1984-1988. Print.

5. Yimin Zou Laboratory, UCSD Division of Biological Sciences.

6. Zou, Yimin. “An Update on Spinal Cord Injury.” Neuroscience Bulletin. 1 August 2013, 29(4): 399–401. Print.

WRITTEN BY RAHUL LODHAVIA

Figure 2 During development of the embryonic spinal cord, the presence of Wnt proteins is observed. In adult invertebrates, these proteins exist in low concentrations. However, Wnt protein levels are high in adults after the spinal cord has been injured.

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Rahul is a Physiology and Neuroscience major from Thurgood Marshall College. He will graduate in 2015.

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n i a P e h t e k a M

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Skin ailments and wounds can be unusually painful. These irritations affect the barrier which separate our body from the outside world.

Thus, the protection of the epidermal layer should be considered of the utmost importance. Recently, eight genes which contribute to wound mitigation in the fruitfly have been uncovered and the human genome contains similar versions of these eight genes. This discovery could have profound implications in the field of wound healing. Who would have thought that flies could be the key to providing relief?

Vaishali Talwar staff writer

The image of a fruitfly does not normally conjure pleasant fly embryos and preparing them for experimentation thoughts. Flies are generally associated with dirt, garbage, when they came across mutant fruit fly embryos with infectious diseases and other such unappealing matters. sterile wounds. “A lot of work has been done in the field People are inclined to think that flies are more prone to of septic and infectious wounds, for obvious reasons. cause pain than to make it go away. However, research done However, not much is known about aseptic wounds,” by the McGinnis lab, UC San said Dr.William McGinnis, Diego, shows that the fruitfly, “When an injury breaks the a professor of biology as well Drosophila is probably the key epidermal barrier, this causes the dean of the The Division of to under-standing the genetics Biological Sciences. “Therefore, a pathway to transcriptionally looking at these fly embryos behind wound healing. The team of scientists at the McGinnis lab activate a number of genes with aseptic, sterile wounds discovered eight wound healing piqued our interest.” present in the nearby cells to naturally genes present in the fruitfly as Dr. McGinnis, along with regenerate the missing cells Michelle Juarez and Rachel well as a roadmap as to how these genes work. These genes Patterson, is an author of the and facilitate barrier repair.” paper on this discovery. The are responsible for the healing team was interested in finding out which genes played an of aseptic wounds, wounds that are not contaminated by important role in the healing of the wound as well as the bacteria or microorganisms. The human genome has counterparts or analogues of these eight genes. This pathway which activated these genes. discovery, therefore, could have a profound impact on Why the Fruitfly? aseptic wound healing in humans.

The fruit fly has been used as a model organism in a Surprisingly, the team had not originally set out to number of experiments for the past 100 years. This is locate these eight genes. Researchers had been examining because its genome bears a remarkable resemblance to the human genome, thereby providing endless possibilities for science. The Drosophila epidermal barrier consists of a single cell layer that secretes an impermeable, multilayered cuticle at the apical, or exposed, surface.

This impermeability is achieved through the cross linkage of protein and chitin polymers.

When an injury breaks the epidermal barrier, this causes a pathway to transcriptionally activate a number of genes present in the nearby cells to regenerate the missing cells and facilitate barrier repair. Since this pathway is evolutionarily conserved between Drosophila and Figure 1 A serine protease injection, or a hydrogen peroxide mammals, the research team thought that Drosophila injection, shows the wound response gene activation in all the would be a model organism for studying the process of epidermal cells. Both serine protease and hydrogen peroxide lie epidermal wound healing. upstream of the gene activation in the wound healing pathway.

Illustration by Grace Park

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FEATURES The Wound Healing Pathway

Understanding the cause of the gene activation was essential in pinpointing the genes involved in wound healing. Gene activation is the process of expressing the gene at the correct time. Serine proteases, or proteins which help cleave peptide bonds, were known to be involved in the process. However, their place in the pathway of gene activation was unknown. When the team injected a serine protease inhibitor into the embryos, the wounds that were previously inflicted on the embryos themselves, did not heal. Because of this, it became clear that a serine protease was required to activate those genes, and therefore needed to be upstream, or positioned before the genes, in the pathway. From previous studies and papers published on this subject, it was also known that mammalian wound healing pathways required hydrogen peroxide.

To find the position of hydrogen peroxide in the pathway, the researchers injected the embryos first with serine protease and then with hydrogen peroxide.

Interestingly, the wounds did not heal. However, when hydrogen peroxide was injected before the serine protease, the wounds successfully healed. The team came to the conclusion that hydrogen peroxide activated an enzyme which came upstream of the serine protease in the pathway.

FEATURES Flotillin-2 (aka as reggie-1 spread of epidermal wound response gene activation)

Figure 4 The in-situ hybridization method was used to help pinpoint the genes responsible for the epidermal wound healing in fruit fly embryos. A DNA or RNA probe helped to point out the mutated genes and the normal genes by binding to the non-mutated genes.

Figure 3 The coloring of the embryos shows the difference in the activation of a certain gene flotillin 2 in the wild type embryo of the fruitfly and in the mutant embryo. In the mutant, the over expression of flotillin 2 causes barely any transcriptional activation to wound response.

Figure 2 The wound response activation pathway consists of a multi-step process. Hydrogen peroxide activates the enzyme which activates the serine protease, which in turn activates the genes involved in the wound healing process.

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In order to carry on with the research, the team had to choose which type of serine protease was to be injected in the fruit fly embryos. Injecting the correct serine protease would enable the scientists to locate the genes involved in aseptic wound healing. “After a lot of ‘guesswork’ and ‘trial and error’ we came to the conclusion that trypsin would be the best fit, because once injected, it did not compromise the integrity of the epidermal cell barrier,” Dr. McGinnis stated.

In addition, trypsin didn’t promote apoptosis or cell death. Thus it was a perfect substitute for the real serine protease which was secreted endogenously, by the body itself, to promote wound healing. The identity of this real serine protease however, remains unknown.

Pinpointing the Genes

Once the components of the wound healing pathway were known, scientists set out to locate the genes which sqonline.ucsd.edu

had been transcriptionally activated by the pathway. “We knew that about 40 genes were involved in the process of wound healing in general,” said Dr. McGinnis. “So we used a brute force approach to narrow down the list to these eight, which was the toughest part of our research.”

The team spent a significant time period performing experiments and screening a large number of subject flies to see how many of them were actually affected by a genetic mutation. About one mutant in forty was affected by said mutation. The team then performed insitu hybridizations to locate the mutated gene.

These in-situ hybridizations involved using a labeled complementary RNA or DNA probe to localize

a specific RNA or DNA sequence or gene. This RNA or DNA probe would not bind to a mutation. Thus, without a probe attachment, a mutation in a particular gene could be immediately recognized. By recognizing the mutation, researchers were able to pinpoint the gene containing this mutation as well as the deficiency in an organism that the mutation caused. In this way, researchers were able to come to the conclusion that a particular gene was responsible for a specific function or phenotype which showed the deficiency.

In this case, the in-situ hybridizations identified the genes which were involved in wound healing processes.

Once the 40 genes had been located, the team noticed that eight of these genes actually regulated the process and activated the transcription factors required for aseptic wound healing. This activation of transcription factors in turn, caused re-epithelialization, cuticle regeneration and remodelling. However, these eight genes were not the only ones involved in the above functions. These were the only genes which gave conclusive results. A number of mutations however, were not explained by the eight genes. “We definitely know that there are more genes involved. We just need to perfect our methods and find them,” said Dr. McGinnis.

A Step Forward

The scientists intend to take this research a step further by finding more about the wound healing pathway as well as the genes involved in wound healing. Once the epidermal regeneration pathway in Drosophila has been established, one can look at it in mammals in order to apply these concepts to skin ailments in humans.

“600 million years ago, mice, humans and the fruitfly probably shared an aquatic worm like ancestor with the same control pathway,” said Dr. McGinnis. “Therefore wound healing processes in all three would be the same.”

The implications of the successes of this study are hopeful. Once the wound healing pathway in humans has been discovered, it could be possible to see a profound and positive impact on aseptic wound healing, thereby, making our pain ‘fly’ away.

References

1. McDonald, Kim. “Discovery of Wound Healling Genes in Flies could mitigate Human Skin Ailments.” www.ucsdnews.ucsd.edu University Communications and Public Affairs. 24 April 2013.

Web.17 July 2013.

2. Patterson RA, Juarez MT, Hermann A, Sasik R, Hardiman G, et al. (2013) Serine Proteolytic Pathway Activation Reveals an Expanded Ensemble of Wound Response Genes in Drosophila.

PLoS ONE 8(4): e61773. doi:10.1371/journal.pone.0061773 3. William McGinnis Laboratory, UC San Diego Figure 5 The fluorescent green color highlights a protein gene responsible for the activation of the transcriptional wound response in the fruitfly. The transcriptional response repairs epidermal breaks.

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WRITTEN BY VAISHALI TALWAR

Vaishali is a General Biology major from Thurgood Marshall College. She will graduate in 2016.

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It takes sunlight, fertile soil, and water to grow a beautiful garden.

Interestingly, when you water your garden, the water from the hose is the same as tap water, which contains salt ions. This same water is being used to irrigate massive expanses of crop field farmlands. Salt ions come to rest in the harvest soil and cannot be easily removed.

Therefore, new plant engineering techniques are being studied and utilized to build up the salt tolerance of plants.

Schroeder is a Distinguished Professor and Co-director of UC San Diego’s New Center for Food and Fuel for the 21st Century.

“As the intense drought periods in recent years have shown, irrigation could become even more important in the future,” Dr. Ulrich Deinlein emphasized. “Since most of the usually cultivated crops are rather salt sensitive, increasing soil salinization represents a major Contaminated Land threat for agriculture and food security. Therefore it is Earth has about 7% infertile, areas with high salinity crucial to understand the mechanisms underlying the that cannot be used for agricultural purposes. On top plant salt stress response in order to use this knowledge to of that, over 30% of the world’s obtain more salt tolerant crops.” Dr. irrigated crop production is “We are delving into the Deinlein is a post-doctoral scientist limited by salinity stress in unknown in these stresses working on plant salt tolerance in the crops, a problem that costs Dr. Schroeder’s lab.

California farmers over $1 billion and trying to make new Water irrigation is vital for per year. Researchers from Dr. fundamental advances. efficient agriculture. However, Julian Schroeder’s Lab at the UC water is increasing soil It’s exciting and there is a irrigation San Diego Biological Sciences salinity. Irrigation water carries trace Division are working to identify lot more unknown territory amounts of salts that remain and molecular mechanisms involved to be discovered.” accumulate in the soil over time and in plant salt tolerance. Identifying will not disappear when the water these mechanisms may help in developing plants evaporates. “Soil salinization is a result of irrigation, fossil salt and crops that can adapt to and tolerate imperfect deposits, mineral weathering, or seawater contaminations growing conditions. or appliance of fertilizers,” said Dr. Deinlein.

Dr. Schroeder’s lab investigates abiotic environmental Irrigation water is usually derived from natural stresses and how plants respond to drought, soil salinity sources, such as lakes or rivers, and is not purified or filtered. and elevation of CO2 due to climate change in plants. “In Another source of irrigation water is recycled drainage my lab, we focus on fundamental basic discovery research or waste water, which is purified microbiologically and and then we communicate it to other researchers and chemically. However, this purification does not deionize industry labs,” Dr. Schroeder explained. “We are delving the salt content. “Once there are high salt contents in into the unknown in these stresses and trying to make soils, it is not possible to remove them via chemicals,” Dr. new fundamental advances. It’s exciting and there is a lot Deinlein stated. more unknown territory to be discovered.”

With the constantly increasing population around the world, resources such as freshwater and rich soil are becoming even more scarce and precious. It may be urgently important to reconsider how foods will be grown to feed the growing population. New advances in plant breeding and biotechnology can help to achieve a more efficient and sustainable agriculture.

Lily Huang staff writer

Salt in the Soil

Globally, agriculture relies on irrigation to water secure crop yields. “California’s Central Valley as well as many arid areas like Australia utilize water irrigation on a regular basis,” Dr. Julian Schroeder explained. Dr.

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Illustration by Bianca Chong

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Identifying Salt Tolerant Plants

It is crucial to identify ways to counteract the Na+ over-accumulation of salt in plant leaves, because the saline toxicity is detrimental to photosynthesis and plant metabolism. If a plant does not have a mechanism to control salt tolerance, the photosynthetic process

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Figure 1 High-affinity potassium transporters (HKT transporters) can help retrieve Na+ in plants. Plants take in nutrients and water from its roots to the xylem which carries the nutrients and water to the leaves. The HKT transporters in the xylem retrieve and pull out the Na+ from the plant.

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FEATURES is disturbed, leading to growth arrest. A common phenotype of a severely salt stressed plant is leaf chlorosis.

The leaves of the stressed plant turn into a sickly yellowish or white-greenish color, indicating a lack of chlorophyll. Symptoms of anthocyanin accumulation such as red spots on the leaves also indicate that the plant is stressed. Reducing Na+ transport to leaves and controlling its accumulation is one important way to prevent plant salt stress.

Arabidopsis thaliana, an easy to cultivate, small flowering plant with a relatively small genome. They found that Arabidopsis had only one HKT gene, named “class 1 HKT gene,” making the hunt for the HKT gene function more amenable. Schroeder’s laboratory and his former post doc Dr. Nobuyuki Uozumi found that the Class 1 HKT transporters are expressed in the living cells surrounding the xylem veins and retrieve excess Na+.

Finding the Mechanism: HKT Research

In this research Schroeder and his colleagues found that land plant HKT transporters fall into two classes. The two classes of HKT transporters have differences in the structure of their cation selectivity filters. Specific ‘class 2’ HKT transporters are usually less Na+ selective than their Class 1 counterparts. Class 2 HKT transporters mediate cation influx into roots. Genome intron structure and sequence of the structure of these two HKT transporters are different.

“Plant plasma membrane transporters in the HKT family transport sodium (Na+) and potassium (K+) and play an essential role in salt tolerance,” explained Dr. Schroeder. “Our research showed that the ‘class 1’ HKT transporters are more Na+ selective and protect plant leaves from salinity stress by prohibiting toxic sodium over-accumulation in leaves.”

Further research showed that the same mechanism mediates improved salt tolerance in a more tolerant rice cultivar. An analogous, ancestral form of the HKT1; 5

Purifying and deionizing irrigation water is a possible option in order to reduce high salinity levels in the soil. However, disadvantages including high cost and need for a final resting place of the remaining salts are conflicting this approach. A good alternative would be to grow plants that are efficient at removing salt.

Dr. Schroeder’s laboratory identified High-affinity Potassium Transporters (HKT transporters) as a possible mechanism to invest in for salt retrieval in plants. HKT transporters retrieve and pull out the salt (Na+), which will prevent sodium over accumulation in plants and toxicity in the photosynthetic leaf tissues, as seen in Figure 1. Specifically, HKT transporters retrieve Na+ from the xylem, the the woody stem part of a plant.

The xylem is a complex tissue that carries nutrients and water from roots to leaves. A plant takes up water and nutrients from the soil through its roots and transports them to the xylem.

Within a crop species, there are certain varieties that have higher salt tolerance levels than others. One crop may grow better in saline soil than another same species crop due to its unique genetics and mutated DNA sequences. An over-expressed gene can cause the crop to become more salt tolerant and help it to live efficiently despite poor potassium environments.

A plant generally needs potassium (K+) to help ionically regulate many plant specific processes such as growth regulation and protein synthesis. In Dr. Schroeder’s laboratory, a potassium deficient yeast mutant line was found that can grow on low K+ soil medium, because it contained a certain gene from the wheat root cDNA (expressed gene) library, which is a library that contains only those DNA sequences that are transcribed into mRNA. The cDNA is generated by the enzyme reverse transcriptase, which has the ability to use the information in an RNA to generate a complementary DNA. Researchers in Dr. Schroeder’s laboratory isolated and identified this certain gene as a part of the HKT transporter gene family and named it HKT1. Then Schroeder and co-workers at UCSD found that HKT transporters also transport the cation sodium (Na+).

To find out why a transporter would transport an element that is less beneficial, his lab studied the HKT transporters in the popular plant model system of

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A Class of Their Own

Figure 2 Dr. Schroeder and his team are delving into the unknown about plant stresses and trying to make new fundamental advances for healthy plants like this one. There is still much to be uncovered about this study.

gene from a more Na+-tolerant wheat line was successfully crossbred into the commercial durum wheat by scientists in Australia, which helped to increase grain yields on saline soil by 25% in the field, illustrating the immense potential of this mechanism. “This is an example of how focused basic research in the plant Arabidopsis, the lab guinea pig of the plant research world, can accelerate crop improvement,” Dr. Schroeder explained.

Figure 3 Plants with salt stress showed visible physical changes. Placed side by side, these stressed and normal plants differentiated by colored tags show obvious differences in their growth.

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A Combination Method

Plants also have another natural mechanism called sequestration, found in the plant vacuoles, that can help with salt tolerance. Vacuoles could be described as the waste containers of plant cells which serve as deposition organelles for toxins, such as Na+, or as storage for osmotic compounds (Figure 4).

The HKT transporters, and the transporters that sequester sodium into vacuoles have great potential to improve salt tolerance of crops. “Thus combining [pyramiding]HKT transporter traits with vacuolar Na+ sequestration mechanisms provides a potentially powerful platform for molecular breeding and transgenic approaches to improve the salinity tolerance of crops,” said Dr. Schroeder.

However, combining these two mechanisms may not be enough to combat all of the possible abiotic stresses in plants, but it’s a start to fixing Na+ stress. “Drought stress is another big challenge that today’s agriculture has to face. Usually abiotic stress is not occurring with one single factor such as salinity. For example drought often takes effect together with salinity and therefore more complex solutions are needed,” Dr. Deinlein stated. “In my opinion the development of integrated approaches combining technical and biological efforts are very promising.”

Engineered Plants in Agriculture

Dr. Schroeder and Dr. Deinlein can also see the relevance of their research for biofuel approaches. “Since engineering of salt tolerant plants is related to higher biomass production when facing abiotic stress, this topic is also relevant for biofuel production,” said Dr. Deinlein.

Figure 4 The sequestration mechanism found in the plant vacuoles can help with salt tolerance. The vacuoles serve as waste containers where excess Na+ can be deposited.

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Figure 5 There is a visual difference between a stressed plant with functional HKT transports and one with non-functional HKT transports. The wild type (WT) normal plant with the functional HKT transporter genes looks healthy. The mutant plant with non-functional HKT transporters looks unhealthy with minimal growth and pale white-green leaves.

Some people have concerns of ingesting the engineered crop plants, but the crops that we consume today were traditionally cross-bred over centuries, which is also a form of genetic modification. “Every crop that is cultivated today was somehow genetically engineered by traditional breeding or crossing techniques over centuries,” explained Dr. Deinlein. “Our basic research leads to the identification of molecular mechanisms determining a plant’s fitness and that can be exploited by traditional breeding as well as by advanced biotechnological approaches, including molecular marker assisted breeding.”

Dr. Schroeder elaborates that we can possibly cross the HKT bred crops with commercial varieties to increase crop yields, “By doing that the breeders in Australia can increase yields by twenty-five percent.”

HKT transporters are not only found in wheat crop plants, but they are also found in rice plants. Researchers in Asia are looking to cultivate greater yields of rice from the salinic rice paddy fields. “The International Rice Research Institute (IRRI) in the Philippines is looking into rice varieties to identify the genes that are accounting for higher tolerance,” stated Dr. Schroeder.

Myanmar (Burma) and Bangladesh’s rice varieties generally have to be more salt tolerant due to their close proximity to the Indian Ocean and the subsequently more saline soil. “The rising sea waters in regions like

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Figure 6 These healthy looking plants have HKT transporters with high salt tolerance. There are more mechanisms to be discovered and understood about HKT transporters to find ways to help plants grow healthier and more robustly.

Bangladesh are compounding the huge losses caused by salinity on food security,” Dr. Schroeder stated.

The Future of World Sustainability

On how we can help to improve sustainable food and fuel production, Dr. Deinlein has a positive outlook on improving the future of sustainable food and fuel production. “With a steadily growing world population and the subsequently increasing demand for sustainable food production we are facing huge challenges. I believe that our basic research can in the long term help to explore use of lands that are currently considered as infertile, for agriculture,” stated Dr. Deinlein.

After discovering many methods for sustainable farming, we now need to learn how to share the wealthy, bountiful harvest. “We have to investigate our global efficiency of food use. For example, worldwide immense amounts of food are wasted on a daily basis whereas many people die of hunger in some parts of the world,” concluded Dr. Deinlein.

At the same time Schroeder states “Food independence and growing your own food has historically paved the way to wealth and upward mobility in many if not most countries.”

With these advancing techniques in plants, the agriculture industry may stand a chance for growing enough food to feed the world. The world’s crops may be so efficient one day that anyone from anywhere with any kind of conditioned soil and salt ion concentration will be able to grow a beautiful garden of their own.

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References

1. Gassmann, W., Rubio, F. and Schroeder, J.I. Alkali-cation selectivity of the wheat root high-affinity potassium transporter HKT1. The Plant Journal 10: 869-882 (1996).

2. Schachtman, D. and Schroeder J.I. “Structure and, transport mechanism of a high-affinity potassium K+ uptake transporter from higher plants.” Nature, 370: 655-658 (1994).

3. Schroeder J.I, Delhaize E, Frommer WB, Guerinot ML, Harrison MJ, Herrera-Estrella L, Horie T, Kochian LV, Munns R, Nishizawa NK, Tsay YF, Sanders D. Using membrane transporters to improve crops for sustainable food production.

Nature. 497.60-66 (2013).

4. Rubio, F., Gassmann, W. and Schroeder, J.I. Sodiumdriven potassium uptake by the plant potassium transporter HKT1 and mutations conferring salt tolerance. Science 270: 1660-1663 (1995).

5. Uozumi, N., Gassmann, W., Cao, Y. & Schroeder, J.I. Identification of Strong Modifications in Cation Selectivity in an Arabidopsis Inward-Rectifying Potassium Channel by Mutant Selection in Yeast. J. Biol. Chem. 270:24276-24281(1995).

6. Uozumi, N; Kim, EJ; Rubio, F; Yamaguchi, T; Muto, S; Tsuboi, A; Bakker, EP; Nakamura, T; Schroeder, JI. The Arabidopsis HKT1 gene homolog mediates inward Na+ currents in xenopus laevis oocytes and Na+ uptake in Saccharomyces cerevisiae. Plant Physiology 122(4): 1249-59 (2000).

7. Julian Schroeder Laboratory, UCSD Division of Biological Sciences.

WRITTEN BY LILY HUANG

Lily is a Molecular Biology major from Roger Revelle College. She will graduate in 2015.

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The

The California coast is changing. Years of human predation have put increasing selective pressure on coastal species, and we are witnessing a dramatic shift in their life patterns. Once large and abundant, mollusks are now small and scarce. This pattern may mark the beginning of a major change in coastal ecosystems.

Shrinking

To outsiders and residents alike, the city of San Diego is synonymous with amazing beaches and the beautiful California coast. UC San Diego is surrounded by an incredible collection of coastal reserves and attractions, most notably the nearby Scripps Coastal Reserve, or “the cliffs” as it is known by students. Coastal and rocky intertidal ecosystems like these harbor a vibrant and diverse array of organisms, and among them, several species of mollusks that are frequently harvested recreationally as food sources. Although someone collecting these local delicacies may not look past his or her immediate action, the over-harvesting of these mollusk species in the local coast significantly impacts the life patterns of the species, and as a result, the dynamic of the whole ecosystem.

One observed phenomenon that is the result of overexploitation is a dramatic decrease in organism size, a “downsizing” in nature. Evolution, Behavior, and Ecology professor Kaustuv Roy, in a recent research endeavor, tracked the evolutionary history of Lottia gigantea, a favorite for recreational harvesters. What the researchers found was a significant negative shift in organism size throughout the years. This disturbance in the local ecosystem is a harbinger of a much bigger issue, as coastlines worldwide experience over-harvesting.

Shore

Humphrey Lin staff writer Figure 1 The Lottia gigantea keeps a distinct “garden” free from invasive species. The scratch marks on the rock indicate the range of the Lottia’s territory.

Illustration by Jamie Yoon

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Figure 2 The graphs on the left (red) indicate the average size of Lottia in protected areas. The graphs on the right (gray) show the statistics from heavily exploited areas.

A Good Day on the Coast The 1970s marked the beginning of a new predation pattern for mollusks on the California coast. Recreational harvesting, though much of it illegal, dominated the routines of coastal life. “Much of the harvesting started in the 70s,” Dr. Roy said. “Since then, the ecosystem looks nothing like it used to.” One of the primary targets was abalone, which, once abundant, is now virtually absent from the ecosystem. A subtler problem, however, exists for many of the other shallow water species. On an ordinary day, one might look upon the coast and see an abundance of biodiversity. “Lots of the species are still there…there isn’t a lot of extinction,” Dr. Roy said. “But what we’re seeing is much smaller individuals.” Ecologists have noticed that compared to protected sites in which harvesting is strictly prohibited, sites that experience significant harvesting activity host individuals that are much smaller than their protected counterparts. To track the patterns and consequences of this downsizing, Dr. Roy’s lab focused on the species Lottia gigantea.

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Figure 3 The absence of Lottia in coastal ecosystems like this allows for the proliferation of invasive species.

Lottia gigantea

Since the decline of abalone on the Southern California coast, harvesters have turned to this rock-dwelling mollusk. The Lottia gigantea is a broadcaster species— male and female gametes are released into the water, and the hatched larvae swim around and eventually settle on rocks. Once settled, the Lottia is a very territorial species.

Each one has a distinct “garden” free of competitors.

An interesting part of the Lottia biology is that it is a hermaphroditic species. From birth to adolescence, every individual is male, and when the organism reaches about 75% of its maximum body size, it switches sex and becomes female—these are the ones the harvesters target. With such gender-specific selection, it is expected that the sex ratio would change; as females of the population get hunted, the small males should dominate the population. This, however, was not what Roy’s lab observed. “An average female lives 15-20 years, but under such selective pressure, no one lives that long…they get eaten if they do,” Dr. Roy said. “The whole biology starts to change as a result of top-down predation.” Roy and his team observed that individuals are turning into females at much smaller body sizes. This phenomenon is observed in many coastal ecosystems around Southern California, even in protected ones that do not strictly enforce harvesting policy.

This shift causes several ecosystem changes: first, the smaller females do not maintain as large a territory, and thus allow proliferation of barnacles and mussels on rock.

Second, although more organisms now exist, due to the less territorial nature of smaller females, total biomass has drastically decreased.

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Dr. Roy and his lab combined classic field biology techniques—capture and release and mitochondrial tagging—with a new, less common practice known as historical ecology. In order to document size shift over time, current data is not enough; the team must gather data from decades, or even centuries in the past and compare the numbers with current observed trends.

“The goal is to find ‘what is the trajectory of change?’,” Dr. Roy said. “We need more than a static study; we need to know the dynamics of the system over time.”

For current data, the Roy lab focused on five areas along the California coast: San Diego, Orange County, Santa Barbara, Ventura, and Los Angeles. Sites range from areas heavily impacted by harvesting to strictly protected areas in San Diego, including Torrey Pines State Reserve, Scripps Coastal Reserve, Ocean Beach, and Cabrillo National Monument in Point Loma. The researchers captured and measured Lottia in these sites, and used microsatellites to track gene flow between these areas.

For historical data, the team consulted various institutions with access to relevant data in the past.

“Historical ecology uses non-traditional data sources— old newspapers, old literature, old reports,” Dr. Roy said.

“There are fairly large databases available in museum collections and historical sites.” The lab was able to gather century-old reports from places as the University of California Museum of Paleontology, Natural History Museum of LA County, the California Academy of Sciences, and the Smithsonian in Washington, DC. By comparing these data sets, the lab was able to create models of size change over time.

In addition to Lottia, several other species of mollusks were documented in the same fashion. The data all point toward one trend—significant reduction in organism size.

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The lab also found, however, that heavily protected areas as Cabrillo National Monument show no significant size shift.

A Complex Issue

Although the effects of mollusk harvesting are now apparent, in order to predict future trends in coastal ecosystems, many other factors must be considered. “The problem with the coast is that there are a lot of things happening at the same time,” Dr. Roy said. “There is synergy amongst everything that’s going on…temperature change, ocean acidification, ocean nitrification, human harvesting…none of them happen in isolation.” The effects of human harvesting and organism response must be viewed through the scope of long-term ecosystem change. In order to track such longitudinal changes over time, the lab once again employs similar data synthesis techniques, but this time much larger in geographic and temporal scope.

Dr. Roy currently studies coastlines in Chile and Japan, where similar ocean temperature and acidity as well as different trends in human harvesting isolate harvesting as a variable. The lab also uses fossil records found in Torrey Pines State Reserve and other museum collections to gauge the ocean conditions 125,000 years ago. Fossil records are sent to other labs which specialize in paleothermometry, or determining ocean temperatures in the past by comparing the ratio of oxygen isotopes.

The overarching goal is ultimately to create a predictive model that accounts for all the anthropogenic disturbances. With most of the world’s major cities located on the coastline, disturbances in the coastal ecosystem can have far-reaching consequences for the human population. Coastal ecosystems provide many important services as food and regulation of global climate, and without an accurate predictive model, it is hard to enforce a policy that protects these systems.

Dr. Roy’s Lottia study is a small step in understanding the dynamics between human impact on the environment and its subsequent responses. His lab seeks to further understand these dynamics by studying other relevant factors. “A lot of the trajectory we take depends on the interest of the students,” Dr. Roy said. “I’d love to see more students get involved in this endeavor.”

References

2000 Figure 4 Another example of an ecosystem in which Lottia is now absent—no distinct gardens exist, but rather, the rocks are covered completely by other species.

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Figure 5 Over time, selective pressure from human harvesting has drastically shifted the average size at which Lottia Gigantea change gender. Large females are no longer present in areas of heavy exploitation.

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1. Fenberg, Phillip, et al. “Range limits and geographic patterns of abundance of the rocky intertidal owl limpet, Lottia gigantea.”

Journal of Biogeography, 38(2011), 2286-2298. Print.

2. Kaustuv, Roy. Personal Interview 20 November 2013.

WRITTEN BY HUMPHREY LIN

Humphrey is a Bioengineering: Biotechnology major from Earl Warren College. He will graduate in 2016.

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Known 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. The Research section highlights the work of these undergraduate researchers, whose contributions constantly push forward the boundaries of science.

Photo taken by Siddharth Atre near Pune, Maharashtra, India.

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Insectivorous Birds Visit Resting Monkey Troops to Monitor Associated Food Source David Dannecker and Mary Jade Farruggia Insectivorous birds periodically check on groups of mantled howler monkeys (Alouatta palliata), because the movement of the monkeys flushes insects from the leaves, making them more available to birds.

This study addressed the question of whether or not this was a deliberate behavior by observing bird populations around groups of howler monkeys in Santa Rosa National Park, Costa Rica. In our study we documented bird sightings in our observations of both moving and nonmoving groups of monkeys, as well as areas with no monkeys, with results indicating a significant association of birds with groups of monkeys. The implications of this study reveal how the behavior of one species can shape the behavior of another through indirect means.

For the full-length research manuscript, please pick up a print issue of SQ Volume 11 from the following locations: Biological Sciences Student and Instructional Services Office Pacific Hall, Room 1128, Revelle Campus, UC San Diego Biomedical Library UC San Diego School of Medicine

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For the full-length research manuscript, please pick up a print issue of SQ Volume 11 from the following locations: Biological Sciences Student and Instructional Services Office Pacific Hall, Room 1128, Revelle Campus, UC San Diego Biomedical Library UC San Diego School of Medicine

WRITTEN BY DAVID DANNECKER AND MARY JADE FARRUGGIA

David and Mary Jade are Ecology, Behavior, and Evolution majors. David is from Earl Warren College. He will graduate in 2014.

Mary Jade is from Eleanor Roosevelt College. She will graduate in 2015.

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Ability of the Cane Toad (Chaunus marinus) to Associate Artificial Lights with Food Mary Jade Farruggia Cane Toads, Chaunus marinus (Anura, Bufonidae) have demonstrated attraction to artificial lights at night, because the lights attract more insects for the toads to eat. This study addresses the question of whether or not this is a learned behavior. In San Luis Monteverde, Costa Rica, I tested five toads never before exposed to artificial lights and two toads with past exposure to artificial lights by rewarding them with food when they arrived at an artificial light placed in a training structure. The results of this classical conditioning study indicate a clear ability of C. marinus to learn to associate light with a food reward. They also offer an explanation as to why cane toads thrive in urban areas, and consider the implications of artificial light in changing population dynamics in the toads’ natural habitat, and in the urban areas that they now inhabit.

For the full-length research manuscript, please pick up a print issue of SQ Volume 11 from the following locations: Biological Sciences Student and Instructional Services Office Pacific Hall, Room 1128, Revelle Campus, UC San Diego Biomedical Library UC San Diego School of Medicine

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For the full-length research manuscript, please pick up a print issue of SQ Volume 11 from the following locations: Biological Sciences Student and Instructional Services Office Pacific Hall, Room 1128, Revelle Campus, UC San Diego Biomedical Library UC San Diego School of Medicine

WRITTEN BY MARY JADE FARRUGGIA

Mary Jade is an Ecology, Behavior, and Evolution major from Eleanor Roosevelt College. She will graduate in 2015.

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Microbial Islands: Soil Microbe Diversity in Canopy Soil Patches Josh Kenchel Decaying organic matter forms patches of soil in trees, known as “canopy soil”, which are isolated from the ground. This study sought to determine whether patterns of microbe diversity in canopy soil patches are consistent with patterns of macrospecies diversity on islands. Via microscopy, I recorded microbial morphospecies abundance in ground and canopy soil. Canopy soil contained significantly lower alpha diversity than ground soil and the similarity between the microbial communities of those pairs decreased as the distance between them increased. Ground-canopy pairs were significantly more similar than pairs of ground soil samples and pairs of canopy soil samples. I found no significant difference in species evenness or beta diversity between ground soil and canopy soil. The data indicate that canopy soil patches limit the number of species that can inhabit them. However, the separation between ground and canopy soil may not actually form a barrier to microbial movement.

For the full-length research manuscript, please pick up a print issue of SQ Volume 11 from the following locations: Biological Sciences Student and Instructional Services Office Pacific Hall, Room 1128, Revelle Campus, UC San Diego Biomedical Library UC San Diego School of Medicine

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RESEARCH

For the full-length research manuscript, please pick up a print issue of SQ Volume 11 from the following locations: Biological Sciences Student and Instructional Services Office Pacific Hall, Room 1128, Revelle Campus, UC San Diego Biomedical Library UC San Diego School of Medicine

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For the full-length research manuscript, please pick up a print issue of SQ Volume 11 from the following locations: Biological Sciences Student and Instructional Services Office Pacific Hall, Room 1128, Revelle Campus, UC San Diego Biomedical Library UC San Diego School of Medicine

WRITTEN BY JOSH KENCHEL

Josh is a Biochemistry and Cell Biology and Ecology, Behavior, and Evolution double major from Roger Revelle College. He will graduate in 2014.

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Foraging and Feeding Preferences of Co-occuring Carollia Bat Species on the Shared Resource Piper Fruit Ariana Ananda, Maria F. Obando Quesada, Ph.D., and Susan Whitehead, Ph.D. Bats in the genus Carollia are specialists on the fruits from the genus Piper and serve as the primary dispersers of Piper throughout the neotropics. At La Selva Biological Station in Costa Rica, three species of Carollia coexist: C. castanea, C. perspicillata, and C. sowelli. In this study, we explored whether the three Carollia species exhibit differential feeding preferences for various species of Piper, which would provide one potential mechanism for reducing competition and allowing these species to coexist despite similar niche requirements. Pair-wise choice tests were conducted with individual bats in flight cages that compared each species’ preferences for Piper sancti-felicis, the most abundant Piper species at La Selva, against one of six other Piper species. Results showed that Carollia feeding preferences were similar for most Piper species tested; however C. sowelli did differ from C. castanea in its preference for P. colonense. Coexistence theory suggests that different species cannot cohabitate without resource partitioning. Because all three species of Carollia appear to compete for the same limited food resources, this implies there are other mechanisms that are allowing for Carollia’s coexistence at La Selva. Different foraging habits, such as emergence time, food handling rate, and proportion of Piper in the diet may be contributing factors allowing these three closely related species to coexist.

For the full-length research manuscript, please pick up a print issue of SQ Volume 11 from the following locations: Biological Sciences Student and Instructional Services Office Pacific Hall, Room 1128, Revelle Campus, UC San Diego Biomedical Library UC San Diego School of Medicine

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For the full-length research manuscript, please pick up a print issue of SQ Volume 11 from the following locations: Biological Sciences Student and Instructional Services Office Pacific Hall, Room 1128, Revelle Campus, UC San Diego Biomedical Library UC San Diego School of Medicine

WRITTEN BY ARIANA ANANDA, MARIA F. OBANDO QUESADA, AND SUSAN WHITEHEAD

Ariana is a Wildlife Conservation and Photography major from John Muir College.

She will graduate in 2014. Dr. Quesada is a professor in Escuela de Biologia in the Universidad Nacional de Costa Rica. Dr. Whitehead is a faculty member in the Department of Ecology and Evolutionary Biology at the University of Colorado, Boulder.

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BREVIAS BREVIAS

BREVIAS

BREVIAS The UC San Diego Division of Biological Sciences offers a unique contiguous BS/MS program in which current undergraduates can expand their education and receive a Master’s degree within 3-5 quarters following completion of their Bachelor’s degree. The Brevias section presents a set of competitively selected manuscripts from students in the Master’s portion of the BS/MS program.

Photo taken by Dilara Onur at the San Diego Zoo Safari Park.

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Effects of Trophic Interactions and Climate on CO2 Flux from Reservoirs

Emily Adamczyk, Ecology, Behavior, and Evolution | PI: Jonathan Shurin, Ph.D. Background Atmospheric carbon dioxide (CO2) concentrations are rising due to the burning of fossil fuels and land use changes, thereby increasing global temperatures. Artificial reservoirs are particularly important to the flux of CO2 from freshwaters because large amounts of organic carbon accumulate in their sediments [1]. It is estimated that they emit twice the quantity of CO2 compared to natural lakes [1, 2]. The ecological processes that occur within these reservoirs determine whether they release or absorb CO2. Primary producers such as phytoplankton utilize CO2 for photosynthesis and are consumed and/or compete with fungi, invertebrates, bacteria and viruses [3]. Little is known about how these ecological interactions influence CO2 flux, as they vary spatially and temporally.

Research and Methods We are currently studying how seasonal changes in abiotic and biotic factors, such as zooplankton, phytoplankton, chytrid fungi, bacteria, dissolved organic carbon, and water temperature, influence CO2 flux from three local reservoirs. We collected samples from Lakes Murray, Miramar, and Poway on a weekly basis for abiotic variables (conductivity, pH, salinity, % dissolved oxygen, wind speed), and biotic variables (the abundance of planktonic invertebrates, fungi, bacteria, and chlorophyll-a). Using the data we collected, we calculated the partial pressure of CO2 (pCO2) which indicates its exchange between the water and the atmosphere [1]. pCO2 and wind speed was used to calculate CO2 flux between the water and the atmosphere. We measured chlorophyll-a using a fluorometer to determine phytoplankton abundance. We examined bacteria and chytrids using DAPI stain and calcofluor white, respectively, and used a fluorescence microscope to determine their abundance. Dissolved organic carbon (DOC) was analyzed using a Total Organic Carbon Analyzer at the Scripps Institution of Oceanography. Data were analyzed using ANOVA models with lake and date as predictors.

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Results Bacteria abundance exhibited a seasonal trend, with the lowest biomass during summer, and highest during winter. This seasonal pattern of bacterial abundance differed among lakes (Figure 1c, P=0.018) and was inversely correlated with water temperature (Figure 1a, r = -0.66). Phytoplankton biomass (chlorophyll-a) did not vary seasonally but concentrations were consistently highest in Lake Murray (Figure 1b, P<0.001). pCO2 (ppm) exhibited a seasonal trend where the concentrations rose during winter (Figure 1c, P<0.001). DOC concentrations were significantly different among reservoirs (P<0.001), with the highest concentrations in Lake Murray and the lowest concentrations in Lake Miramar (Figure 1d).

Discussion pCO2 rose during the winter in two of the three reservoirs but remained undersaturated compared to atmospheric values, indicating the reservoirs were absorbing CO2 from the air (Figure 1c). This is unlike most reservoirs, which are oversaturated with carbon and release large quantities of CO2 into the atmosphere [1, 2]. DOC concentrations varied at each lake but not over time (Figure 1d). There were seasonal trends in bacteria abundance (Figure 1a), in contrast with phytoplankton biomass

which did not vary seasonally (Figure 1b).

Bacterial biomass was inversely correlated with water temperature, indicating that bacteria proliferate in colder habitats. Additionally, bacteria abundance was not correlated with DOC concentrations (r=-0.012) suggesting that the increase in bacteria abundance during the winter was a result of a factor other than allochthonous organic input or in situ primary production.

The growing human population is affecting the global carbon cycle by diverting more water from rivers and storing it in reservoirs.

Our results suggest that reservoirs built for storing drinking water in semi-arid ecosystems absorb more atmospheric carbon than they are releasing, meaning that the reservoirs contain less CO2 compared to the air. Our ultimate goal is to determine how the seasonal variation in ecological processes and species interactions influence the release of atmospheric carbon and what that means for long-term changes in the global carbon cycle.

References [1] J. J. Cole et al. Plumbing the global carbon cycle: integrating inland waters into the terrestrial carbon budget. Ecosystems 10, 172-185 (2007).

[2] V. L. St. Louis et al. Reservoir surfaces as sources of greenhouse gases to the atmosphere: A global estimate. BioScience 50, 766-775 (2000).

[3] J. Urabe et al. Within-lake and watershed determinants of carbon dioxide in surface water: A comparative analysis of a variety of lakes in the Japanese Islands. Limnology and Oceanography 56, 49 (2011).

Figure 1 a. Number of bacteria per liter by date.

Bacteria abundance is low during the summer and increases during winter months (date: P<0.001), and is inversely correlated with water temperature (r= -0.66). b. Chlorophyll-a concentration (μg/L) by sampling date. There are no seasonal trends in chlorophyll-a, but Lake Murray has consistently higher chl-a concentration than the other lakes (lake: P<0.001). c. pCO2 by sampling date. pCO2 varies with time and is higher in the winter and lower in the summer (date: P<0.001). The seasonal pattern also varies among lakes (lake*date: P=0.034). d. DOC concentrations by sampling date. DOC concentrations were significantly different among reservoirs (P<0.001), with the highest concentrations in Lake Murray and the lowest concentrations in Lake Miramar. sqonline.ucsd.edu

Mechanisms of Thy-1 and its Effects on Signaling Pathways Regulating Myofibroblast Differentiation in Pulmonary Fibrosis Jeeyeon Kim, General Biology | PI: James Hagood, M.D. Background Idiopathic pulmonary fibrosis (IPF) is an incurable and destructive disorder characterized by active fibrogenesis [1]. Fibrogenesis, the process by which fibroblasts (scar-forming cells) proliferate and accumulate excessive extracellular matrix (ECM) molecules, is a mechanism of normal wound healing, but in fibrosis this process is impaired, causing excessive scarring in the lungs. One of the distinct features of IPF is the development of myofibroblastic foci, which indicate areas of active fibrosis. Myofibroblasts are a distinct population of contractile fibroblasts that are considered to be main sources of ECM production in scar tissue and express alphasmooth muscle actin (α-SMA) [1, 2].

Transforming growth factor (TGF)-β is a protein that controls proliferation and cellular differentiation. Patients with IPF have been shown to activate latent TGF-β, regulating fibroblast to myofibroblast differentiation [3]. A member of a skeletal muscle-specific transcription factor (MRF) family called MyoD is also known to initiate fibroblast to myofibroblast conversion [1]. Thy-1 (CD90), a glycosylphophatidylinositol-linked outer membrane leaflet glycoprotein, is a fibrosis suppressor. It functions as a cell adhesion molecule and modulates cellular signaling pathways [3]. Thy-1 (+) fibroblasts are found in normal human lung while the majority of myofibroblasts in fibroblastic foci of IPF are Thy-1 (-) [1]. Prolonged and severe pulmonary fibrosis was also seen in rat lungs transiently stimulated with TGF-β.

Research and Methods In this experiment, we sought to determine the degree to which Thy-1 regulates myofibroblastic differentiation of fibroblasts.

Rat fetal lung cells (RFL-6), fibroblasts which lack Thy-1 expression, were transfected with full-length murine Thy-1.2 (CD90.2) cDNA (making them Thy-1 positive) or empty vector (still lacking Thy-1). Cells were grown and then stimulated with 5ng/ml TGF-β at different time points to observe the expression levels of proteins characteristic of the myofibroblastic sq.ucsd.edu

phenotype. Immunoblotting was conducted using anti-α-SMA (Biocare) and anti-MyoD1 monoclonal antibody (GeneTex). AntiGAPDH polyclonal antibody (GeneTex) was used to normalize expression data.

Results Baseline α-SMA expression is increased in Thy-1 (+) fibroblasts, however response to TGF-β and increased expression of α-SMA is more prominent in the Thy-1 (-) subset in rat lung fibroblasts. It is important to note that Thy-1 (-) TGF-β stimulated cells demonstrated a rising response to TGF-β over time as compared to the Thy-1 (+) cells, which showed no noticeable difference between unstimulated and activated cells (Fig. 1A). In the MyoD incubation, there was also increased intensity in Thy-1 (-) cells stimulated with TGF-β for 24h as compared to the level of intensity observed in Thy-1 (+) cells undergoing stimulation for the same amount of time (Fig. 1B). Unstimulated Thy1 (-) cells had no indication of the presence of α-SMA or MyoD, which was expected because both are markers of myofibroblastic differentiation (Fig. 1A, B).

Discussion Studies have shown Thy-1 to inhibit lipid raftassociated signaling via Src-family kinase (SFK) and focal adhesion kinase (FAK) pathways [4, 5]. Recent work has also disclosed the interaction of Thy-1 with the extracellular matrix receptors alpha-v integrins and syndecan 4 at the cell surface, modulating cell-cell

and cell-matrix interactions and mechanical coupling, to inhibit TGF-β activation and myofibroblastic differentiation [6]. Global targeting of the myofibroblast phenotype offers the best possibility for effective IPF therapy, and the study of mechanisms of Thy-1 working as a “phenotype switch” in lung myofibroblasts may lead to improved clinical therapeutics.

References [1] Sanders YY, Kumbla P, Hagood JS (2007).

Enhanced Myofibroblastic Differentiation and Survival in Thy-1(-) Lung Fibroblasts. Am J Respir Cell Mol Biol 36: 226-235.

[2] Phan SH (2003). Fibroblast phenotypes in pulmonary fibrosis. Am J Respir Cell Mol Biol 29: S87-S92.

[3] Rege TA, Hagood JS (2006). Thy-1 as a regulator of cell-cell and cell-matrix interactions in axon regeneration, apoptosis, adhesion, migration, cancer, and fibrosis. The FASEB Journal 20: 1045-1054.

[4] Lagares D, Busnadiego O, Garcia-Fernandez RA, Kapoor M, et al. (2012). Inhibition of Focal Adhesion Kinase Prevents Experimental Lung Fibrosis and Myofibroblast Formation. Arthritis & Rheumatism 64: 1653-1664.

[5] Rege TA, Pallero MA, Gomez C, Grenett HE, Murphy-Ullrich JE, Hagood JS (2006). Thy-1, via its GPI anchor, modulates Src family kinase and focal adhesion kinase phosphoylation and subcellular localization, and fibroblast migration, in response to thrombospondin-1/hep I. Experimental Cell Research 312: 3752-3767.

[6] Avalos AM, Valdivia AD, Munoz N, Herrera-Molina R, et al. (2009). Neuronal Thy-1 induces astrocyte adhesion by engaging syndecan-4 in a cooperative interaction with avb3 integrin that activated PKCa and RhoA. Journal of Cell Science 122: 3462-3471.

Figure 1 (A) α-SMA – There is a noticeably steady increase in the presence of α-SMA (a myofibroblastic marker) in Thy-1 (-) cells, while Thy-1 (+) cells have similar band intensity for α-SMA in both the untreated and the TGF-β stimulated samples. (B) MyoD – Stronger band intensity is seen at the last time point of TGF-β stimulated Thy-1 (-) cells compared to the 24h band in Thy-1 (+) cells.

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FEATURES

FEATURES

Photo taken by Siddharth Atre near Pune, Maharashtra, India.

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SENIOR HONORS THESES

The Senior Honors Theses section highlights the achievements of the accomplished undergraduate researchers in the graduating class of 2014.

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Application of proteomic mass spectrometry to the characterization of Mycobacterium smegmatis bacteriophage Chiara Ricci-Tam, Warren College, Molecular Biology major PI: Joe Pogliano, Ph.D., Division of Biological Sciences, Molecular Biology Section

SENIOR HONORS THESES The Division of Biological Sciences Senior Honors Theses Program (BISP 196) is open to undergraduate

biology majors who have an overall and 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 2013-14 academic year.

As one of the most abundant and genetically diverse life forms, bacteriophages hold great potential for advancing our understanding of bacterial-phage interactions and for treating bacterial infections in these times of rising antibiotic resistance. Mycobacteriophages-viruses that infect such bacterial hosts as Mycobacterium tuberculosis and Mycobacterium smegmatis are of particular interest, and efforts are underway to characterize their diverse structures, functions, and replicative processes. Here, we use protein mass spectrometry to examine the proteomes of both sequenced and newly isolated M. smegmatis bacteriophages, with the goal of characterizing and classifying them on the basis of conserved proteins identified from genomic studies. Tandem mass spectrometry was conducted on tryptic digests of prepared phage lysates, and peptides were identified with ABSCIEX Protein Pilot 4.0 software. The spectra were analyzed using a protein database compiled from the published genome sequences of M. smegmatis phages in the NCBI repository. Proteomic analysis of multiple sequenced phages allowed us to annotate putative gene products with likely roles in the replication and structure of the phage particle. This approach is effective as a rapid, high-throughput for characterization of phage proteomes and permits the enhanced annotation of the putative gene products of sequenced phages.

Role of Pioneer Transcription Factor FOXA2 in Various Stages of Pancreas Development Dieter K. Lam, Warren College, Biochemistry and Cell Biology major PI: Maike Sander, M.D., UC San Diego School of Medicine, Department of Pediatrics

Maternal High Fat Feeding and Fetal Fat Accumulation in Mice Zhuyu “Julia” Guo, Eleanor Roosevelt College, Human Biology major PI: Jianhua Shao, M.D., Ph.D., UC San Diego Department of Pediatrics

Maternal obesity or excessive weight gain during gestation increases offspring birth weight in humans.

High birth weight is a strong indicative factor for obesity and metabolic defects during adulthood.

Due to the limitations of some tissue mass measurement techniques of newborn and fetal fat, it is not clear whether this increase in birth weight is attributed to the increase in body fat or other tissues. By using the mouse model, this project investigated the effects of maternal over-feeding on fetal growth and fat development. High fat diet (60% calories from fat) or regular chow is provided to 2-3 month old C57BL/6 dams during gestational periods. Fetuses were collected at several time points during pregnancy through C-section and neonatal samples were collected within 12 hours after birth. Water is first removed from the samples through heating them in an oven at 85 °C overnight until constant mass. Then, fat is extracted from the samples by solvating in petroleum ether using a Soxhlet chemical extraction apparatus. Our results revealed that the average body composition of newborns to chow-fed dams were 84.35±0.15 % water, 0.59 ± 0.05% fat, and 15.06 ± 0.14% non-fat tissues.

Comparing body fat of newborns, there was a slight increase in neonates of high fat diet-fed dams (n=6, 7.8±0.7 vs 19.9±4.5mg; p=0.06) but did not reach to statistical significance. In addition, compared to newborns from chow-fed dams, neonates of high fat diet-fed dams also displayed a slight increase in non-fat tissue mass (n=6, 196.5±6.2 vs 209.8±6.7mg; p=0.22) and water content (n=6, 1099.5±24.5 vs 1185.8±37.1mg; p=0.11). This study is part of an ongoing project in our lab. More samples are required to conclude the effects of maternal over-feeding on fetal fat development in mice.

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Forkhead box protein A2 (FOXA2) is known to play an important role in priming condensed chromatin in preparation of binding by other regulatory transcription factors. Specifically, FOXA2 is known to be a pioneer transcription factor, which differs from normal transcription factors by its ability to bind its target sequences on nucleosomes or compacted chromatin without the need for any cooperative binding. FOXA2 is expressed throughout most stages of pancreas development, and is proposed to be a crucial player in opening specific regions of condensed chromatin for subsequent binding by other regulatory factors during pancreas development. To define the role of FOXA2 during pancreatic differentiation, we must gain a better understanding of when and where FOXA2 binding occurs and whether it is required for initiation of pancreatic gene expression.

To address these key questions, we employ ChIP-seq and shRNA mediated knockdown at various stages of pancreatic differentiation of human embryonic stem cells (hESCs). Through these experiments and subsequent analysis, we hope to disentangle the role of FOXA2 in greater detail.

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The Role of HTR7 Gene Regulation in Influencing SSRI Response in Bipolar Disorder Donel Purcella, Revelle College, Physiology and Neuroscience major PI: John R Kelsoe, Jr, M.D., Department of Psychiatry, Institute for Genomic Medicine, UC San Diego

Bipolar Disorder (BD) is a serious brain chemical disorder characterized by alternating euphoric and depressive states associated with suicidality. The disorder responds to medications belonging to the Selective Serotonin Reuptake Inhibitor (SSRI) family. Some BD patients respond well to SSRI therapy, others respond poorly.

Currently, there is no method to pre-screen patients for SSRI response. Therefore, patients must endure medication trials followed by efficacy evaluation, which often delays effective treatment. In a previous study, Single Nucleotide Polymorphisms (SNPs) associated with SSRI response were genotyped in the HTR7 promoter region.

In this study, two types of BD subjects were identified based on two criteria. The first criterion was either- good or poor response to SSRI therapy. The second criterion was homozygotic alleles associated with the HTR7 promoter region, either a wild-type reference allele or an alternative one with a previously identified SNP. The two types of subjects were identified as either good responders homozygotic for the reference allele or poor responders homozygotic for the alternative allele. The hypothesis of this study is that there is a difference in HTR7 regulation in variations of the HTR7 promoter. In order to evaluate this difference, DNA fragments from both subject types were individually subcloned.

These subclones were then evaluated by luciferase-based reporter assay for modulation of HTR7 gene expression.

The results of this study will provide insight into a genetic link between Bipolar Disorder and SSRI response with the potential to develop future drug therapy pre-screening methods.

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Discovering Essential Genes for the Day-Night Cycle in Cyanobacteria Anish Pal, Thurgood Marshall College, Biochemistry and Cell Biology major, Business minor PI: Susan Golden, Ph.D., Division of Biological Sciences, Molecular Biology Section

Dissecting the Nuclear Import Mechanisms of a Transcription Factor in Axon Regeneration Phoenix Ying, Thurgood Marshall College, Physiology and Neuroscience major, Psychology minor PI: Yishi Jin, Ph.D., Division of Biological Sciences, Neurobiology Section

Our world is heavily dependent on liquid fossil fuels that are both limited and environmentally harmful.

The model photosynthetic cyanobacterium, Synechococcus elongatus exhibits tremendous potential as a biofuel production organism as it has already been engineered to produce multiple fuel products while requiring few nutritional inputs beyond CO2 and sunlight. A major setback in using cyanobacteria as an alternative liquid fuel source is the lack of information about the organism in its environmentally relevant day-night cycle. We are using the innovative approach of Tn-seq on S. elongatus to conduct the first genome wide screen on cyanobacteria in the day-night cycle. Tn-seq utilizes transposon mutagenesis combined with high throughput sequencing to conduct a genome wide screen and elucidate genes important to the day-night cycle. As a first step, we have experimented with four different transposons and optimized the conjugation process in order to build a mini-library that has been verified by sequencing. This indicates that our technique and procedure are successful, and should be scalable to a fully functional mutant library. Once the library is created these genetic tools can be easily reused for genome wide screens under other scientifically and industrially important conditions such as predation and environmental stress.

Reorganization of the Dentate Gyrus Neural Network in Medial Temporal Lobe Epilepsy and its effect on Learning and Memory Tatyana Goldring, Revelle College, Human Biology major PI: Jill K. Leutgeb, Ph.D., Associate Professor, UC San Diego Division of Biology, Neurobiology Section

Past research has proposed that the dentate gyrus (DG) plays an important part in pattern separation, the process of differentiating between similar inputs patterns, a network computation thought to be critical for episodic memory formation. Patients with medial temporal lobe epilepsy (MTLE), characterized by chronic seizures due to the lack of neuronal inhibition, exhibit memory deficits, which might be caused by the anatomical changes frequently characterized in the DG of these patients. These changes include synaptic reorganization of the neuronal network, and mossy fiber sprouting from principle neurons of the DG. Ongoing research in our lab has suggested that an increase in the anatomical reorganization of the DG is associated with a loss of pattern separation in epileptic animals. To further investigate the effects of this pathological reorganization on memory processing, I explored the impact a reorganized dentate network has on the ability of the DG to distinguish between two different novel environments. Local field potentials and extracellular action potentials were recorded from populations of dentate neurons while rats, with chronic epilepsy, were foraging for food in a series of familiar square and circular environments and then in a novel environment to test changes during learning. Comparisons of the rate and pattern of DG granule cell activity in control and epileptic animals were assessed and the differences/similarities were analyzed to determine whether there is a correlation between impairments in distinguishing novel environments and MTLE.

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Axon regeneration is critical for functional recovery after nerve injury; however, the molecular mechanisms of this capability remain poorly understood. Caenorhabditis elegans is an excellent model organism in which to examine conserved molecular mechanisms involved in nerve regeneration. Recent studies have revealed the importance of a transcription factor known as CCAAT/enhancer-binding protein-1 (CEBP-1) in axon regeneration. We have proposed a tentative model that upon injury CEBP-1 undergoes retrograde transport to the nucleus, thereby triggering pro-regenerative pathways that accelerate axon regrowth. Supporting this idea, we have identified a member of the Importin alpha protein family as a putative binding partner of CEBP-1. Importin proteins regulate the retrograde transport of cargoes from the axon to the nucleus, allowing for the import of transcription factors into the nucleus. To better understand the mechanisms of injury signal transport from the injured site to the nucleus, we are examining the position of nuclear localization signals on the CEBP-1 protein itself. Moreover, we are investigating the relationships between CEBP1 and various importin proteins, as well as the function of importin proteins in the nervous system. Understanding of the cellular and molecular mechanisms behind the function of CEBP-1 may lead to greater comprehension of the mechanisms involved in axon regeneration. As these functions are elucidated, insight may be gained as to their application in nerve regeneration across species.

Identification of Rab GTPase roles in muscle cell Transverse Tubule formation and remodeling Wilson Huang, Warren College, General Biology PI: Amy Kiger, Ph.D., Division of Biological Sciences, Department of Cell and Developmental Biology

Muscle cells rely on increases in cytoplasmic calcium levels for successful contraction. Transverse (T)-tubules are a network of tube-like invaginations from the plasma membrane that mediate this process by spreading depolarizing signals to activate calcium release from the sarcoplasmic reticulum. While the physiological significance of T-tubules is well understood, the mechanisms behind their formation and remodeling are unknown. We discovered that during Drosophila metamorphosis, the T-tubule network in abdominal muscle cells undergoes remodeling, as characterized by disassembly during the late larval-pupal transition and rapid reassembly in late pupal stages. Additionally, we have described specific roles in this T-tubule remodeling program for several genes involved in endosomal trafficking and with homologs associated with human myopathy. Thus, flies provide an ideal system to study the mechanisms underlying T-tubule structure. To identify additional T-tubule regulators, I screened the Rab GTPases for T-tubule localization and required functions. The Rabs are a protein family with diverse roles in coordinating cellular membrane trafficking processes. We speculated that one or more of the Rabs likely are involved in T-tubule development.

Using muscle-targeted RNAi knockdown and expression of fluorescently-tagged Rab proteins, I identified several candidates.

Most notably, I demonstrated that Rab35 localizes along T-tubules and is also required for T-tubule reassembly during pupal muscle cell remodeling. Rab35 has been implicated in endocytic recycling, further pointing to a possible pathway framework and significance of regulated endosomal trafficking in T-tubule remodeling. Importantly, our findings can enhance knowledge of how Rab mutations may be linked to human myopathy and heart disease.

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Localization and Trafficking of ATP-binding Cassette Transporters in the Sea Urchin, Strongylocentrotus purpuratus, embryo Rose Hill, Revelle College, Biochemistry and Cell Biology major PI: Amro Hamdoun, Ph.D., Assistant Professor of Marine Biology at Scripps Institution of Oceanography, UC San Diego

ATP-binding cassette (ABC) transporters are conserved membrane-spanning proteins that efflux structurally diverse compounds, and thus their cellular functions are varied and incompletely known.

Differences in primary structure account for some variability, but it is not fully understood how localization and trafficking, which dictate subcellular distribution, affect transporter function. The goals of this project were: 1) to characterize the differences in ABC transporter localization within sea urchin embryos as relates to predicted function and 2) to assess the mechanisms governing trafficking of ubiquitous ABC transporters. Urchin embryos express at least twenty ABC transporters, including those with presumed developmental (ABCC5a) or protective (ABCB1a) functions. I first determined the tissue-specific localization of ABCC5a by immunohistochemical analysis, and found that it is expressed in aboral non-skeletogenic mesenchyme cells, supporting a developmental function. To address the second goal, I co-expressed fluorescently tagged ABC transporters and Rab11-GTPase, a marker of apical recycling endosomes, via microinjection of embryos with synthetic mRNAs. Using confocal microscopy, I visualized the trafficking of newly synthesized transporters to the plasma membrane after inhibiting anterograde ER-Golgi membrane traffic with brefeldin-A. Previous reports indicate Rab11 is required for localization of ABCB1a to apical membranes in fertilized embryos, but my preliminary results demonstrate that in blastulae, Rab11-positive vesicles do not co-localize with ABCB1a or related ABCB4. Continued testing will identify vesicle populations responsible for the transport of these proteins to the plasma membrane, and elucidate how localization and traffic patterns influence protein function on embryonic and cellular scales.

Role of Atypical Protein Kinases in Axon Regeneration Natalie Warsinger-Pepe, Revelle College, Molecular Biology major PI: Daniel Gibbs, Ph.D., Assistant Professor, Department of Neuroscience at the UC San Diego School of Medicine

The identification of cell intrinsic factors that can modulate the regenerative potential of both central and peripheral nerve axons is of particular interest for enhancing axon growth and functional recovery following traumatic injury to the nervous system. In this study, we identify a novel role for atypical protein kinase C isoforms PKM-ζ and aPKC-λ, which have recently been shown to regulate axon specification in developing neurons. Our experiments indicate that PKM-ζ and aPKC-λ may also regulate axon growth in adult neurons, making them potential targets for regenerative therapies.

Overexpression or knockdown of PKM-ζ was tested in primary adult DRG cultures grown on growth permissive (laminin) or inhibitory (myelin) substrates, and neurite extension was quantified using automated image analysis. Overexpression of PKM-ζ reduced neurite extension on laminin, whereas knockdown of PKM-ζ enhanced neurite extension on myelin. These results suggest PKM-ζ is functioning as a negative regulator of neurite outgrowth in adult DRGS, as reduction of PKM-ζ allows for outgrowth of adult neurites in an inhibitory environment.

Complementary experiments testing the effect of altering aPKC-λ expression levels in adult DRGs, as well as in vivo experiments studying the role of PKM-ζ and aPKC-λ in axon regeneration after optic nerve injury are underway. These studies will help identify a functional role for PKM-ζ and aPKC-λ in enhancing axon regeneration in the peripheral and central nervous systems, and assess their therapeutic potential for nervous system injuries.

Circadian Regulation of Metabolism under Diurnal Cycles in Cyanobacteria Chase Barber, Warren College, General Biology major PI: Susan Golden, Ph.D., Division of Biological Sciences, Molecular Biology Section

Circadian rhythms are of major biological importance to most organisms and are important for proper timing of cellular events in coordination with daily light-dark changes. The photosynthetic cyanobacterium Synechococcus elongatus is a model organism for the circadian clock. Currently, there is interest in photosynthetic organisms to produce biofuel, pharmaceutical products, and industrial chemicals. On an industrial scale, photosynthetic organisms are grown outdoors, which are subject to light-dark cycles that impact product yields through alterations in metabolism. Experiments have not been conducted on how the clock regulates glycogen − an energy storage molecule − in these organisms and how environmental cycles integrate with clock control. Glycogen degradation is important for night-time survival and glycogen content is regulated by the circadian clock. The transcriptional regulator RpaA is the primary output from the S. elongatus circadian clock and has over 170 gene targets. Mutants in rpaA display poor glycogen degradation at night and are sensitive to light-dark cycles. We hypothesize that these phenotypes are due to the rpaA mutant’s mis-regulation of glycogen metabolic genes, rather than a direct effect of the lack of RpaA protein. To test this hypothesis we have cloned genes that are known RpaA transcriptional targets, have decreased expression in an rpaA mutant, and are important in glycogen metabolism. We expressed these genes in an rpaA mutant background using the highly expressed psbAI promoter. Elucidating how the circadian clock controls glycogen metabolism in environmentally relevant light-dark cycles is important if we are to optimize biomolecule production on a mass scale.

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The Anatomical Changes Associated with Impaired Behavioral Pattern Separation in Chronic Model Temporal Lobe Epilepsy Raafat Kuk, Muir College, Physiology and Neuroscience major PI: Jill K. Leutgeb, Ph.D., Laura A. Ewell Ph.D., Division of Biological Sciences, Neurobiology Section Other Advisers: Anelah McGinness

Pathological synchronization of electrical signals that result in involuntary body movement is known as seizure. Multiple consecutive seizures are known as epilepsy. A brain area with recurrent circuits thus prone to epilepsy is the medial temporal lobe (MTL). Within the MTL, the hippocampus plays a role in episodic, working, and spatial memory. A specific sub region of the hippocampus, the dentate gyrus (DG), is known to support the process of pattern separation. This computation describes the ability of brain circuitry to distinguish between two similar, yet non-identical cues. Studies have shown that epilepsy may result in axonal sprouting and cell loss in the DG, suggesting that DG function may be perturbed due to epileptogenesis. Ongoing studies from our lab have suggested that behavioral pattern separation is impaired in a rat model of MTL epilepsy. We hypothesize that the anatomical changes in the DG may be responsible for the behavioral deficit.

To conduct our study, control Long-Evans male rats and rats with epileptogenesis (induced by a low-dose, IP- kainate injection) were tested for pattern separation on an 8 arm radial maze, and a behavioral deficit in the induced rats was observed. To test the correlation between axonal sprouting and behavioral deficit, the tissue from tested rats was Timm stained and microscopically visualized. To test the correlation between neuronal loss and behavioral deficit, immuno-histochemistry and stereology were used. Our results showed that neither sprouting nor cell loss in the dentate gyrus were associated with the behavioral deficit.

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Group A Streptococcus M2 Protein Interaction with C4BP and Interference by Hyaluronic Acid Capsule Garrett Brooks, Eleanor Roosevelt College, Physiology & Neuroscience major PI: Victor Nizet, M.D., Professor of Pediatrics and Pharmacology, School of Medicine

Streptococcus pyogenes, or group A Streptococcus (GAS), is an important human pathogen that contributes to more than half a million deaths annually.

GAS pathogenicity is attributed to the expression of many virulence factors, including M protein and a hyaluronic acid capsule, that diminish the host immune response. GAS hyaluronic acid (HA) encapsulation, encoded by the hasABC synthase operon, contributes to host immune response evasion by precisely mimicking the structure of human hyaluronic acid. M proteins are surface-bound molecules that promote GAS colonization through increased epithelial cell adherence and internalization. Some M protein subtypes have been shown to promote resistance to opsonophagocytosis mediated by the binding of C4BP, a human complement-inhibitory protein. AP2 is a GAS isolate that expresses M2 protein, but due to a mutation to the CovR/S regulator, also expresses high levels of HA capsule.

Although M2 protein is known to bind C4BP, wild type AP2 showed no C4BP binding, suggesting that AP2 hyper-encapsulation interferes with the interaction between M2 protein and C4BP. To test this hypothesis, the HA capsule was enzymatically cleaved using hyaluronidase, which resulted in increased C4BP binding. Additionally, a mutant strain deficient in capsule expression (AP2ΔhasA) showed enhanced C4BP binding. Together, these data demonstrate an inverse correlation between capsular expression levels and C4BP binding in GAS.

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Evidence for consumption of krill in pre-breeding Antarctic fur seals (Arctocephalus gazella) Marcus Bobar, Thurgood Marshall College, Biochemistry and Cell Biology major PI: Carolyn Kurle, Ph.D., Assistant Professor, Division of Biological Sciences, Ecology, Behavior, and Evolution Section Other Advisers: Mike Goebel, Ph.D., Antarctic Ecosystem Research Division, Southwest Fisheries Science Center, NOAA

Antarctic fur seals overwinter in warmer waters north of the Antarctic Convergence. There, pregnant seals forage continuously, storing fat for their return to Antarctic breeding sites in summer. Once on these seasonally ice-free islands, they nurse their young with milk generated from fat stores obtained by ingestion of a broad range of prey species. Stable nitrogen isotope (δ15N) analysis of the protein in the seals’ milk allows us to estimate the foraging ecology and movement patterns of these seals. While living and foraging in the more northern areas, the seals primarily consume several species of fish with higher δ15N values, leading to correspondingly higher δ15N values in the seals’ tissues. When south of the Antarctic Convergence, seals often feed on abundant Antarctic krill that have lower δ15N values, which would also be reflected as lower δ15N values within the seals’ tissues.

It is not yet known whether Antarctic fur seals travel from their overwintering, northern waters directly to their breeding islands fueled entirely by fat stores accumulated in winter, or whether they forage as they migrate, consuming krill in the process. The goal of this project is to use stable isotope analysis to determine if the seals forage on krill before giving birth.

The results will provide evidence for the importance of krill in the diet of Antarctic fur seals prior to pupping and breeding and the potential for conflict with krill harvesting by humans.

Neonatal Enteropathogenic Escherichia coli Infection Causes Long Term Behavioral Defects and Long Term Alterations in the Microbiota Kevin Huynh, Eleanor Roosevelt College, Biochemistry and Cell Biology major PI: Melanie Gareau, Ph.D., Assistant Adjunct Professor, Kim Barrett, Ph.D. Dean of Graduate Studies, Department of Medicine, Division of Gastroenterology

Increasing evidence suggests that the intestinal microbiome exerts a major influence on mental health and behavior. In humans, repeated exposures to enteric bacterial infections that cause diarrheal disease in early life are associated with cognitive defects in adulthood, despite resolution of diarrhea with oral rehydration solution (ORS) treatment. Previous work from our lab demonstrated that cognitive defects caused by the enteric pathogen Citrobacter rodentium were stress–induced. Currently, there is no effective model that emulates the cognitive defects in adulthood associated with early life diarrheal diseases.

Here we proposed to establish a model of neonatal infection to study adult behavioral changes. Mice infected with enteropathogenic Escherichia coli (EPEC) at post-natal day 7 were found to exhibit baseline memory defects in adulthood but did not exhibit anxiety-like behavior when compared to sham infected neonates. qPCR analysis of fecal samples from adult, previously EPEC-infected mice demonstrated long term changes in the composition of the microbiota compared to uninfected controls. Significant decreases were observed in segmented filamentous bacteria (SFB), E. rectale, and Bacteroides populations in EPEC inoculated mice relative to sham mice, whereas the population of Enterobacteriaceae increased. Taken together, these results demonstrate that early-life infection with enteric bacterial pathogens can have detrimental cognitive effects and cause long-term dysbiosis in mice. We speculate that the changes in the microbiota may underlie long-term post-infectious behavioral defects.

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HDGF-related Protein 3 Regulates Oligodendrocyte Myelination in the Central Nervous System Shereen Georges, Thurgood Marshall College, Physiology and Neuroscience major PI: Fred H. Gage, Ph.D., Adjunct Professor, Division of Biology, Neurobiology Section

In the central nervous system, axons are myelinated by oligodendrocytes (OLs). However, though our understanding of OL biology is increasing, the signals that emanate from the axons and trigger myelination initiation are still poorly understood. Previous studies have found that HRP3II, a newly identified isoform of the hepatoma-derived growth factor (HDGF) family, shows peak expression in the axons of spinal motor neurons before and during the myelination period. Furthermore, overexpression of HRP3II increased Schwann cell proliferation and myelination, suggesting its importance for regulating the local pool of Schwann cells. Therefore, our goal is to determine whether HRP3II also plays a critical role in the myelination of neurons by OLs in the central nervous system (CNS) using a recently developed CNS myelination model. Mouse embryonic stem cell derived cortical neurons were cultured onto microfluidic devices. We overexpressed HRP3II using two different constructs: GFP-HRP3II Fusion Protein and GFP-HRP3II inner ribosomal entry site (IRES). After co-culturing the neurons with OLs, the devices were stained and imaged. We identified OLs based on their morphology and found that the average number of MBP+ OLs is significantly larger with both HRP3II overexpression constructs compared to controls, suggesting its role in the proliferation and/or maturation of OLs. After further quantitative analysis, we found that both overexpression constructs increase the percent of myelinated axons significantly compared to the neurons containing normal levels of HRP3II; and this effect is not selective for GFP+ axons only, supporting the idea of some type of factor that can act at a distance.

Effects of Streptolysin O in Drosophila Development and Roles of H1F1 Kyle M. Yuquimpo, Thurgood Marshall College, Human Biology major PI: Jesus Rivera-Nieves, M.D., UC San Diego Health Sciences, Department of Medicine, Division of Gastroenterology

Interleukin-37 (hIL-37) expression in mice protects from DSS colitis. We aimed to determine whether hIL-37 may play a protective role in adaptive-immune mediated model of Crohn’s Disease (TNFdeltaARE), that develops ileitis and arthritis. Ilea from untreated TNFdeltaARE (n=5) and TNFdeltaARE/hIL-37g (n=8) mice were collected for histology. To determine whether colonic injury might be required for a therapeutic effect, TNFdeltaARE mice (n=3) and TNFdeltaARE/ hIL-37tg mice (n=6) were treated with 4% dextran sodium sulfate (DSS) to induce colitis. Weights were monitored daily. WT mice and TNFDARE mice served as controls. Mice were sacrificed and intestine collected for evaluation of colitis severity. Statistical analysis was performed using Graphpad Prism. Error bar indicates standard error mean (SEM). Significant differences were calculated using unpaired two-tailed T-test. Ileitis severity at basal conditions showed no difference between TNFdeltaARE and TNFdeltaARE/hIL-37tg. Moreover, no differences were observed in CD4, CD8, B220, or CD11c cells isolated from the mesenteric lymph node or spleen. Although significant change in weight was observed upon DSS treatment at day 6 and 7 between TNFdeltaARE and TNFdeltaARE/hIL-37tg mice, we did not observe any changes in ileal scores. Our results suggest that hIL-37 does not attenuate ileitis in TNFdeltaARE/hIL-37tg mice under basal conditions, although treatment with DSS seems to reduce the inflammation. However the absence of significance may be due to the small sample size, and an increased sample size in future experiments may provide clearer evidence of a potential role for hIL-37’s in attenuating adaptive-immune mediated inflammation.

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The Effects of Adolescent Ethanol Exposure on Neurophysiology and Behavior Diana Kneiber, Revelle College, Human Biology major PI: Cindy Ehlers, Ph.D., Professor of Psychiatry, UC San Diego School of Medicine

Studies have shown that drinking during adolescence is a risk factor for alcohol related problems during adulthood. The 2012 National Survey on Drug Abuse and Health reported that current alcohol use among teens aged 12 to 17 was 12.9% and binge drinking was 7.25%. However, the mechanism by which early exposure to drinking leads to an increased risk for alcohol dependence in adults is not clear. Here we show that high degrees of voluntary drinking during adolescence can significantly enhance levels of alcohol self-administration during adulthood. In our study male Wistar rats were given a 2-bottle choice between 20% ethanol and water during adolescence to model voluntary drinking in adolescent humans. As adults, these rats as well as control rats who did not have the opportunity to drink during adolescence, underwent operant lever press training to measure their motivation to drink alcohol as adults. We found that rats that voluntarily drank ethanol during their adolescence were significantly more likely to drink more ethanol when presented ethanol as adults than control animals. Similarly, significant differences were observed in the amount of alcohol consumed by each rat over all operant sessions between the control group, a subgroup of rats that only drank small amounts of ethanol during adolescence and a group that drank large amounts of ethanol during adolescence. This study shows that the voluntary consumption of alcohol during adolescence can have an impact on the amount of alcohol consumed during adulthood. Further research is needed in this model to identify the mechanisms mediating the increase in adult alcohol drinking.

The Effects of Caveolin on Aging in C. elegans and Establishment of a Liquid Culture Medium Model Grace Grogman, John Muir College, Human Biology major PI: Hemal H. Patel, Ph.D., Department of Anesthesiology Other Advisors: Eileen Shi, Heidi N. Fridolfsson, Meghna Mehta, Ekta Mehta, Jan M. Schilling

Caveolins are scaffolding proteins found in highly ordered membrane domains rich in lipids. These lipid ordered domains are cholesterol and glycosphingolipid enriched signaling microenvironments that allow for adaptation to external stress. The caveolin proteins (Cav-1, -2, -3 in mammals and Cav-1, -2 in C. elegans) interact with and organize various signaling molecules within these domains. Increased caveolin expression in mice protects against cardiac and neuronal stress and increases organismal health and resiliency with age. This project seeks to analyze more global effects of caveolin on aging by using C. elegans as a model species in which Cav-2 is overexpressed (OE).

We hypothesize that overexpression of Cav-2 will affect worm aging and function. We performed phenotyping experiments in our N2 wild-type and Cav-2 OE worm strains. These included lifespan, fertility, morphology, and energy utilization assays. We show that Cav-2 OE worms display an increased lifespan, increased fertility, and more efficient mitochondrial respiratory function.

Therefore, we conclude that overexpression of Cav-2 in C. elegans facilitates stress adaptation at the whole organism level. This observation underscores the role of caveolin in stress adaptation and has implications to characterize its important role across species. Another goal of this project was to establish a liquid culture medium model to allow completion of function biochemical/ biophysical studies in C. elegans that require large quantities of starting material. The liquid culture model has been successfully established to dramatically scale up sample size. Studies are underway to replicate lifespan and functional endpoints. We conclude that caveolin may be a highly conserved stress adaptive molecule and C. elegans may be an ideal model species to further explore the role of caveolin in biology.

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Isolation and Characterization of a Bacteriophage 201φ2-1 Protein Joanna Coker, Roosevelt College, Human Biology major PI: Joe Pogliano, Ph.D., Division of Biological Sciences, Molecular Biology Section

A number of Pseudomonas bacteriophage with large genomes (>300Kb) have recently been described, but little is known about the genes required for their propagation. Three of these phages – ΦKZ, ΦPA3, and 201φ2-1 – are closely related and make up the ΦKZ phage family. ΦKZ and ΦPA3 infect the human opportunistic pathogen Pseudomonas aeruginosa, and bacteriophage 201φ2-1 infects Pseudomonas chlororaphis, an agriculturally-important organism that produces anti-fungal molecules. I have been using a combination of cell biology, genetics, and proteomics to study bacteriophage 201φ2-1 in order to identify novel mechanisms these large phage use to replicate. Proteomics data of 201φ2-1 has shown that during infection, the phage protein gp105 is produced earlier and in greater amounts than any other phage protein. Given its high relative abundance and early expression timeframe, gp105 probably plays an important role during phage infection. However, analysis of the protein gp105 with multiple databases revealed no homologs of this protein other than similar ΦKZ and ΦPA3 proteins.

This protein’s function during phage infection is therefore unknown. To gain insight into this, I am examining the localization pattern of gp105 in the host cell and its interactions with phage DNA during infection. I have constructed a fusion protein of gp105 to GFP, which will allow me to track the position of gp105 throughout the infection process using time-lapse microscopy.

From these data, I hope to deduce the function of gp105 and its homologs during infection and gain insight into the infection processes of 201φ2-1 and its ΦKZ relatives.

Analysis of Pyruvate Carboxylase and Pyruvate Dehydrogenase Kinase Functions in Diet Induced Obesity and Cardiac Lipotoxicity Stephanie Tsoi, Revelle College, Human Biology major PI: Rolf Bodmer, Ph.D. and Soda Diop, Ph.D., Postdoctoral Fellow Department of Pediatrics and Pathology

Obesity has become an increasingly prevalent problem on both global and national level due to higher caloric consumption and decreased activity level. Obesity has dangerous side effects including heart dysfunction, inflammation, and diabetes. Humans share anatomical and genetic characteristics with Drosophila melanogaster, which allows this fly species to be a good model for studying the genetics behind high-fat diet (HFD) induced obesity and the associated heart dysfunction. Previous studies in the laboratory have shown that HFD feeding leads to obesity and cardiac liptoxicity via deregulation of the TOR (Target of Rapamycin) - PGC-1 (PPARγ Co-activator 1) axis. A microarray analysis was performed to identify downstream targets of PGC1. My project consisted of analyzing the role of two of these genes: PDK (Pyruvate Dehydrogenase Kinase) and PC (Pyruvate Carboxylase), both regulators of glucose metabolism. I tested each gene through loss-of-function mutations and analyzed the effect on fat content. I found that both PDK and PC mutants exhibited decreased fat content under both diet conditions (NF and HFD). Additionally, I used the UAS Gal4 system to do a heart-specific knockdown of each of these genes individually.

My preliminary results show that the increased heart rate and concomitant decreased heart period caused by HFD is abolished under PC knockdown. This data suggests that PC and PDK play key roles in the mechanism of HFD induced obesity and heart dysfunction, downstream of PGC-1. To confirm this data, future research will include genetic epistasis experiments with PGC-1 and other members of the insulin-TOR pathway.

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Investigating the Molecular Regulation of CD100-Induced Dendritic Epidermal γδ T Cell Rounding William J. Kuo, Muir College, Biochemistry and Cell Biology major PI: Wendy L. Havran, Ph.D., UC San Diego Department of Medicine, Division of Dermatology Other Advisors: Deborah Witherden, Ph.D., The Scripps Research Institute, Department of Immunology and Microbial Science

Dendritic Epidermal T Cells (DETC) are the prototypical dendritic γδ T cells resident in the epidermis. DETCs make multiple contacts with neighboring keratinocytes via dendritic protrusions that monitor for signs of damage, upon which DETCs retract their dendrites, adopt a rounded morphology, and secrete effector molecules—including keratinocyte growth factors—that facilitate wound repair. It has been shown that the DETC CD100 (Semaphorin 4D) receptor interaction with its Plexin B2 ligand on neighboring keratinocytes is required to facilitate DETC rounding in response to keratinocyte damage. However, the molecular regulation of CD100-induced DETC rounding has not been fully defined. Here we demonstrate that the CD100 molecular signaling pathway involves the activation of RhoA-GTPase, a molecular switch involved in both cellular migration and cytoskeletal dynamics. Immunoblot analyses of RhoA-GTPase were performed following stimulation of CD100-expressing CHO cells with CD100 antibody. Our results indicate a rapid and transient RhoAGTPase activation peaking at five minutes after CD100 antibody addition, followed by a similarly rapid and transient inactivation of RhoA-GTPase beyond the five minute time point. Uniquely, this is the first demonstration of RhoA-GTPase activation via semaphorin signaling. To further investigate the importance of RhoA-GTPase activation, in vivo experiments utilizing RhoAGTPase inhibitors to block CD100-regulated activation are currently in progress. Additional experiments in the future will be directed towards the analysis of various RhoA-GTPase downstream effectors, including ROCK and LIMK. We anticipate that these experiments will help decipher DETC biology, resulting in an improved understanding of immune defects contributing to chronic, non-healing wounds in patients.

Role of TRAIL in NK Cell-Mediated Control of Murine Cytomegalovirus in C57BL/6 Mice Bryan McDonald, Eleanor Roosevelt College, Human Biology major PI: Chris Benedict, Ph.D., La Jolla Insitute for Allergy and Immunology

Natural Killer (NK) cells play an important role in the early antiviral response to murine cytomegalovirus (MCMV) infection. C57BL/6 (B6) mice mount a strong NK response to CMV as a result of the interaction between MCMV glycoprotein m157 and NK-activating receptor Ly49H, leading to reduced acute viral replication. TNF-related apoptosis inducing ligand (TRAIL) interacts with its death receptors (DR) to promote antiviral defenses through apoptosis regulation.

CMV encodes many immune modulatory genes which aid in blocking host immune defense. Our recent work has demonstrated that the MCMV m166 protein restricts cell surface expression of TRAIL-DR in infected cells, thus protecting them from TRAIL-mediated apoptosis. An MCMV mutant lacking m166 gene expression is severely attenuated for replication in vivo, especially in the liver of BALB/c mice. In this study we sought to determine the contribution of NK cell TRAIL in early MCMV control in B6 mice. MCMV-m166stop replicates to the same extent at early times as WT MCMV, indicating that TRAIL on B6 NK cells plays little to no role in viral control. We verified the presence of four NK cell developmental subsets in the B6 liver that have differential TRAIL surface expression. We show that IFN-I signaling influences constitutive TRAIL expression in naïve NK subsets. We finally demonstrate that MCMV orf m166 is sufficient for inhibiting TRAIL-DR surface expression. These results indicate that in B6 mice where NK cell responses are very robust, despite the presence of TRAIL on liver NK cells, TRAIL-dependent NK cell mediated apoptosis is not a major contributor to early MCMV control.

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Methods for Quantifying the Stability of Spatial Firing Patterns of Hippocampal Place Cells in One Spatial Dimension Christopher C. Cannova, Sixth College, Molecular Biology major PI: Stefan Leutgeb, Ph.D., Division of Biological Sciences, Neurobiology Section

We sought to investigate how the spatial and temporal firing patterns of rodent hippocampal CA1 neurons are altered after the major inputs from the medial entorhinal cortex had been disrupted. Upon such disruption, we found that the existence and stability of spatial firing patterns was greatly diminished, yet shorter periods of temporally stable spatial firing were still present. To conduct our investigation, then, we developed various methods to detect and extract these periods of transiently stable spatial firing in one spatial dimension. In this paper, we describe these methods and use statistical procedures to quantify their success. Of these methods, we find that performing principal component analysis on a temporal sequence of one-dimensional rate maps is the most successful. In particular, we find that the proportion of the variance explained by their first principal component most successfully classifies periods of transiently stable spatial firing. Finally, we provide an algorithm for the extraction of these periods and describe how these methods can be used to quantify the stability of spatial firing more generally.

Characterization of the Interaction Between the Translational Repressor Complex GIGYF2-4EHP and the AU-rich Element Binding Protein Tristetraprolin Myanna Olsen, Warren College, Human Biology major, Psychology minor PI: Jens Lykke-Andersen, Ph.D., Division of Biology, Molecular Biology Section

Gene expression is tightly regulated by different post-transcriptional control pathways. One pathway in particular involves the RNA-binding protein tristetraprolin (TTP), which binds to AU-rich elements (ARE) in the 3’ untranslated region of proinflammatory- and cancer-associated mRNAs and promotes their decay by recruiting degradation factors. Recently, the LykkeAndersen lab (by mass spec in collaboration with the Bennett Lab) revealed a novel interaction between TTP and the translational repressor complex GIGYF2-4EHP. Though TTP is well known to be involved in mRNA decay, its role in translational repression is poorly understood. Whether TTP-mediated mRNA decay and translational repression are inter-dependent is also unknown.

To better understand the functional implications of this novel interaction between TTP and GIGYF2-4EHP, first the domains of TTP essential for binding GIGYF2 and 4EHP were mapped out by mutagenesis of TTP. Flag-tagged GIGYF2 and 4EHP and myc-tagged TTP variants were co-transfected into 293T cells and co-immunoprecipitated, and revealed the N-terminal domain of TTP to be sufficient for binding to the GIGYF2-4EHP complex, whereas the C-terminal domain and RNA-binding domain were not sufficient for binding. We also showed this interaction to be independent of other known motifs in TTP, such as the CNOTinteracting motif essential for binding the deadenylase complex, suggesting that TTP binds GIGYF2-4EHP independently of the deadenylase complex. More detailed mutagenesis will be carried out to identify the amino acids necessary for this protein-protein interaction and will allow further elucidation of the relationship between decay and translational repression by TTP.

The LysE Superfamily of Transport Proteins Involved in Cell Physiology and Pathogenesis Brian Tsu, Thurgood Marshall College, Molecular Biology major PI: Milton Saier, Ph.D., Division of Biological Sciences, Department of Molecular Biology

We have characterized the LysE superfamily of transmembrane transport proteins which catalyze export of amino acids, lipids and heavy metal ions. Statistical means were used to expand this superfamily to include newly identified families specific for (1) tellurium, (2) iron/lead, (3) manganese, (4) calcium, (5) nickel/cobalt, (6) neutral amino acids, and (7) peptidoglycolipids. Internal repeats were identified, and multiple alignments, phylogenetic trees, average hydropathy, amphipathicity and similarity plots, and conserved motifs provided evidence that all members of the superfamily derived through evolution from a single ancestral 3-TMS peptide via intragenic duplication. The common origin of these families implies that they share common structural, mechanistic and functional attributes. The transporters of this superfamily play important roles in ionic homeostasis, protection from excessive cytoplasmic heavy metal/metabolite concentrations and cell envelope assembly. Thus they influence the physiology and pathogenesis of numerous microbes, making them potential targets of drug design.

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STAFF Editor-in-Chief Kevin Perez Executive Editor Sameeha Khalid Special Sections Editor Arjun Patel Editor-at-Large Milli Desai Vicky Hwang Senior Research Editor Nicholas Kotsyubko

Junior Research Editor Amelia Wong Features Editor Areana Park Production Editor Firooz Kabir

REVIEW BOARD Technical Editor of Content Jasmine Chau Sveta Mohan

Publicity Chair Sushmitha Divakar

Features Design Editor Sejal Patel

Web/Publicity Committee Goonjan Agarwal Natalie Chen Olivia Meyer Jasmine Tan Cindy Yang

Special Pages Design Editor Sora Chee

Head Illustrator/Photographer Justine Liang

Review Board Manager Florence Lambert-Fliszar Jacky Lu

Staff Illustrators Jamie Yoon Bianca Chong Grace Park Elizabeth Cai Wenpei Li Nicole Pollack

Research Design Editor Nikhil Kanthi

Web Editor Brianna Egan Staff Writers Lily Huang Vaishali Talwar Humphrey Lin Rahul Lodhavia

STAFF ADVISOR

Associate Dean for Education Gabriele Wienhausen, Ph.D.

Manager, Do/Bio Center Hermila Torres

Neurobiology Kathleen French, Ph.D. Jill Leutgeb, Ph.D. Ecology, Behavior, and Evolution Heather Henter, Ph.D.

Jessica Yu Jingtong Wang Julia Brown Julia Oh Kevin Chau Kristen Jensen Kyle Jackson Kyle Yuquimpo Lauren Waller Lynn Song

Maryana Yurchshyna Mary Jade Farruggia Mitchell Zhao Neil Srinivas Shirleen Cheng Sowon Kim Stephen Howell Sue Kim

Director, Writing Center Madeleine Picciotto, Ph.D. Director of Science Communications, Division of Biological Sciences Kim McDonald ACMS Web and Graphic Designer Elaine Fleming

Staff Photographers Yaamini Venkataraman Lucy Chian Jonathan Gao

FACULTY ADVISORY BOARD Cell and Developmental Biology Emily Troemel, Ph.D. Steve Wasserman, Ph.D. Jim Wilhelm, Ph.D. Martin Yanofsky, Ph.D.

Alan Du Allison Greene Aneesa Chowdhury Arushi Atluri Brandon Meng Ellis Juhlin Evelyn Yang Gary Le Hanna Tran Ivonnie Shih

ACKNOWLEDGEMENTS

Molecular Biology Eric Allen, Ph.D. James Golden, Ph.D. Pictured: (bottom to top and left to right) Gabriele Wienhausen, Kevin Perez, Sameeha Khalid, Hermila Torres, Cindy Yang, Amanda Shelton, Michaela Go, Sejal Patel, Sora Chee, Anna Alvarado, Areana Park, Justine Liang, Dilara Onur, Rubeena Basra, Jasmine Chau, Sushmitha Divakar, Brianna Egan, Sveta Mohan, Florence Lambert-Fliszar, Safwan Haque, Nicholas Kotsyubko, Arjun Patel, Mandeep Bajwa, Jacky Lu

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