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Saltman Quarterly Division of Biological Sciences University of California, San Diego 9500 Gilman Drive La Jolla, CA, 92093-0376 (858) 534-5635

Undergraduate Biological Research Journal UCSD Division of Biological Sciences

Volume 10 2012-2013 Nos. 1, 2 & 3 sq.ucsd.edu

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The photograph of the monarch in the cocoon was taken on a tree at Balboa Park. Usually milkweed is the prefered plant for the life stages of the monarch, but this tree is also a favorite of the monarch caterpillar. One can witness the entire life cycle in the wild on these butterfly bush trees. There are tiny larval caterpillars, fatter ones about to crystallize, and evidence of emerging butterflies all around this single tree in San Diego.

This cocoon will soon finish its metamorphosis into a monarch butterfly, as one can begin to see through the translucent cocoon.

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Photo taken by Ariana Ananda

LETTER FROM THE EDITOR

T

his year marks the production of the 10th volume of Saltman Quarterly (SQ). As we celebrate more than one decade of SQ journals, we also celebrate one decade of our unique role as a facilitator of science communication.

At UC San Diego, we welcomed Pradeep Khosla as our eighth chancellor, and along with a new chancellor, we welcomed new visions for our university.

During his Investiture speech, Chancellor Khosla quoted the founding father of UC San Diego, Roger Revelle, in saying that “the way to the future may derive from honoring the past”. The SQ program indeed revolves around this quote, as we continuously try to expand upon the visions of Dr. Saltman.

Dr. Saltman understood the passion that undergraduate students have for biology. He strove to provide means for students to be integrally involved in biological research, even at the undergraduate level.

As we provide a means for students to have their own research published in our journal, we involve students in the process of disseminating knowledge—from students reviewing research manuscripts to designing the journal to distributing the journals for all students to read. And through this dissemination of knowledge, we as students encourage our own peers to learn about

and develop a passion for biological sciences.

SQ continues to broaden its reach with Dr. Saltman’s legacy in mind. This year’s publication includes Master’s students’ short scientific contributions called brevias.

We also expanded our social media reach and created a new interactive media website that is the online version of our journals—where one can access and share each of our research manuscripts and features articles on the web. A highlight of this year was when SQ students had the opportunity to meet Dr. Bruce Beutler, UC San Diego alumnus and 2011 Nobel laureate in Physiology and Medicine, on his visit to our campus. When we presented the SQ journal to Dr. Beutler, we are proud to say that he was very impressed by the quality of the publication and he said that the journal is “very beautifully put together”. I invite you to read my conversation with Dr. Beutler at the end of this journal to find out why he thinks the ability to communicate is the biggest indicator of success in science.

I am honored to introduce to you Volume 10.

As the Division of Biological Sciences undergoes an expansion in its own way to no longer have impacted biology majors, it is my hope that SQ will carry on Dr. Saltman’s visions to make biology accessible to all students for many more decades to come.

Pictured on the cover: The cover photo was taken by Hanwen Xie at the San Diego Zoo on June 23, 2012.

Generously underwritten by

Photographers featured in the Table of Contents include: Dilara Onur, Kriti Gupta, Ariana Ananda, Lauren Mutschler, Fred Qafiti, and Yasmeen Elsawaf

and supported by

Milli Desai Editor-in-Chief 2012-2013

The Saltman Family

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.

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of TABLE C Features

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Research

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Brevias

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Senior Honors Theses Dr. Beutler’s Interview Dr. Saltman Dedication

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Staff

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47 51

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The Monster Behind the Fever Mandeep Bajwa

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Studying Your Saliva Andrew Lum

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A Crucial Piece to the Cholesterol Puzzle Michaela Go

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CONTENTS Areana Park

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Epigenetics Chris Probert

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research

Sweeter Than Honey

features

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Effects of Algal Diversity on Productivity, Light Capture and Grazing Resistance GarďŹ eld Kwan

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Size-selected Harvesting of a Common Iguana Species Elizabeth Miller

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Photo taken by Yasmeen Elsawaf on a dock in Deland, Florida.

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Hailed as one of the top research institutes in the country, UC San Diego is constantly expanding the field of biology. The Features section highlights the groundbreaking work accomplished at UCSD.

a diverse collection of

FEATURES

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The Monster Behind the

Fever

Mandeep S. Bajwa staff writer

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FEATURES It is mysterious that a healthy child could suddenly develop serious heart problems, but such is the case when Kawasaki Disease (KD) has taken its toll on a child’s body. When a child develops a fever, parents usually react by simply giving medicine to reduce the fever, check under the bed for monsters, and put the child to bed. Unbeknownst to the caretakers, a monster may be hiding not under the bed but in the child’s heart.

The Monster Surfaces For Dr. Jane Burns, Director of the Kawasaki Disease Research Center at UCSD/Rady’s Children’s Hospital, this mystery of Kawasaki Disease and strong desire to help the children affected is what has always driven her forward for the 30 years she has been working with the disease. “The eyes cannot see what the mind does not know” is a Hindu proverb that Dr. Burns uses when talking about the history of the diagnosis of KD. In the past doctors were puzzled by the symptoms that include fever coupled with swollen, hands, feet, and lymph nodes in the neck, along with bloodshot eyes, red rashes on the skin, bleeding lips, and a strawberry tongue (Figure 1). But only recently were these telltale symptoms of Kawasaki Disease outlined and the first cases diagnosed.

Dr. Burns has been working with this disease from the very beginning of its recognition in the U.S. starting as a third year resident in 1981 at the University of Colorado School of Medicine where she witnessed the heartbreaking condition firsthand. A very ill baby was flown into Denver from Wyoming to the hospital where Dr. Burns was working. The high fever and rash was unresponsive to treatment confusing the residents and doctors. An infectious disease fellow who visited the patient with Dr. Burns suspected Kawasaki Disease and this truly mesmerized her because this was now the third

time she had seen patients with this curious disease. At the time, there was no cure available for the child and after 21 days in the hospital the baby died. If a KD patient is not properly managed the possibility of damage to the arteries covering the heart rises, which may cause aneurysms putting the child at risk for complications, even heart attack and death. When Dr. Burns was able to perform an autopsy on the child she was amazed to see beaded aneurysms dotting the little heart confirming the diagnosis of Kawasaki Disease.

In a heartwarming move, the family, who had had no contact with Dr. Burns until after the death of their baby, had gone around their town collecting money and brought this money handing it to her saying, “We want you to do research on Kawasaki Disease.” That is exactly what she has done along with so much more. Dr. Burns has peered into the depths of this disease working alongside historians, filmmakers, microbiologists, cardiologists, and people from many other walks of life to try to solve the mysteries of KD.

Pathogenesis and Treatment

In Japan, 1 in 150 children will contract KD and in the United States 5000 to 6000 new cases are diagnosed each year with possibly many more cases being attributed to other diseases and missed. 4For children in the United States and Japan, KD is the leading cause of acquired heart disease in children. When a child develops KD, the coronary arteries surrounding the heart are inflamed. The endothelial lining of the arteries recruit neutrophils from the flowing blood stream which transverse the cell layer and migrate into the vessel wall. Neutrophils recruit other cells such as T cells, which cause further inflammation in the vessel wall.1 If the disease is not recognized and the child not treated, then one in four children will develop coronary artery aneurysms. The coronary artery balloons out causing a swirling of blood cells that can form a blood clot that could result in a heart attack.

In order to treat the disease, administration of intravenous immunoglobulin (IVIG), which is essentially a solution of human antibodies, acts to deregulate the inflammation through poorly understand mechanisms. After treatment, if abnormal dilation of the arteries has occurred, scarring regenerates the near normal diameter of the artery. In those who go untreated, about 25% of these children will develop aneurysms, but if the disease is recognized within the first ten days of fever, IVIG treatment can substantially reduce the risk of aneurysms to Figure 1. Kawasaki Disease, discovered in Japan in 1960, is a widespread inflammation of the less than 5%. 1Even if a child recovers from body’s medium-sized blood vessels that affects mainly children under the age of five. The cause is 6 KD without aneurysms, there is the chance that unknown. But the symptoms, which also include fever, are striking. the damage done to the arteries over time could sq.ucsd.edu

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FEATURES cause scarring and impede blood flow causing a lack of nutrition and oxygen supply to the heart, termed myocardial ischemia, which can occur decades later in adulthood.2 The narrowing could also increase the risk of heart attack.2

At the Forefront of Discovery It all started with the Polymerase Chain Reaction (PCR) that is used to amplify a specific DNA sequence and is used in some cases for pathogen discovery. Dr. Burns was able to work with some of the creators of PCR like John Sninsky and Henry Ehrlich when the technique was still new. She used this technique to attempt to discover a pathogen associated with Kawasaki Disease but the study failed to yield an answer. Nonetheless, the use of PCR to study KD is the first example of many to showcase how Kawasaki Disease has in many cases and continues to be the first topic of molecular study to utilize a newly discovered technique thanks to Dr. Burns’ unyielding desire to experiment.

In 1996, Burns used the study of single nucleotide polymorphisms (SNP), which were only beginning to be investigated, to identify possible genetic relationships to KD. It was known that the TNF-α cytokine was elevated in patients presenting the symptoms of Kawasaki Disease. This cytokine is a chemical messenger made by the body to promote inflammation and immune cell activation, which are known to play a role in KD. This observation led to genetic studies that located polymorphisms, which are essential changes in the genetic code of the DNA, in the promoter region of the TNF-α gene, which

might cause it to be over-expressed. This in turn led to a clinical study that was the first to study the effectiveness of infliximab, which is a monoclonal antibody that binds to the TNF-α cytokine and blocks its actions. It was found that infliximab successfully eliminated the fever present in the children with KD and was helpful for children who were resistant to IVIG treatment.3 A more recent study completed by researchers in Japan in collaboration with Burns at UCSD utilized sibling pairs with KD. In this study the ITPKC pathway has a mutation that is associated with immune cell activation may be of special importance in those children who are IVIG resistant and require other treatments. It was shown that in this pathway a molecule called calcinuerin is a regulator of the gene creating IL2 that acts to increase inflammation.4 If the amount of IL2 could be decreased then the inflammation in KD patients could be reduced. The UCSD group recently reported the use of Cyclosporin A, a specific inhibitor of calcinuerin, as effective therapy for resistant KD patients4 (Figure 2).

These two studies only touch the surface of the work Dr. Burns has done. Along with her colleagues she continues to test new techniques in hopes of gaining clinical treatment options and developing a better understanding of this mysterious disease. Burns considers herself extremely lucky to have her feet in both the science lab and the treatment of children. She calls it “science with a purpose”, and the purpose is to help the kids.

“She calls it ‘science

with a purpose’, and the purpose is to help the kids. ”

Figure 2. Calcinuerin and NFAT regulate the synthesis of IL-2, which normally causes inflammation. Cyclosporin inhibits Calcinuerin and lowers production of IL-2 thereby decreasing inflammation. This entire pathway is influenced by the ITPKC pathway, which has been found to have a mutation that causes immune cell activation and inflammation.

Courtesy of Dr. Chisato Shimizu, Burns Laboratory.8

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The Monster that Flies with the Wind

Burns teamed up with climate scientist Daniel Cayan from Scripps Institution of Oceanography at UCSD. Burns noted that there was an increase of KD incidence in the spring and summer months and a decrease in the fall and winter months. In Dr. Burns’ study, medical records indicated KD occurrence in Japan, Hawaii, and San Diego occur in similar peaks from November through March.5 In Japan it was shown that KD fluctuates in a consistent pattern throughout the Japanese archipelago.4 Working with a climate team from Barcelona headed by Xavier Rodo, analysis of satellite data suggested that there are wind patterns associated with the months of KD incidence. A northwest wind pattern sweeps from central Asia and over Japan and then travels from Japan to Hawaii eventually reaching the West Coast.5 Northwest winds are associated with the winter/spring months and with a higher incidence of KD and southeast winds are associated with lower incidence (Table 3).

This suggests that a unique agent, possibly a microbe, may travel in the winds.5 Air samples were collected in the upper troposphere and they contained numerous bacterial and fungal organisms that could possibly cause KD. Although clinical samples collected from KD patients have not revealed a consistent organism, a comparison between DNA of organisms collected in air samples could be crosschecked with immune response of KD patients to point to the agent.5 If these wind patterns are shown to carry the vector of KD transmission, it will be the first time that a human pathogen has been recognized to travel thousands of miles over an ocean without the help of a man-made machine.6 sq.ucsd.edu

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Figure 3. Graph shows analysis of the wind vector over Japan (blue) and number of KD cases (red). The number of KD cases in Japan is slowly rising, for unknown reasons, but is strongly correlated with the average velocity of winds coming from the northwest-the direction of central Asia.6

Looking Forward

References

Kawasaki Disease is a relatively unknown disease to many parents and its symptoms mirror many other common childhood conditions such as scarlet fever or viral infections. Dr. Burns is an active community educator about KD through the annual Kawasaki Disease Parent Symposium hosted each year at UCSD. This program allows parents to become aware of the telltale symptoms of the disease and also outlines the newest research and advances made in relation to Kawasaki Disease. Dr. Burns believes this program is a way of giving back to the parents who have allowed their children to participate in various research studies, which is critical to furthering their understanding of this mysterious disease. She has also worked on documentaries about KD and the response by healthcare professionals in places such as India in hopes to further educate doctors around the world about the treatment of KD. This mysterious affliction continues to befuddle doctors with many cases being misdiagnosed and overlooked. Dr. Burns and her colleagues’ research dives into the unknown and continually tries to uncover the monster hiding in the heart may it be viral, bacterial, or an environmental toxin. Dr. Burns says she feels extremely grateful to have worked with and continue to work with some of the brightest minds in science and she continues to rope creative minds in with the mystery of KD. With all of the effort that she and some many of her colleagues devote to this disease, it seems that the light is about to uncover the monster that has evaded them for so long.

1. Newburger, J. W., M. Takahashi, M. A. Gerber, M. H. Gewitz, L. Y. Tani, J. C. Burns, S. T. Shulman, A. F. Bolger, P. Ferrieri, R. S. Baltimore, W. R. Wilson, L. M. Baddour, M. E. Levison, T. J. Pallasch, D. A. Falace, K. A. Taubert and E. Committee on Rheumatic Fever, and Kawasaki Disease, Council on Cardiovascular Disease in the Young, A.erican Heart Association (2004).

“Diagnosis, treatment, and long-term management of Kawasaki disease: a statement for health professionals from the Committee on Rheumatic Fever, Endocarditis, and Kawasaki Disease, Council on Cardiovascular Disease in the Young, American Heart Association.” Pediatrics 114(6): 1708-1733.

2. Daniels, L. B., M. S. Tjajadi, H. H. Walford, S. Jimenez-Fernandez, V. Trofimenko, D. B. Fick, H. A. Phan, P. E. Linz, K. Nayak, A. M. Kahn, J. C. Burns and J. B. Gordon (2012). “Prevalence of Kawasaki disease in young adults with suspected myocardial ischemia.” Circulation 125(20): 2447-2453.

3. Burns, J. C., et al. “Infliximab Treatment of Intravenous ImmunoglobulinResistant Kawasaki Disease.” J Pediatr 153.6 (2008): 833-8. Print.

4. Onouchi, Y., et al. “Itpkc Functional Polymorphism Associated with Kawasaki Disease Susceptibility and Formation of Coronary Artery Aneurysms.” Nat Genet 40.1 (2008): 35-42. Print.

5.Burns, J. C., D. R. Cayan, G. Tong, E. V. Bainto, C. L. Turner, H. Shike, T. Kawasaki, Y. Nakamura, M. Yashiro and H. Yanagawa (2005). “Seasonality and temporal clustering of Kawasaki syndrome.” Epidemiology 16(2): 220-225.

6. Frazer, Jennifer. “Infectious Disease: Blowing In The Wind.” Nature 5 Apr. 2012: 21-23. Print.

7. Rodó, X., J. Ballester, D. Cayan, M. E. Melish, Y. Nakamura, R. Uehara and J. C. Burns (2011). “Association of Kawasaki disease with tropospheric wind patterns.”

Sci Rep 1: 152.

8. Kawasaki Disease Research Center UCSD

WRITTEN BY MANDEEP SINGH BAJWA. Mandeep Singh Bajwa is a Human Biology major from Earl Warren College. He will graduate in 2014.

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Areana Park staff writer

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Background When we think of honeybees, we usually associate them with pollination and honey. With such fuzzy, cumbersome bodies, it is hard to take them seriously. That is, until one is trapped in close vicinity to us. Then their proximity is quite alarming. But other than that, when was the last time you ever thought carefully about how much bees affect our lives? With closer examination, it becomes apparent that the work of honeybees is extremely important within human society. The honey that they create is used as natural flavoring and sweeteners in our food, whether eaten directly or applied in our cooking. Honey is regarded by nutritionists as a “perfect protein” because of its molecular structure, and contains many medicinal properties that are effective in treating a wide range of maladies from sore throats to diabetic ulcers. But perhaps most importantly, honeybees work hard to help plants continue their cycle of growth with pollination, making them essential to human food production.

It makes sense that honeybees are very important to our economy, health and future scientific discoveries. Unfortunately, due to a variety of reasons, the healthy of honeybee colonies has slowly declined.

Dr. James Nieh, Vice Chair of Section of Ecology, Behavior, and Evolution, has conducted important research on honeybee health and was able to find an important correlation between the effect of pesticides and the negative way in which these pesticides affect honeybee behavior and communication.

food sources is extremely important to the well-being of their colony.

Bumblebee behavior was closely monitored through a special viewing monitor which allowed researchers to observe the bees directly.

Honeybee Decline Unfortunately, over the past decade, honeybee colonies have slowly collapsed and numbers have declined. Though this decline is due to a number of reasons, one of the causes is due to the negative effects of pesticide on honeybee behavior. We use pesticides in our crops to get rid of pests and harmful insects, but in doing so we often harm the insects that help the harvest, like the honeybee.

Imidacloprid, an important component of many pesticides, was found to have a direct affect on honeybee foraging behavior.

Dr. James Nieh has studied honeybee behavior and the effect of imidacloprid extensively. With careful research and experimentation, Dr. Nieh found that pesticides negatively affect honeybee behavior on a molecular level and cause dysfunctional behavior that affects the well being of the entire colony.

Picky Eaters Through Dr. Nieh’s research focuses on honeybees, the behavior of bumblebees were also monitored and catalogued in the Nieh Lab.

Key Communication Scientifically, the honeybee colony is an interesting system to study. Honeybees have a sophisticated yet simple communication system within their colony. They are one of few insects that have a highly evolved and complex social life. Like humans, honeybees are extremely social creatures with highly evolved representational communication. A good example of one of these types of evolved communication is the waggle dance. When food is scarce, worker bees (who are all female) communicate the location of a quality food source through recruitment. A worker bee recruits its nest mates by communicating the location of a potential high quality food sources by implementing a waggle dance, an incredible communication system that maps out the source of potential food through movements. The waggle dance is just one of the ways honeybees relay information, but how they obtain the information of the location of high quality sq.ucsd.edu

“The most amusing and also admirable thing about bees, for me, is their tenacity at getting a job done. A forager will often continue to forage at a food source non-stop, despite inclement conditions,” Dr. Nieh said. So what happens when something interferes with that tireless and motivated behavior? Through extensive experiments, Dr. Nieh was able to study the effects of imidacloprid on honeybee behavior.

“Imidacloprid blocks the nicotinic acetylcholine receptor, which is found on insects like the honeybee, leading to its cascade of effects in honeybee behavior. It is a key ingredient in many insecticides and in high doses can be lethal,” he explained. Dr. Nieh tested the effects of imidacloprid on learning and memory in bees. The purpose of his experiments was to find out whether or not it would affect a bee’s ability to measure distance, perceive quality of a sugar solution, and communicate that information to the rest of the colony.

This discernment of quality in sugar or “sucrose responsiveness” (SR) plays a role in how forager bees divide their labor. Within the colony, foragers specialize in nectar while others specialize in collecting pollen or water. Depending on their role in the colony, bees will have a certain standard for the concentration of sugar that VOL 10 • SALTMAN QUARTERLY

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FEATURES accept fewer available food sources. Flight and foraging activity of honeybees exposed to imidacloprid were unchanged, but the amount of waggle dances was notably decreased. Overall, honeybees that were exposed to imidacloprid became picky and uncommunicative to the detriment of the health of the colony.

Honeybees and the Future Dr. Nieh’s work has shed light on the neural control of honeybee behavior and ecology and has huge ramifications for the future. “The health of honeybees is important to our economy and agriculture. In California alone, a healthy almond crop brings in 2 billion dollars a year. The most valuable effect of beekeeping is pollination and the decline of communication due to the negative effects of pesticides has a multi-billion dollar impact on agricultural pollination,” said Dr. Nieh. Dr. Nieh’s insightful research brings hope for the development of a chemical alternative that will help stave off honeybee death and encourage healthy colonies for the future of agriculture.

Top: Dressed in a beekeeping suit for protection, a student refills the sugar and water jars located near the bee hives. Bottom: Honeybees were gently secured in holders before being marked with red paint in order to keep track of ongoing experiments.

they expect in their food source. “Foragers specializing in collecting nectar will, not surprisingly, have developed a sweet tooth and will not extend their proboscis, or mouthparts, to accept low concentration sugar solutions. On the other hand, foragers that collect pollen or water would accept very low concentrations of sugar,” said Dr. Nieh.

Therefore, to test the effects of imidacloprid on sucrose responsiveness (SR), bees from three colonies were individually captured and fed with a sucrose solution containing a minute concentration of imidacloprid. After allowing the imidacloprid to be fully ingested in the gut, the bees were then harnessed and restrained in stainless steel tubes and presented with different sugar concentrations ranging from 0-50%. A response was recorded when the bee fully extended its mouthparts to accept and taste the solution. To test the effects of imidacloprid of the foraging behavior of worker bees and their subsequent communication to nest mates, bees were first exposed to feeders of different sugar concentrations a short distance away from colony to represent a highly desirable food source no matter what the concentration. Specimens were individually captured and fed sucrose solutions of imidacloprid, marked and then re-released. Sucrose solutions with concentrations ranging from 0-50% were provided and the number of dances the forager bees performed after they visited these food sources was recorded.

Through extensive and careful experimentation, Dr. Nieh and his colleagues discovered that exposure to the chemical imidacloprid increased the sucrose concentration expected by honeybees from their food source. Because the honeybees expected high sucrose concentrations due to their exposure to imidacloprid, there was a decreased efficiency in foraging behavior causing the honeybees to

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Top: In the observation booth, students tracked the movements of the worker bees, keeping a sharp lookout for the waggle dance. Bottom: Dr. Nieh explains the purpose of the samples to the student. During his research, Dr. Nieh often worked with many undergraduate biology students and continues to do so today.

WRITTEN BY AREANA PARK. Areana Park is a Biochemistry and Cell Biology major from Eleanor Roosevelt College. She will graduate in 2015. sq.ucsd.edu

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Studying your Saliva Image from staffnurse.com

Understanding the Vast Oral Microbiota Andrew Lum staff writer sq.ucsd.edu

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Background Face it--we’ve all gone to the dentist with high hopes, only to receive a lecture about the importance of flossing our teeth while they jab at our gums, stirring up pain and guilt. There’s even more of a chance that someone you know has experienced the dreadful effects from one or more forms of gum infection, whether swelling due to mild gingivitis or a case of receding gums that is common with severe periodontal disease. A little blood from flossing may seem minor at first, but an unaddressed infection of the gums can rapidly lead to a long list of serious dental related problems including bone loss, tissue damage, and the worst case scenario--tooth loss. While no one wants to be walking around at an early age with gaps in their smile, vulnerability to the bacterial build up causing periodontal disease can begin as soon as you finish that sandwich you ate for lunch. Much research has been conducted on the pathogenesis and etiology of this bacterial disease but there remains a level of uncertainty about the role of other microbial inhabitants such as viruses. Currently, Dr. David T. Pride is leading a team of pathologists and immunologists at the UC San Diego School of Medicine in learning how interactions between bacteria and viruses carry a vast amount of information pertaining to human oral cavity conditions and susceptibility to diseases such as periodontitis.

The oral cavity is inhabited with pathogenic microorganisms that can be affected by anything that might enter the mouth. As a result, the micro-abrasive surface of our teeth is extremely susceptible to the bacterial colonization and secretion of chemicals which help break down our lunch. When we forget to brush, or when we put off flossing, the accumulation of bacteria starts to form a bio-film commonly known as plaque. As plaque hardens due to neglect and time, it becomes tartar which is known as calcium build- up. Days turn into weeks, and flossing seems to only make your gums bleed

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more; so while you put your floss back in the cabinet, tartar forms below your gum line and causes inflammation during the onset of gum infection. While much is known about the bacterial contribution towards developing periodontal disease, little is known about the role coexisting viruses may have on determining what type of bacteria exist, and how the bacteria might behave in the oral cavity.

Viruses are infectious agents that exist in almost all environments, develop continuously into new forms, and thrive wherever bacteria are present. As a result bacteria have become well acquainted with their neighboring infectious viruses. One way in which the interactions between viruses and bacteria are being studied by Dr. Pride is through the analysis of clustered regularly interspaced short palindromic repeats (CRISPRs), which is a form of an adaptive immunity toward previously encountered viruses and plasmids. Patterns among these sequences, have the potential to reveal composition differences among hosts, as well as the rate of evolution between generations of bacteria, both of which contribute to the overall understanding of the driving force behind periodontal disease.

Analysis of CRISPR Repeats During their initial study of human salivary viruses in 2010, saliva from four healthy human subjects was collected over an 11-17 month period so that bacterial CRISPR sequences could be analyzed alongside a library of 16S rRNA gene sequences. 16S rRNA is a highly conserved gene among bacteria and archaea that is useful for identifying bacteria. Of additional importance was the evaluation of CRISPR spacers, which are short segments of viral DNA that have been incorporated into the space between each CRISPR. CRISPR repeat loci and spacers analyses were conducted using amplifications of primers specific to sequences present in several common oral bacteria such as Streptococcus mutans.

Collectively, the data is suggestive of a continual interaction between oral bacteria and viruses in order to maintain a stable bacterial population.

Differences in spacers between individuals suggests that exposure to different viral populations influence the composition of each CRISPR system. Dr. Pride and his team have used this information to formulate two important phenomena within the CRISPR system which may contribute to further understanding of the bacteria-virus interactions. The first idea is the presence of a core set of shared CRISPR spacers over time. Preservation of these core units likely reflects the pressure from repeated exposure of the same virus types.

The second phenomenon is rapid variation in sq.ucsd.edu

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CRISPR Sequences Across Individuals

the CRISPR repeats over time which brings up the possibility of new bacterial strains arising in the oral cavity. As this initial study assessed just a single CRISPR repeat out of many, further analysis will enable them to visualize how a specific household environment can influence an individual’s risk of developing periodontal disease through repeated exposure to certain viral communities.

Virus Identification In 2011 Dr. Pride revealed the surprising presence of lysogenic bacteriophages in human saliva. Unlike lytic bacteriophages, which result in the death of their infected bacterial host, lysogenic bacteriophages integrate their genetic material into the host DNA before replication occurs. This finding of bacterial viruses was contrasted against their original belief that the oral cavity was primarily host to human viruses. Furthermore, lysogenic bacteriophages reveal their role in spreading genes among bacteria or a, “Reservoir of bacterial genomic material,” as Dr. Pride phrases it.

The composition and role of viruses was determined using a series of blastX analyses which align similar genomic sequences with the purpose of identifying genes against a known library of proteins.

This time, saliva from five individuals was collected over a 3 month period for use in virome sequencing and assignment of contigs based on blastX analysis. BlastX analyzes DNA sequencing based on alignment so that contigs, overlapping regions of consensus DNA can be assigned. Initial comparison of virus composition between subjects indicated the presence of shared homologs between all subjects, throughout the entire experiment.

In the oral cavity, several virulence factor homologs, were found among the analyzed viromes. These virulence factors, involved in platelet binding, iron scavenging, lipopolysaccharide biosynthesis, cell wall antigenic variation, and DNA methylation reveal a secondary role for viruses. Aside from simply targeting bacteria, these viruses represent a bank of infections genes, potentially enabling their target bacteria with pathogenic traits that may contribute to periodontal disease. In addition, each subject revealed 10% of their viral contigs to contain integrase protein homologs, suggesting the presence of lysogenic viruses. This integrase is similar to the enzyme used by retroviruses such as AIDS to incorporate foreign DNA into the host cell genomic material. While viral BlastX analysis determined that there was a complex diversity of viruses present in each subject, bacteriophages were the most abundant. These unique viral qualities differ greatly from the properties that were previously identified in viruses from human respiratory and gastrointestinal tracts, and as a result will require close attention in the future. sq.ucsd.edu

Dr. Pride’s most recent research on virus-bacteria relationships in the oral cavity has analyzed 13,977 streptococcal CRISPR sequences against 2,588,172 virome reads to further study how bacterial immunity is reflective of current and previously encountered viruses.

This study involving saliva from four humans over 17 month periods contributes to his initial findings by concluding that CRISPR spacers and their corresponding viruses coexist in humans at the same time.

In contrast to their first study which compared CRISPR sequences in bacteria to a library of known bacteria, this compared CRISPR sequences and viruses within the same individual.

Viruses from saliva were collected and sequenced, while CRISPR spacers were evaluated metagenomically from two repeat sequence families (Streptococcus gordonii and Streptococcus thermophilus), instead of analyzing a single group of bacterial CRISPRs. Interestingly, there were new CRISPR spacers that arose, which corresponded to specific virome matches. This revealed bacterial ability to adapt to new local viruses. CRISPR spacers were also tested for matches against other virome reads outside of the oral cavity in an individual, but failed to show any significant matches. However, CRISPR spacers did display matches with virome reads of other individuals, indicating the potential for CRISPRs from one individual to neutralize viruses of another individual.

While bacteria and their viruses in other ecosystems have been studied extensively, this is the first analysis of CRISPR-virus relationships in humans that has unearthed critical understandings of what shapes the microbiota existing within humans. Thus far, data points to the idea that two families of streptococcal oral microbiota contain a developed immune response, designed to deal with the vast oral viral community present in our saliva and encountered in the environment. This has implications for how bacterial communities deal with viruses in the oral cavity, by opening doors for studying the effect of disarming viral communities and how oral microbiota can develop into the disease causing pathogens that lead to periodontal disease. Much of this is currently being tested in what Dr. Pride calls, “household studies, involving passive resistance” to viral communities.

Future Dr. Pride hopes his work will someday help reveal the baseline level of microbial factors necessary for developing infections such as periodontal disease. His use of CRISPR sequences to compare virome and 16S rRNA reads have opened up a library of information that was previously unstudied. Although diagnostic tools for marking viruses specific to periodontal disease is a long way off, this research has uncovered a deep viral-bacterial relationship that has major implications for our oral health. While this doesn’t solve the issue of flossing on a daily basis, it sheds light on one of the least understood human ecosystems and provides a target for future diagnostic tests to detect infectious diseases caused by bacteria.

WRITTEN BY ANDREW LUM. Andrew Lum is a Biochemistry and Cell Biology major from Earl Warren College. He will graduate in 2013.

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a crucial piece to the Cholesterol puzzle Michaela Go staff writer

Illustration by Abby Diamond

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Background When you go for a checkup and get a blood test, chances are your doctor will take a look at your cholesterol levels to make sure they aren’t too high. Cholesterol is an essential lipid to human metabolism; however, having high levels of cholesterol is also linked to atherosclerosis. Atherosclerosis is the formation of plaques on the inside of your arteries, which subsequently causes them to narrow and harden. These plaques may even grow large enough to restrict blood flow or completely block arteries that provide blood to crucial organs causing tissue damage to extremities or organs, stroke, or heart attack. According to the National Heart Lung and Blood Institute, “coronary heart disease (atherosclerosis of the coronary arteries) is the #1 killer of both men and women in the United States.” Fortunately, breakthrough research led by UCSD researcher Christopher Glass, M.D., Ph.D., is paving the way towards new and much needed treatments for atherosclerosis.

Comparison between a normal artery and an artherosclerotic artery.

An Attractive Angle Dr. Glass’s involvement in atherosclerotic research really started with his fascination with the macrophage cell and its diverse functions in immunity and homeostasis. Macrophages are cells that consume foreign or dangerous identified cells and matter. As macrophages consume other cells, they also consume the cell’s store of cholesterol.

Most of the time, these cells have effective means of metabolizing and getting rid of excess cholesterol. Macrophages that do not properly metabolize the extra cholesterol accumulate cholesterol in the form of “foamy lipid droplets,” giving these cholesterol-filled cells the name “macrophage foam cells.” These macrophage foam cells are one of the most abundant cell types within atherosclerotic lesions.

He began studying the molecular biology of the macrophage, but since these cells play such an important role in atherosclerosis, putting his work in the context of “one of the most common diseases of our society…was a very attractive angle.”

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Linking Inflammation and Atherosclerosis There is a definite correlation between inflammation of the arteries and accumulation of macrophages with high cholesterol and atherosclerotic lesions. This correlation lead many scientists to believe that the accumulation of cholesterol within macrophages caused macrophages to express more pro-inflammatory genes; in other words, to be more “activated” and cause inflammation. However Glass’ research shows that, unexpectedly, the opposite is actually true.

When induced to fill up with cholesterol, these macrophages were actually deactivated—their pro-inflammatory genes were turned off.

Thus, Glass believes that something else, not cholesterol, is driving the inflammatory response and thwarting these macrophages’ ability to inhibit inflammation. “Exactly how the increase of cholesterol is causing inflammation in my mind is still an unanswered question.”

The reason inflammation in the context of atherosclerosis is such an important topic of research is because while inflammation is usually self-limiting, it may become continuous and cause chronic inflammatory diseases. In this case, the body recognizes plaques deposited in the arteries as abnormal and launches an inflammatory response. The inflamed arteries then further promote atherosclerotic lesions launching a positive-feedback cycle of inflammation and plaque formation.

Glass set out to identify the molecule accumulating inside the macrophage foam cells that was responsible for inhibiting inflammation. Further research identified this molecule as desmosterol, a precursor in the cholesterol biosynthesis pathway that is eventually converted into cholesterol. Because desmosterol is able to inhibit the inflammatory response, Glass hopes it can be used as a basis for a novel treatment aimed toward the prevention of inflammation and the propagation of atherosclerotic lesions.

Unexpected Findings Macrophages were generated in mice by feeding them a diet high in cholesterol and fat and additionally knocking out the mice’s LDL receptors, which bind free floating low-density-lipoproteins (LDL).

Knocking out the LDL receptors helped to facilitate even higher cholesterol levels and accumulation of macrophage foam cells within the mice. Analysis of the macrophages’ genes, transcription factors and lipids revealed significant changes in almost all of the lipid classes and gene expression. Genes linked to inflammation and cholesterol synthesis were unexpectedly downregulated. This downregulation of pro-inflammatory genes was very shocking because the accumulation of macrophage foam cells were supposed to activate inflammation.

This discovery was so surprising that after presenting the data to a group of colleagues, someone suggested that the samples may have been mixed up, “because the suppression of inflammation was so counter to what everyone was expecting.”

Desmosterol turned out to not only be important in inhibiting inflammation, but is also very important in cholesterol level regulation.

During their analysis, Glass and his team followed two important proteins that regulate cholesterol biosynthesis, Sterol Regulatory Element-Binding Proteins (SREBPs) and Liver X Receptors (LXRs).

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FEATURES other hand, helps rid the cell of too much cholesterol. It also turns off cholesterol biosynthesis to prevent the synthesis of new cholesterol when cholesterol levels are already high. Glass and his colleagues found that desmosterol, the last intermediate in the biosynthesis pathway, was the molecule responsible for both down regulating cholesterol synthesis and upregulating the LXR pathway. This was surprising because they didn’t expect the responsible molecule to be an intermediate of cholesterol biosynthesis—a pathway that should be turning off. Glass’ explanation for this unexpected behavior is that during the accumulation of cholesterol within the cell, only the last step of cholesterol biosynthesis is turned off instead of the whole pathway. This still hinders cholesterol biosynthesis, but because desmosterol is prevented from turning into cholesterol, it allows desmosterol to accumulate.

What’s Next Now at the position of finding out that desmosterol is responsible for stopping the synthesis of new cholesterol, getting rid of excess cholesterol, and inhibiting inflammation, Glass and his team hypothesize that somehow the LXR pathway must be becoming inactivated in the artery wall. If this is indeed the case, they want to find out how this is happening and are setting up a number of experiments to look at the inactivation of the LXR system. They are also looking into making desmosterol mimetics (small desmosterol-like molecules) that will have the same function as normal functioning desmosterol. They hope that this can be beneficial in the context of hypercholesterol and atherosclerosis.

Current cholesterol medications aim to prevent atherosclerosis by

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merely lowering cholesterol levels. However, Glass’s discovery has the potential to give rise to a new medication that additionally hinders the inflammatory response, prevents excess cholesterol biosynthesis, and promotes cholesterol excretion. At this point in time, Glass says although he thinks he knows how the desmosterol mimetic will affect the macrophage, he cannot accurately predict what will happen in the liver, which is really the organ that controls total cholesterol levels. “And that’s really one of the questions that we want to look at either using desmosterol mimetics or other approaches to tweak this pathway in vivo.” Although their research is still at the basic science level, Glass believes that these findings have the potential to be translated into new therapies.

“These findings really changed the way I thought, and that’s what a discovery is…a finding that changes the way you think about things.”

References 1.www.ncbi.nlm.nih.gov/pmc/articles/PMC1810472/ 2.http://www.heart.org/HEARTORG/Conditions/Inflammation-and-Heart-Disease_UCM_432150_Article.jsp

WRITTEN BY MICHAELA GO. Michaela Go is a Biochemistry and Cell Biology major from Muir College.

She will graduate in 2014.

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Epigenetics Changing How We Interpret Genomes Chris Probert staff writer

Photo from VisualLogs.com

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ENCODE When Eric Lander and Craig Venteur stood before a crowd of reporters in Washington D.C. on June 2nd, 2000 to announce that their respective groups had completed a draft of the completed human genome, it was heralded as one of the greatest scientific discoveries of the past century. The Human Genome Project (and the genome projects of other model organisms) has transformed biology; we have now boldly entered the “genomic era,” where one can peruse the entire DNA sequences of many organisms from their internet browser. But even before the first champagne bottles had been uncorked in celebration of this milestone of international scientific collaboration, it became clear that an organism’s sequence of DNA bases, taken alone, does not provide information about when, where, or how genes are expressed in patterns that allow the creation of that organism.

Given these large unanswered questions, the National Institutes of Health organized another largescale genomics project: the Encyclopedia Of DNA Elements (ENCODE). The project’s broad goal is to identify all DNA elements in the human genome and determine their roles in regulating gene expression. Dr. Joe Ecker, an ENCODE participant and a professor at the Salk Institute, likens the information the project seeks to a “recipe” that he hopes will show how cells take “the raw ingredients” of genomic DNA, and turn them into “masterpieces” of differentiated tissues, organs, and

entire organisms. By Dr. Ecker’s metaphor, the Human Genome Project took the first step towards this goal by providing us with the “ingredients list,” or genome. ENCODE now seeks to understand how epigenetic “recipes” control where and when we express our “ingredients list” of genes.

DNA Methylation

The ENCODE project’s broad goal of understanding the “recipes” of gene expression has been broken into many sub-projects, each of which seeks to understand the role of one particular process.

Dr. Ecker’s group has been focused on the process of methylation, or the reversible addition of a –CH3 group to Cytosine bases in a DNA sequence [figure 1]. Using new, high-throughput sequencing approaches, the group first began studying the process in Arabidopsis thaliana, a plant related to mustard that is a common model organism, where they found large-scale differences in methylation both between different tissues within one plant and between different individual plants. They followed up this study with research in human samples that showed genomic methylation on a base pair by base pair scale. Their results demonstrated that, similarly to plants, the extent of variation in methylation between humans is much larger than previously thought, and that the presence or absence of methylation at particular loci strongly correlates to differences in gene expression. The group have termed this information about methylation at various loci and its effects on gene expression a “methylome” (think “methyl” + “genome”), and made it publically available to browse and download via the Salk website (http:// neomorph.salk.edu/human_methylome/) [figure 2].

As methylation does not change the DNA sequence of an organism, it is “epigenetic,” or a form of heritable variation that is not reflected in just the coding sequence of a DNA molecule. Sequencing a DNA molecule, even with modern DNA sequencers, does not provide any information on the level of methylation within the molecule. As part of their contribution to the ENCODE project, the Ecker lab has been investigating ways to determine which particular cytosine bases within a DNA strand are or are not methylated, providing data with base pair resolution. The group’s main approach uses ChIP-Seq, a combination of Chromatin Immuno Preciptation and high throughput Sequencing [figure 3]. The process relies upon antibodies specific to methlyated cytosine, which attach to the bases and allow their separation and detection with DNA sequencers. Dr. Ecker’s lab continues to test other novel methods for assaying methylation in DNA, which have led to the methylomes of many cell types Figure 1. Epigenetic modifications to the human genome include cytosine methylation published by other ENCODE participants.

“ENCODE now seeks to understand how epigenetic ‘recipes’ control where and when we express our ‘ingredients list’ of genes.”

and chromatin modifications.

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Figure 2. The Ecker Laboratory’s “Human Methylome Browser” allows users to access vast data sets combining information on methylation and gene transcription.

Discoveries Several discoveries have already been made by ENCODE researchers. One example is how ENCODE data have shattered notions about the roles of non-protein coding DNA. From the earliest drafts of eukaryotic genome assemblies, scientists have been puzzled by the large part of the genome (80-95%, depending on the organism) that does not fall on protein-coding regions. Some scientists have gone as far as labeling these regions of the genome “junk DNA.” But through high-throughput sequencing and screening techniques, ENCODE has shown that a staggering 75% of the genome is indeed transcribed at some, and that at least 80% of the entire genome plays a vital role in metabolic function of stem cells. In short, these findings show that “junk” DNA is not junk at all – it’s both regularly transcribed and also vital for normal cell function.

The ENCODE project is far from complete; the project’s director estimates they have less than 5% of all the data they hope to collect. But with the combination of new, high-throughput sequencing technologies and the many scientists working together to better understand the processes surrounding epigenetic regulation of gene expression, the project appears to have a bright future.

Researchers envision one day being able to pinpoint events that cause epigenetic modifications that have ramifications on human health. Perhaps behavioral or phenotypic patterns in parents can help explain complicated patterns in offspring, like the onset of cancer or autism. It is clear that the story of epigenetic gene regulation is far from complete, but that many exciting discoveries lie ahead. sq.ucsd.edu

Figure 3. ChIP-Seq approaches can be applied to identify sites of methylation within eukaryotic genomes. Methylated Cytosine bases (labeled “CH3”) are identified by beaded antibodies with specificity for modified Cytosines. The beads (and attached DNA) are precipitated, then DNA is separated and sequenced.

WRITTEN BY CHRIS PROBERT. Chris Probert is a Computer Science major from Thurgood Marshall College.

He will graduate in 2014.

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Photo taken by Dilara Onur at La Jolla Cove sq.ucsd.edu

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looking toward the future with

RESEARCH The Research section highlights the driven undergraduate researchers at UC San Diego, whose work constantly expands the boundaries of science.

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RESEARCH Effects of Algal Diversity on Productivity, Light Capture and Grazing Resistance Garfield T. Kwan Algal biofuel represents an attractive option to satisfy the growing demand for economically viable renewable energy. Currently, algal biofuel is most economically grown as a monoculture in a raceway structure. However, ecological principles posit that a diverse community may be more productive and resilient in the face of environmental challenges. We experimentally tested the hypothesis that growing a diverse group of algae in polycultures would increase biomass production, nutrient absorption, and grazer resistance. Algae were grown in combinations of 1, 2, 5, and 10 species. Observed algal biomass, dissolved phosphate, and the grazer Daphnia’s survival were recorded.

We found that peak biovolume occured in the five species treatment group, suggesting that increased biodiversity when grown in combination could potentially increase biofuel lipids. Dissolved phosphate and Daphnia were generally lowest in cultures with 5-10 algal species compared to one or two species, although large differences between species combinations suggest that random combinations of algae taxa will not offer stable and productive yield. This experiment suggest that a combinations of the most productive species may allow biofuel to be produced at greater rates, increasing its potential for commercialization, and ultimately replace fossil energy.

Introduction Algal biofuel represents an attractive option to satisfy the growing demand for economically viable renewable energy. Fossil fuels are a limited resource that release carbon into the atmosphere when they are burned; whereas biofuels are renewable and remove carbon dioxide when they are grown, offsetting carbon released during combustion and closing the carbon loop. Biofuel can be produced by growing sugar cane, corn, algae or other organisms; however, the relative values of different bioenergy crops are a source of much debate.

Biofuels are already a large part of the global energy economy. For instance, Brazil utilizes sugar cane to generate cheap biofuel for its economy, reducing gasoline emissions by about 90 percent (Biofuels: the promise and the risk, 2008). The United States cannot grow sugar cane as readily or easily, therefore, uses corn to create its own source of biofuel. However, using corn as a biofuel source directly competes with food for land, fertilizer, and water. This results in upward pressure on world corn prices.

It was estimated that the amount of cornbased ethanol required to fill the tank of a sport utility vehicle (100 liters of ethanol) could feed one person for a year (240 kg of corn) (Biofuels: the promise and the risk, 2008). Price increases in corn can thus cause significant welfare losses to developing nations, such as Mexico, a major importer of America’s corn. Overall, these dilemmas may be partially or wholly resolved through the use of algal-based biofuel.

Algal biofuel has several distinct advantages over corn-based biofuel. First, algae can be grown on non-arable land and therefore does not compete with food sources. By developing an algal-based biofuel industry, corn can be left to feed humans,

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either directly or indirectly through livestock, rather than engines. Second, algal biofuel yields more fuel per hectare compared to other biofuel producers (fig. 1). Algae can produce 2-50 times more biomass per unit area compared to terrestrial crop plants, easing demands for land with food crops and native ecosystems (Shurin et al. in review).

Algae can also be grown in alternate water sources such as seawater or wastewater, decreasing competition with farmland and fresh water. (Borowitzka and Moheimani 2010, Shurin et al. in review). Algae have many potential advantages over terrestrial crop plants as sources of bioenergy; however, a number of technical and economic challenges remain to be resolved before this promise can be realized.

Algal biofuel is most economically grown as a monoculture in raceway structure (fig 2) that is constantly circulated to prevent

sinking and shading by dense populations.

However, because the top of the raceway is exposed to air, algae are left vulnerable to viral, bacterial, and fungal contamination as well as invasion by grazers such as rotifers and crustaceans (Shurin et al. in review). The raceway environment is similar to a natural pond system, but while most ecosystems have a diverse group of species, algal raceways strive to maintain only a single strain of algae.

Density dependent population regulation due to competition, predation, parasitism, and disease may become particularly important in algal biofuel ponds where the densities of cells are very high (Cohen et al 1991, Cain et al. 2008).

Ecological principles posit that a diverse community may be more productive and resilient in the face of environmental challenges. Different species may have different requirements for resources such as

Figure 1. Fast-growing algae yield more fuel per unit area than other biofuel producers (Service 2011). sq.ucsd.edu

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Figures 2 and 3. (Left) Image of an algae raceway. (Right) Algae raceways are continuously churned. Used with permission from Carbon Capture.

nutrients and light, allowing suites of species to convert the available resource base into biomass more efficiently. In addition, natural enemies such as predators or pathogens may selectively attack some prey or hosts, so that other species may compensate for the loss of any prey that is controlled by consumers. Tilman et al. (2006) compared energy productivity of diverse grasslands with that of corn and soybean crops and found that the net energy yield (the output minus the inputs) were far greater with native plants. There are several reasons to suspect that diverse communities may be both more efficient in producing biomass and more resilient against invasion, contamination and predation.

We experimentally tested the hypothesis that growing a diverse group of algae in polycultures would increase biomass production, nutrient absorption, and grazer resistance. Biovolume is simply the density of biological matter in the culture. Total algal biomass was measured by counting and measuring cells using geometric equations to estimate their volume. Nutrient uptake by algal cells was measured by the amount of dissolved phosphate left in the media at the end of the experiment, an indicator of how much resources are available for invading algae. Grazer resistance was recorded through the survival of Daphnia, one of many common grazers of algae with a global distribution.

Materials and Methods In our experiment, we grew combinations of 1, 2, 5 and 10 species of algae and recorded the observed algal biomass, dissolved phosphate, and Daphnia survival.

Each sample had a unique combination of sq.ucsd.edu

species that was assigned by random draws from the full 10 species pool. Replicates consisted of 100 ml semi-continuous cultures grown in Wright’s Cryptomonas (WC) Medium. Each of the 31 unique species combinations was crossed with the addition of one neonate of the crustacean grazer Daphnia pulex on day 25 and each treatment combination was replicated three times. The experiment was conducted in an environmental chamber at 19.5¹1C and on a 12-hour artificial daylight cycle and was concluded after 29 days when the algae were collected for cell counts and measurements in a hemacytometer under an inverted microscope. Algal growth over time was observed by chlorophyll-a concentration in the cultures and was measured with a Turner Triology Laboratory Fluorometer. Dissolved phosphate was analyzed according to Wetzel & Likens (1991) and also measured with a Turner Fluorometer with an absorbance module and an 880 nm filter (Shurin et al. in review). Daphnia survival was recorded by observing swimming movements under a microscope.

Results The main results of the experiment are seen in figures 6, 7, and 8. The maximum biovolume was observed in a single five species combination (number 21 in fig 6).

Furthermore, we observe that variability in algal biovolume also decreases as diversity of species increases. Note that the y-axis of the biovolume figure is in log10 scale In light of this, the variability observed in the one and two species treatment is large compared to that among the five and ten species treatments. However, only one five species combination exceeded the maximum

biovolume produced by the single most productive monoculture (Chlamydomonas reinhardtii).

Dissolved phosphate, the amount of left in the media that was not taken up by the algal cells during the experiment, generally declined as diversity of species increases.

However, there were large differences among different combinations with the same numbers of species (fig 7). Daphnia survived poorly in cultures with 5-10 algal species compared to one or two species, although again there were large differences between species combinations apart from diversity (fig 8).

Discussion We found that biovolume production generally increased when more species of algae were grown together, but that only the single most productive mixture out-performed the most productive monocultures. We observed that most species combinations produced similar amounts or less biomass than the highly productive single species. Instead, the main benefit of growing diverse algae for biofuel production may be in protection from grazers. The survival of Daphnia was greatest in monocultures and very low in polycultures of 5-10 species even though the amount of algal food available to Daphnia was high. Our results show that while diversity may be one approach to optimizing productivity and resistance in bioenergy, combinations of species must be chosen carefully as many mixtures performed no better than single species.

Biovolume We found that peak biovolume production occurred in the five species VOL 10 • SALTMAN QUARTERLY

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RESEARCH treatment groups, suggesting that increased biodiversity when grown in combination could potentially increase biofuel lipids.

The increased diversity was also observed to decrease variability among the different treatments. We may expect the advantage of the polycultures relative to monocultures to be greater outdoors where more variable weather and contamination play a larger role.

(Cohen and Arad 1989, Cohen et al 1991).

Nonetheless, the experiment suggests that growing a diverse culture of algae may lead to more stable productivity.

Nutrient uptake As algal diversity increased, the amount of dissolved phosphate left in the system decreased. The decrease in dissolved phosphate in more diverse polyculture may be the result of higher productivity and more efficient nutrient uptake. If more phosphate is taken up by the culture, then invading algae may have a harder time establishing and ourishing. Therefore, by eliminating invading algae from the culture, the target algae may experience less competition and become more productive.

Grazing Resistance Increased diversity led to decreased Daphnia survival. Although this experiment included only one species of grazer, an unusual situation under more natural conditions, other grazers may show similar responses to algal diversity. Grazers may have a higher mortality rate in polycultures due to the presence of inedible algae species, secretion of toxins, and greater difficulty in foraging when confronted with a mixture of potential prey items, which would slow or stop predators from hunting

effectively (Kratina et al. 2007). Overall, a diverse community may additionally be less susceptible to pathogens and viruses compared to a monoculture.

The ecological concept of niche differentiation suggests that polycultures may be more stable and productive compared to monocultures in environments subject to invasion and variable weather conditions.

The concept of niche differentiation suggests that a diverse community will yield higher-than-average biomass through resource partitioning, or maximizing the use of the resource by minimizing competition (Loreau & Hector 2001). Our results support some of these predictions, indicating that a diverse community may offer higher biomass yield. Perhaps our most surprising result was that the main advantage of diversity seemed to lie in conferring resistance to invasion by consumers as introduced Daphnia experienced much lower survival in mixtures of algae than in monocultures. Further research is necessary to test this concept in a natural, outdoor setting. Furthermore, because biovolume increase may not necessarily indicate algal lipid increase, further research is necessary to understand whether there is a correlation between the observed biomass productivity to biofuel yield. Additional research in growing cultures in an unsterile, natural condition may be beneficial for further understanding the impacts of diversity on the effectiveness of grazers, as well as bacterial, viral, or fungal infections.

Conclusion This experiment

suggests

that

Figure 4. Diversity experiment in progress; lighting is on a 12 hour on/o cycle to simulate outdoor lighting.

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Figure 5. Image of Daphnia pulex taken under the microscope. Courtesy of Rachel Abbott.

increasing diversity of algae may lead to higher biovolume, more efficient use of mineral resources, and greater resistance to grazing. This polyculture approach to cultivation resembles a natural ecosystem in that it contains a multitude of species.

Our data indicate that completely random combinations of algae taxa will not offer stable and productive yield. We need to understand and carefully test which combination of algae are most beneficial in order to optimize the use of polycultures for bioenergy.

The economic viability of algae biofuel depends on producing energy at a rate that is economically competitive with fossil fuels.

By finding the most productive species combinations, biofuel can be produced at greater rates, increasing its potential for commercialization. Higher yields lead to more competitive market prices, which will be necessary for bioenergy to replace fossil energy. It was estimated that algal biofuel can potentially be produced at about $100 per barrel, making it competitive with petroleum, which was $91.82 as of December 31st, 2012 (U.S. DOE 2010). Algae biofuel has great potential to end our dependence on diminishing fossil fuel. The prospect for closing the carbon loop may finally become a reality if technologies can improve the efficiency and reliability of production.

Acknowledgments Special thanks to Janelle Bryant, Gabby sq.ucsd.edu

RESEARCH Figure 6. Variation in final algal biovolume between treatmentsThe x-axis indicates each of the 31 randomly generated species combinations, split into single species (1 spp), two species (2 spp), five species (5spp) and 10 species (10). The y-axis measures algal biovolume in natural logarithm scale.

Figure 7.

Differences in dissolved phosphate concentration among treatments.

The x-axis’s notation is as in Fig. 6. The y-axis depicts dissolved phosphate in natural logarithm scale. Dissolved phosphate is a measure of the available, unused nutrients present in the medium (not taken up by algal cells).

Figure 8. Daphnia daily survival by treatment -- The x-axis’s notation is as in Fig. 6. The y-axis measures the percentage of Daphnia survival. In this figure, 0 indicates that no Daphnia survived and 1 indicates that all of the animals survived throughout the duration of the experiment.

Reyes, Rohan Mehta, and Pierre Lau for helping in the project. We would also like to thank CalIT2, UC LEADS, & SDCAB for various funding and presentation opportunities. We are grateful to Dr. David Holway and Zac Hodosevich for multiple revisions of the manuscript and providing valuable suggestions. Collaborating authors include: Shovon Mandal, Rachel Abbott, Kelsey Garrder, Mike S. Deal, Jonathan Shurin.

References

1.Biofuels: The Promise and the Risks. The World Bank, 2008, 70-71. 2009.

2.Borowitzka, M. and N. Moheimani. (2010) Sustainable biofuels from algae. Springer Science+Business Media, 18. 1:13-25.

3.Cain et al. Ecology. Sunderland: Sinauer Associates, Inc. 2008. Print. p240-258.

4.Cohen E. and Arad S. (1989). A Closed System for Outdoor Cultivation of Porphyridium. Biomass and Bioenergy, 18, 59-67.

5.Cohen E. et al. (1991). A Closed System for Outdoor Cultivation of Microalgae. Biomass and Bioenergy, 1, 83-88.

6.Doak D. et al. (1998). The Statistical Inevitability of Stability-Diversity Relationships in Community Ecology. The American Naturalist, 151, 264-276.

7.Duffy J.E. (2002). Biodiversity and ecosystem function: the consumer connection. Oikos, 99, 201- 209.

8.Kratina, P., Vos, M. & Anholt, B. R. Species diversity modulates predation. Ecology 88, 1917-1923 (2007).

9.Lee, RE. (2008). Phycology. Cambridge University Press, Colorado State University.

10.Loreau M. and Hector A. (2001). Partitioning selection and complementarity in biodiversity experiments. Nature, 412, 72-76.

11.Service, R. Algae’s Second Try. Science Magazine, 333, 1238-1239. 2 September 2011.

12.Shurin J.B. (2000). Dispersal limitation, invasion resistance, and the structure of pond zooplankton communities. Ecology, 81, 3074-3086.

13.Shurin, J.B. et al. (In Review) Industrial-strength ecology: Tradeoffs and opportunities in algae biofuel production. Ecology Letters.

14.Smith V.H. et al. (2010). The ecology of algal biodiesel production. Trends in Ecology & Evolution, 25, 301-309.

15.Solovchenko, AE, Chivkunova, OB, Maslova, IP. (2011). Pigment composition, optical properties, and resistance to photodamage of the microalga Haematococcus pluvialis cultivated under high light. Russian Journal of Plant Physiology 58:1220.

16.Tilman, D, and J. Hill. (2007). Corn can’t solve our problem. The Washington Post 25 March. Web. 12 December 2012.

17.Tilman, D., Hill, J. & Lehman, C. Carbon-negative biofuels from low-input high-diversity grassland biomass. Science 314, 1598-1600 18.U.S. DOE (2010). National Algal Biofuels Technology Roadmap. U.S. Department of Energy, Office of Energy Efficiency and Renewable Energy, Biomass Program.

WRITTEN BY GARFIELD KWAN. Garfield Kwan is a Marine Biology major from Muir College. He is graduating in 2013.

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RESEARCH Size-selected Harvesting of a Common Iguana Species (Ctenosaura similis): Possible Impacts on Population Structure, Sexual Dimorphism, and Social Dynamics.

Elizabeth C. Miller Size-selected harvesting, where hunters preferentially take large size classes of a population, is well documented in many exploited species.

The effects of size-selected hunting pressure include: reduced growth rate, smaller size at sexual maturity, changes in social hierarchies, and decreased number and quality of offspring. This study evaluated the impacts that size-selected harvesting could have on Ctenosaura similis (ctenosaur) populations in the Guanacaste Province of Costa Rica. C. similis populations were compared in three sites protected from hunting with populations in three nearby unprotected sites. From the photographs of 95 individuals, I estimated snout-vent length, head length, and body height. Males in hunted areas that were identifiable by secondary sex characteristics were smaller across all three measurements taken, while females did not show changes in body or head size. Additionally, more juveniles showed territorial behavior in hunted areas, while juveniles in protected areas were likely displaced by large adults. These results suggest that hunting adults of this species impacts on social behavior and sexual dimorphism.

Introduction Size-selected harvesting occurs when larger size classes, which are the most desirable for meat or trophy hunting, are preferentially hunted over smaller individuals.1 This differential selection pressure causes skewed size class distributions, reduced reproductive output in cases where individual fitness is positively correlated with body size, and behavioral changes in species with size-dependent social hierarchies. These effects have been seen in both wild and experimental populations of species hunted by humans.2-6 Due to the negative impacts on individual fitness, there is potential for slowed population growth and reduction of population size. In order to maintain healthy iguana populations, it is important to evaluate the degree to which animal populations have experienced unnatural selection pressure caused by hunting.

Large iguanid lizards (Iguana, Ctenosaura) have a long history of traditional use in Central America. These iguanas are exported for the fashion and pet trade in addition to local consumption of their eggs, meat, and fat. While iguana farming holds promise for relieving local populations from being hunted, wild iguanas are still heavily exploited.

The ctenosaur, Ctenosaura similis, is common in the dry forests of Costa Rica. Juveniles are ubiquitous in yards and roadsides, but adults at their maximum size are rare in places where they are not protected from hunting. In addition to being economically and culturally important, large individuals are important for the health of dry forests because they eat more fruit than smaller individuals and act as seed dispersers.8-9 Therefore, sizeselected harvesting of ctenosaurs may have a negative impact on dry forest plants; thus making it important to conserve them as part of a healthy ecosystem as well as to maintain the populations for human use.

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The morphology of individuals, especially the head length, body mass, and body length, is sexually dimorphic,15 with males being much larger and having longer heads than females. Secondary sex characteristics of male iguanids develop with increasing age.24 Therefore, older males will show a greater degree of morphological difference from females than younger males.

Male body and head size also influences social rank.16 In females, clutch size is positively correlated with body size.17 Mortality due to natural predation is highest in juveniles and decreases as individuals grow.13 Due to these factors, reproductive success increases with body size in both sexes.

Many studies have demonstrated that lizard sociality is more complex than was once believed10. Like most iguanids, ctenosaurs are territorial and have sizedependent dominance hierarchies that are most pronounced among males.11-13 Though small males may succeed in sneak-mating in a large male’s territory, males may improve their reproductive success by postpone mating until they become large enough to defend a territory or become receptive to females.14 Experiments show that growth rate and aggressive behavior, both of which are factors that influence social rank in male iguanas, are plastic depending on the social and environmental context.18-20 Not much is known about the anthropogenic impacts on aspects of iguana biology besides the reduction of population size from exploitation. In this study I explored the possible effects of size-selected hunting on distribution of size classes, shifts in social behavior, and physical proportions of individuals by comparing individuals in areas protected for conservation with those in hunted areas. I predict that 1) in hunted areas, the majority of individuals will be smaller than those in protected areas; (2) small

Figure 1. A photograph of a large adult male (taken at Junquillal Beach on 11 May 2011), with lines demonstrating the body measurements estimated using ImageJ software.

individuals will show more head-bobbing displays in hunted areas than in protected areas; and (3) the difference in absolute head length between males and females will be lower in hunted areas than in protected areas.

Methods Study Sites This study was conducted in seasonal dry forest and developed sites near the Pacific coast of the Guanacaste Province.

Populations were observed from May 2nd until May 14th, 2011. During this time period, the rainy season had not yet started (May-November) and the breeding season for this species was already over (February to March). Six different sites were surveyed: three protected and three hunted. Protected sites were located in three different sectors of the Area de Conservación Guanacaste (ACG); Each sector sampled was within an area no larger than 1 km2 and contained the main administration and camping area of the park. These included (1) Sector Murcielago (7 km west from the town of Cuajiniquil), (2) Sector Junquillal (4 km north of Cuajiniquil), and (3) Sector Santa Rosa (21 km southeast from Cuajiniquil). The hunted areas were the roads and communities surrounding the ACG which included (1) town of Cuajiniquil sq.ucsd.edu

RESEARCH size within classes roughly follows a normal distribution.

However, in hunted sites the size class distribution is right skewed (Figure 2).

Males in hunted areas were shorter in SVL (t3.85=3.16, Table 1. Numbers of each class of individuals seen in each site. In protected sites (P), many more adults were seen than juveniles, P= 0.036) and had while in hunted sites (H) many more juveniles were seen than adults. More larger individuals display in protected sites, while in smaller body depths hunted sites smaller individuals displayed most often. * # Seen (# with size measurements taken from photograph). ** # Seen (t4.35= 2.86, P = displaying (# smaller than 25cm SVL).

0.042). Male head (10°56'35.86"N, 85°41'9.10"W), (2) a 4 km between individuals. Using ImageJ software length tended to be shorter in hunted sites, road between the town of Cuajiniquil and the (National Institute of Health; http://rsb. though this relationship was not significant entrance of Sector Junquillal, and (3) a 7 km info.nih.gov/ij/), the height of the eye was (t5.52= 2.36, P= 0.06). This suggests that road between the town of Cuajiniquil and the standardized at 0.5 cm and used as a scale males in hunted sites are smaller in size administration building of Sector Murcielago, to measure the head length (from jaw to end and have a smaller body mass as compared with a second 1.7 km road branching off of ear opening), body depth (a substitute for to males in protected sites, though there from this and ending at Playa Cuajiniquil. mass, widest point of trunk from belly to was no significant proportional difference Identification in the Field beginning of crest) and SVL (estimated from (Figure 3). Females and juveniles did not Ctenosaurs were found indiscriminately jaw to end of pelvic girdle) of individuals show a difference in these measurements by walking along roads and surrounding photographed in the field (Figure 1). between protected and hunted sites.

Sexual Dimorphism areas, campgrounds, buildings, and trails. Data Analysis The difference in mean head length Observations were made as early as 0800h Due to low sample size, individuals from and as late as 1700h. Ctenosaurs were the six sites were pooled into two groups: between males and females was 40% smaller photographed as they were found. Individuals hunted and protected. A Welch’s t-test was in hunted sites (Figure 4). The ratio of male were placed into three categories based on used to statistically compare the differences head length to female head length was 1.38 their visible secondary sex characteristics: in SVL, head length, and body depth in protected sites and 1.14 in hunted sites. male (possessing hypertrophied jaw muscles between the two groups. An additional t-test The ratio of male SVL to female SVL was and a long crest), female (not possessing a was used to determine difference in SVL 1.20 in protected sites, compared to 0.99 in long crest but having a large body weight of only head-bobbing individuals between hunted sites. The measurements of males in relative to juveniles), or juvenile (small and the two groups. An Analysis of Covariance hunted sites are closer to those of females thin body). From photographs, individuals (ANCOVA) was used to test for a difference across two sexually dimorphic characteristics. were distinguished from one another based in the proportional relationship of SVL Differences in Display Behavior In protected sites, twenty adults and on location, pattern, color, and tail re- and body depth across the two groups. two juveniles with size data head-bobbed growth. If a ctenosaur ran away before it during the observational period. In hunted could be photographed, it was still recorded Results sites, two adults and nine juveniles displayed. as “seen” though no size data were taken. Abundances Each individual was recorded as “seen” A total of 155 individuals over 44.7 No juvenile in any site was observed headonly once even if it was seen multiple times. hours were observed. Size measurements bobbing in view of an adult male; however, The presence or absence of head-bob were taken from the photographs of 95 juveniles were observed head-bobbing near displays during the observational period individuals (64 in protected sites, 31 in adult females. The mean SVL of individuals by the focal animal was also recorded, hunted sites). This included two deceased observed head-bobbing was smaller in signaling a territorial claim or confrontation. individuals found on roads, a juvenile and a hunted sites compared to protected sites Estimation of Size Measurements male, that were dissected and measured with (t15.77= 4.60, P= 0.0003). In hunted sites, In areas where C. similis has been a measuring tape. These two measurements 43% of individuals smaller than 25 cm SVL hunted, individuals are wary of humans and are included in the data for the hunted site displayed, while 23% of individuals of the very secretive. For this reason, individuals Cuajiniquil, which was the closest site to where same size class in protected sites displayed. were estimated from photographs instead the two individuals were found (though not of capturing them to measure directly. included in data for displaying individuals). Discussion Though C. similis remains common Using a tape measure, snout-vent Within protected sites, size measurements length (SVL) measurements were taken were taken from 30 females, 21 males, in hunted areas, these populations are from two live, captured individuals (eye and 13 juveniles. Within hunted sites, size disproportionately comprised of juveniles. and head length of live individuals were measurements were taken from four males, Juveniles are likely to have home ranges estimated from photographs based on five females, and 21 juveniles (Table 1). hidden from adults due to the risk of known SVL for safety reasons). From these Size Class Distribution and Size cannibalism,21 which may explain why they are more visible in the absence of large adults. measurements, it was determined that the Measurements It is possible that fewer adults were height of the eye, usually 0.5 cm, varied little In protected sites, the distribution of sq.ucsd.edu

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RESEARCH bias. This bias could contribute to the size differences observed in hunted and protected populations.

There is a degree of error associated with the method of estimating size data from photographs.

In some cases, individuals tried to escape before a profile shot could be taken. Size data had to be estimated from slightly tilted images of ctenosaurs in Figure 2. A histogram of size classes in hunted sites (white bars) and these cases. This protected sites (black bars). Data includes males, females, and juveniles. could alter the measurements taken from the photograph.

However, due to the difficulty of capturing ctenosaurs, this noninvasive technique was the best method available for this study. C. similis is considered to be the world’s fastest sprinting lizard species,22 and as mentioned above they are often wary of humans. Future studies with greater available resources Figure 3. The relationship between SVL and body depth in males. White could take advantage squares represent males from hunted sites, and black diamonds represent of trapping methods males from protected sites. There is not much overlap in size between the for more accurate two groups. measurements, seen in hunted areas because of differences though at the risk of causing in predatory avoidance behavior. Adults in stress to the ctenosaurs. protected sites could be easily approached Some small individuals in hunted sites within 1 meter. In contrast, most adults (between 20 and 30 cm SVL) could be seen in hunted sites did not let me approach identified as male based on their secondary them, and had to be observed from far sex characteristics; though I did not observe away. This shows a marked difference in males smaller than 30 cm SVL in protected the recognition of humans as predators areas. An entire size class of males was present between the two groups. It is likely that there where hunting occurred, and not observed in is selection for secretive behavior in hunted areas protected from hunting. One hypothesis populations. In addition, individuals could for this observation is that smaller males be learning to avoid humans by accumulating become more visible in the absence of large negative experiences during their lifespan. dominant males, and the discrepancy is the The cautious nature of large individuals result of sampling bias. If males identifiable in hunted areas is one source of sampling by secondary sex characteristics smaller than

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30 cm SVL exist in protected sites, they are displaced by larger dominant males and have territories in subpar locations. The removal of large dominant males by hunters opens up better territorial opportunities for smaller males; therefore they became more visible A second hypothesis to explain the presence of a smaller size class of males in hunted areas is that the removal of large dominant males acts as a social cue to “turn on” the development of the secondary sex characteristics. Many studies on various iguanid species show that dominance hierarchies affect the development of males.

In many territorial iguanids, the presence of a large dominant male can elevate levels of corticosterone and depress levels of testosterone in subordinate males.18 Even the visual cue of an adult male suppresses the growth rate of juvenile males in the green iguana.23 In a study on Galapagos marine iguanas, the experimental injection of testosterone into subordinate males caused them to act more aggressively and display territorial behavior, showing that there is plasticity in reproductive phenotypes caused by hormone levels.20 Therefore, in the case of C. similis, it is possible that the removal of large males by hunters removes this depressive pressure on the hormones of smaller males and they develop the secondary sex characteristics such as the elongated crest and swollen jaw muscles at a much smaller size. Thus, these individuals are recognizable males even though they are small relative to those in protected sites. To test this hypothesis, individuals in protected and hunted areas of the same size could be captured and sexed using cloacal analysis.24 Males in hunted areas had size measurements of the head and body that resembled those of females in both hunted and protected sites. This decrease in sexual dimorphism can be explained again by the differences in social behavior of males and females. Secondary sex characteristics of male iguanids develop with increasing age.24 Therefore, males that develop secondary sex characteristics at a younger age would not differ in head and body proportions from a female as much as in an older male. It remains yet to be determined if the reduced size of males in hunted areas leads to reduced offspring quality.

The presence of head-bobbing displays was used in this study to signify the ownership of a territory. All individuals, including juveniles, are territorial15 though juveniles may be displaced by large individuals. If a small male displays within the territory of a sq.ucsd.edu

RESEARCH found near the road outside of Sector Junquillal and hatchlings became more abundant closer to the park, with peak abundance within the park (Miller, pers. obs.). Molecular testing could confirm if hatchlings outside the park came from parents living in the park. It is important to understand the role of protected sites in preserving large populations of C. similis, as well as for promoting the health of populations outside of parks.

Acknowledgements I thank Frank Joyce for general project guidance, help with data analysis, assistance in the field, facilitating Figure 4. The differences in head length between males (M) and transportation to my sites, females (F) in unprotected sites. Dimorphism of this trait was and helpful comments on the reduced in unprotected sites. manuscript. Robert Drewes and larger male, the small male would probably Erick McAdam also gave advice. Additional be chased away by the large male. Therefore, thanks to Maria Fernanda Carazo for help in if a juvenile displays, it can be safely assumed the field sighting ctenosaurs. I am thankful that it is not within the territory of a large to the Campos-Castro family for housing male. The increased prevalence of small me for two weeks in the community of El individuals displaying in areas affected by Congrejal, Cuajiniquil while I conducted hunting could be explained by the decreased field work. Equipment such as binoculars number of territories owned by large and cameras were supplied by the University individuals. Smaller individuals are more of California Education Abroad Program. easily able to acquire and defend territories Bob Drewes, Heather Henter, and the in the absence of larger individuals Saltman Quarterly team made helpful that would otherwise displace them. comments. This research was funded in part These trends could be confirmed by by the Friends of the International Center increased monitoring and sampling of at the University of California, San Diego. populations in these sites. It is also important to examine if there are changes in life history References traits in these populations, including growth 1. Fenberg, P.B., and Roy, K. (2008). Ecological Evolutionary Consequences of Size-selective rate, size at sexual maturity, and reproductive and Harvesting: How Much Do We Know? Molecular success with age. A mark-recapture study Ecology, 17(1), 209-20. would reveal any differences in growth 2. Platt, S.G., and Thorbjarnarson, J.B. (2000). rate and size at maturity between protected Population Status and Conservation of Morelet's Crocodylus Moreletii, in Northern Belize. and hunted populations. The number and Crocodile, Biological Conservation, 96, 21-26. survival of these offspring would reveal 3. Conover, D.O., and Munch, S.B. (2002). Sustaining differences in reproductive success caused by Fisheries Yields Over Evolutionary Time Scales. age and size differences of parents in hunted Science, 297(5578), 94-96.

Jennings, S., and Blanchard, J.L. (2004). Fish and protected populations. It is essential for 4.

Abundance with No Fishing: Predictions Based on the health of iguana populations to detect Macroecological Theory. Journal of Animal Ecology, changes in life history traits as a result of 73(4), 632-42. hunting pressure that could compromise 5. Carver A.M., Wolcott, T.G., Wolcott, D.L., and Hines, (2005). Unnatural selection: effects of a maletheir reproductive success. Another future A.H. focused size-selective fishery on reproductive potential area of research is the role of protected areas of a blue crab population. Journal of Experimental as population sources for hunted areas. Are Marine Biology and Ecology, 319(1), 29–41. populations restricted to their home range, or 6. Milner J.M., Nilsen, E.B., and Andreassen, H.P. Demographic side effects of selective hunting in does migration occur? Fitch and Henderson (2007). ungulates and carnivores. Conservation Biology, 21(1), noted that individuals rarely travel more 36–47. than 50 meters from their central refuge.13 7. Wildlife and Rural Development in Latin America.

During observations, hatchling iguanas were (2011) Food and Agriculture Organization of the sq.ucsd.edu

United Nations Corporate Document Repository.

Agriculture and Consumer Protection Department.

Retrieved December 27, 2011, from http://www.fao. org/docrep/V6200T/v6200T0e.htm.

8. Van Devender, R.W (1982). “Growth and Ecology of Spiny-Tailed and Green Iguanas in Costa Rica, with Comments on the Evolution of Herbivory and Large Body Size." Iguanas of the World: Their Behavior, Ecology, and Conservation. Ed. Burghardt, G.M. and Rand, S.A. Park Ridge, NJ: Noyes Publications 9. Traveset, A (1990). Ctenosaura Similis Gray (Iguanidae) as a Seed Disperser in a Central American Deciduous Forest. American Midland Naturalist, 123(2), 402-404.

10. Fox, S.F., McCoy, K., and Baird, T.A.. (2003). Lizard Social Behavior. Baltimore, MD: Johns Hopkins Ups. 11. Henderson, R. W (1973). Ethoecological Observations of Ctenosaura Similis (Sauria: Iguanidae) in British Honduras. Journal of Herpetology, 7(1), 2733.

12. Fitch, H.S., and Hackforth-Jones, J. (1983).

“Ctenosaura Similis.” Costa Rican Natural History. Ed. Janzen, D. University of Chicago, 394-396.

13. Fitch, H.S., and Henderson, R.W (1978). Ecology and Exploitation of Ctenosaura Similis. Lawrence: University of Kansas.

14. Carothers, J. (1984). Sexual Selection and Sexual Dimorphism in Some Herbivorous Lizards. The American Naturalist, 124(2), 244-254.

15. Savage, J.M (2002). The Amphibians and Reptiles of Costa Rica: A Herpetofauna between Two Continents, Between Two Seas. Chicago, IL: University of Chicago.

16. Gier, P.J (2003). “The interplay among environment, social behavior, and morphology: Iguanid mating systems.” Lizard Social Behavior. Ed. Fox, S.F, McCoy, J.K., and Baird, T.A. Johns Hopkins University Press, 278-309.

17. King, R.B (2000). Analyzing the Relationship between Clutch Size and Female Body Size in Reptiles.

Journal of Herpetology, 34(1), 148-50.

18. Phillips, J., Alberts, A., and Pratt, N. (1993).

Differential Resource Use, Growth, and the Ontogeny of Social Relationships in the Green Iguana. Physiology & Behavior, 53(1), 81-88.

19. Baird, T.A, and Timanus, D.K. (1998). Social inhibition of territorial behavior in yearling male collard lizards, Crotaphytus collaris. Animal Behavior, 58, 989994.

20. Wikelski, M., Steiger, S.S., Gall, B., and Nelson, K.N (2004). Sex, Drugs and Mating Role: Testosteroneinduced Phenotype-switching in Galapagos Marine Iguanas. Behavioral Ecology, 16(1), 260-68.

21. Mora-Benavies, J.M (1991). Cannibalism in the ctenosaur lizard, Ctenosaura similis, in Costa Rica.

Bulletin of the Chicago Herpetological Society, 26(9), 197-198.

22. Garland, T.G. (1984). Physiological correlates of locomotory performance in a lizard: an allometric approach. American Journal of Physiology, 247(5), R806-815.

23. Alberts, A., Jackintell, L., and Phillips, J. (1994).

Effects of Chemical and Visual Exposure to Adults on Growth, Hormones, and Behavior of Juvenile Green Iguanas. Physiology & Behavior, 55(6), 987-92.

24. Rivas, J.A. and Avila, T.M. (1996). Sex identification in juvenile green iguanas (Iguana iguana) by cloacal analysis. Copeia, 1996(1), 219–221.

WRITTEN BY ELIZABETH MILLER. Elizabeth Miller is a Ecology, Behavior, and Evolution major from Marshall College.

She graduated in 2012.

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a new section of

Brevias The Brevias section highlights scientific contributions from students enrolled in the Master’s degree program for biology.

Photo taken by Dilara Onur in Page, Arizona

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Effect of Predation Risk on Honey Bee Foraging and Recruitment

Allison Bray; Ecology, Behavior, and Evolution

The risk of predation can affect foraging by honey bees (Apis mellifera) even when predators do not succeed in killing the bee, through a non-consumptive effect.

Bees can alter their behavior in response to a perceived risk, which can reduce foraging efficiency, potentially affecting hive fitness and plant pollination, a key ecosystem service. To better understand these effects it is important to understand how bees detect and react to the presence of a predator. We tested if honey bees, given a choice between a food source with a predator, the praying mantis (Tenodera sinensis), and one without, would avoid the predator. We also tested to see if the introduction of a predator to a feeder would decrease waggle dancing (recruitment) among the bees visiting that feeder.

To test bee avoidance of food sources containing predators, we trained bees to a feeder, and then gave them a choice between a safe feeder, and a feeder with a live tethered predator, a mantis. We used live mantises of varying sizes, measured by instar (mantis

growth stage). We recorded the number of bees that fed at each feeder (each bee was captured and marked). Bees did not avoid younger and smaller 4th and 5th instar mantises, but significantly avoided the larger 6th (73% chose the mantis-free feeder, n=172, p<0.01), 7th (63%, n=149, p<0.01), and 8th (adult) (67, n=161, p<0.01) instars.

Bees may perceive larger mantises as being more risky, likely because larger mantises are more capable of successfully capturing a bee.

To determine if the bees were using visual or olfactory cues to detect predator, we conducted trials with a plastic mantis (visual cues only) and with a hexane extract obtained from mantis exoskeletons on filter paper (olfactory cues only). Bees significantly avoided the plastic mantis (67% chose the mantis-free feeder, n=262, p<0.01) and the mantis extract (74%, n=121, p<0.01). Thus, honey bees are capable of using either type of cue, and most likely use a combination of both.

Foraging behavior could also be affected if the bees altered their communication in

response to a predator. Honey bees recruit nest mates to profitable food sources through a dance that communicates the location and quality of a food source. The more dance circuits, the higher the quality of the nectar.

The presence of a predator may decrease the perceived profitability of a food source, decreasing dancing and recruitment for that source. To test the effect of a predator on recruitment communication, we trained bees from an observation hive to a feeder and marked them. We then recorded the number dance circuits for each returning bee. We then introduced a mantis to the feeder and recorded the number of dance circuits for each bee again (provided that it continued to visit the feeder), and compared dancing using a paired t-test.

Dancing decreased significantly after the introduction of the predator according to a two tailed paired t-test (t=2.68, df=67, p=0.009, Fig. 1), showing that bees do alter their recruitment communication in response to perceived predation risk.

Figure 1. In the majority of cases, predator presence on a food source results in a reduction of the number of dance cicuits performed by a bee (a decrease in recruitment). (N=68 bees, t=2.68, df=67, p=0.009)

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Chronic Stress and Extended-access to Cocaine Produces Escalation of Drug Intake: Involvement of κ-opioid and AMPA Receptors Sharon Chaing; Neuropharmacology

The transition from Thus, the observed decrease recreational cocaine use to drug in p-GluR1S845 was likely due addiction is thought to be a result to a compensatory mechanism of the dysregulation of brain responding to extremely high reward and stress systems. This levels of synaptic glutamate study examined the participation caused by both cocaine of κ-opioid receptor (KOR) withdrawal and stress history. activity in the increase of cocaine Extended-access to cocaine intake produced by either as well as chronic stress are extended-access to cocaine or environmental factors that chronic stress. have each been proven to Part 1: Dynorphin is a cause increased cocaine intake, brain stress neurotransmitter which is symptomatic of drug that activates KORs and is addiction.

Understanding conjectured to produce negative the molecular mechanisms emotional states associated with underlying these contributing cocaine dependence and thereby Figure 2. Mild foot-shock stress induced cocaine escalation (F1,13=1.3; P < environmental factors will increases the motivation to 0.001). (* = P ≤ 0.05; ** = P ≤ 0.01; *** = P ≤ 0.001; **** = P ≤ 0.0001). widen general understanding of addiction. administer cocaine (Edwards and drug addiction.

Western blot analysis of brain tissue References: Koob, 2010).

Norbinaltorphimine (norBNI) is a showed that in the nucleus accumbens (NAc) 1. Edwards, Scott, and George F. KOR antagonist which was injected into core, phosphorylation of GluR1 S845 was Koob. “Neurobiology of Dysregulated a subgroup of rats before allowing them significantly decreased in stressed subjects Motivational Systems in Drug Addiction.” extended-access (6 h/day) to cocaine. The in comparison to non-stressed subjects Future Neurology 5.3 (2010): 393-410. control (saline-injected) rats, but not the after one day of cocaine withdrawal (Figure 2. Kalivas, Peter W. “The Glutamate norBNI-injected rats, showed an increase 3).

Homeostasis Hypothesis of Addiction.”

P-GluR1 S845 potentiates AMPA Nature Reviews Neuroscience 10.8 (2009): in cocaine intake, which is indicative of drug dependence (Figure 1). These findings receptor response to glutamate and is 561-72. indicate a functional role of KOR activity on an index of PKA activity, which is an 3. McFarland, Krista, Christopher C. Lapish, upstream component of dynorphin and Peter W. Kalivas. “Prefrontal Glutamate cocaine dependence.

Part 2: Animals that received production. Withdrawal from chronic Release into the Core of the Nucleus intermittent footshocks (0.5mA, 20 min) cocaine administration as well as stress- Accumbens Mediates Cocaine-Induced during cocaine self-administration sessions induced cocaine seeking have been both Reinstatement of Drug-Seeking Behavior.”

(2 h/day) displayed a significant escalation in separately associated with increased synaptic The Journal of Neuroscience 23.8 (2003): cocaine intake (Figure 2) which suggests that glutamate in the NAc core (Kalivas, 2009; 3531-537. stress is a factor that can contributes to drug McFarland et al, 2003).

Figure 1. In comparison to saline-injected rats (n=4), norBNI-injected rats (n=4) had lower cocaine intake (F1,7=6.989; P=0.0203).

(Produced by Tim Whitfield, Ph.D.) sq.ucsd.edu

Figure 3. Comparison of phosphoprotein level variation between footshock subjects, non foot-shock subjects, and cocaine-naive subjects.

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Are Depressive Symptoms and Disease Severity Associated with Immunosenescence in Cardiac Patients?

Tiefu Cheng, Stoyan Dimitrov, Michael Green, Meredith A. Pung, Paul J. Mills, Laura Redwine, and Suzi Hong; Department of Psychiatry

Heart failure (HF) currently affects 5.7 million Americans, increasing by 550,000 each year [1]. Furthermore, a meta-analysis in 2008 shows worse prognosis for HF patients with depressive symptoms than in patients without depression; up to 58% of elderly in-patients have depression [2]. Evidence of the growing epidemic of heart disease in America begs inquiry into the connection between HF and depression.

Increased circulating inflammatory markers, including inflammatory cytokines and differentiated lymphocytes, have been distinctly linked with both depression and HF symptomology [3-5]. In association with the chronicity of HF, patients that are also depressed may potentially exhibit a chronic inflammatory state that leads to immunosenescence. Senescence of the immune system indicates worsening distinction between self and foreign antigens, dysregulating immunoactivation [6]. Could immunosenescence be the biological link between depressive mood and a progressive heart disease? We hypothesized that disease severity in presymptomatic Stage B HF patients and increased depression levels will both show positive associations with cellular senescent markers in cytotoxic T lymphocytes.

Our study included 35 male patients with Stage B HF, indicating asymptomatic cardiac restructuring [7]. HF severity was measured on scales of increasing circulating B-type natriuretic peptide (BNP) levels and decreasing left ventricular ejection fraction (LVEF) [7, 8]. Depression was assessed via the Beck Depression Inventory (BDI-Ia) and further subcategorized into somatic (BDI-S) and cognitive (BDI-C) components of depressive symptoms [9]. Higher BDI scores indicate greater depressive symptoms.

Senescence is characterized by increased terminally and highly differentiated T lymphocytes and decreased central memory and naïve cells. Flow cytometry analysis allowed categorization of circulating peripheral CD3+CD8+ T lymphocytes, which were as follows: CD45RA+/CCR7+ and CD57-/CD28+ (naïve), CD45RA-/ CCR7+ (central memory), CD45RA-/ CCR7- (effector memory), CD45RA+/

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CCR7- (effector memory RA+/terminally differentiated), CD57+/CD28- (terminally differentiated), and CXCR1+/CX3CR1+ (highly activated/differentiated). Multiple regression analyses controlled for covariates, and statistical significance was determined at p<0.05.

Greater naïve cell marker expression was positively associated with patients who had better cardiac function (higher LVEF, beta=0.35). Percentage of central memory cells was positively associated with more depressed individuals (higher total BDI, beta=0.41, and BDI-S scores, beta=0.48).

Percent highly activated/differentiated T lymphocytes (beta=-0.50) were negatively associated with HF severity (increased BNP levels).

Our results suggest that increased naïve T cells, suggesting stronger adaptive immune function, may be associated with better cardiac function, indicated by greater LVEF. However, severity of HF and depression were also associated with lower percent of differentiated, effector CD8+ T cells in this group of patients. In peripheral blood of these patients, smaller percent of differentiated, effector lymphocytes may be explained by potential increased migration of these cells out of circulation and into cardiac tissues that may lead to disease

Figure. Example density plot of flow cytometry analyses for the characterization of peripheral human CD3+CD8+ T lymphocyte subpopulations based on surface marker expression of CCR7 and CD45RA. Quadrant (Q) 1 shows central memory (CM), Q2 naïve, Q3 effector memory (EM), and Q4 effector memory RA+ (EMRA+) T lymphocytes. This sample shows high percentage of naïve cells, characterized by the CCR7+/ CD45RA+ phenotype.

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progression; the hypothesized migration is likely due to higher surface expression of chemokine receptors in highly differentiated lymphocytes. Our current findings require further study of migratory potential of various T lymphocytes, preferably via longitudinal investigations in HF patients.

This line of work aims to further elucidate the mechanism behind depression and heart disease, contributing to development of meaningful interventions.

References:

1.Roger, V.L., A.S. Go, D.M. Lloyd-Jones, E.J. Benjamin, J.D. Berry, W.B. Borden, D.M. Bravata, et al., Heart disease and stroke statistics--2012 update: a report from the American Heart Association. Circulation, 2012.

125(1): p. e2-e220.

2.Pelle, A.J.M., Y.Y. Gidron, B.M. Szabó, and J. Denollet, Psychological Predictors of Prognosis in Chronic Heart Failure. J Card Fail, 2008. 14(4): p. 341-350.

3.Kupper, N., J.W. Widdershoven, and S.S. Pedersen, Cognitive/affective and somatic/affective symptom dimensions of depression are associated with current and future inflammation in heart failure patients. J Affect Disord, 2012. 136(3): p. 567-76.

4.Redwine, L.S., P.H. Wirtz, S. Hong, I. Pandzic, S. Cammarata, J. Tafur, S.M. Carter, et al., A potential shift from adaptive immune activity to nonspecific inflammatory activation associated with higher depression symptoms in chronic heart failure patients.

J Card Fail, 2009. 15(7): p. 607-15.

5.Wirtz, P.H., L.S. Redwine, S. Linke, S. Hong, T. Rutledge, B.H. Greenberg, and P.J. Mills, Circulating levels of soluble intercellular adhesion molecule-1 (sICAM-1) independently predict depressive symptom severity after 12 months in heart failure patients. Brain Behav Immun, 2010. 24(3): p. 366-9.

6.Cannizzo, E.S., C.C. Clement, R. Sahu, C. Follo, and L. Santambrogio, Oxidative stress, inflamm-aging and immunosenescence. J Proteomics, 2011. 74(11): p. 231323.

7.Hunt, S.A., W.T. Abraham, M.H. Chin, A.M. Feldman, G.S. Francis, T.G. Ganiats, M. Jessup, et al., ACC/ AHA 2005 Guideline Update for the Diagnosis and Management of Chronic Heart Failure in the Adult: a report of the American College of Cardiology/ American Heart Association Task Force on Practice Guidelines (Writing Committee to Update the 2001 Guidelines for the Evaluation and Management of Heart Failure): developed in collaboration with the American College of Chest Physicians and the International Society for Heart and Lung Transplantation: endorsed by the Heart Rhythm Society. Circulation, 2005. 112(12): p. e154-235.

8.Eindhoven, J.A., A.E. van den Bosch, P.R. Jansen, E. Boersma, and J.W. Roos-Hesselink, The usefulness of brain natriuretic peptide in complex congenital heart disease: a systematic review. J Am Coll Cardiol, 2012.

60(21): p. 2140-9.

9.Beck, A.T., Depression Inventory1978, Philadelphia: Center for Cognitive Therapy.

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BREVIAS

G protein-coupled receptor (GPCR) Arrays Identify Physiologically Relevant Targets in Pulmonary Artery Smooth Muscle Cells (PASMC): mRNA to Function Daniel McDonald; Pharmacology

Pulmonary arterial hypertension (PAH) is characterized by increased pulmonary vascular resistance, in part due to pulmonary artery smooth muscle cells (PASMC).

Symptoms of PAH include shortness of breath with exercise, difficulty breathing at rest, dizziness and chest pain due to the excessive strain on the heart. The abnormally high pressure in the pulmonary artery leads to right ventricular hypertrophy, which can ultimately lead to heart failure. PAH has a poor prognosis and currently no cure.

Since cyclic AMP (cAMP) decreases the contraction and proliferation of PASMC, G protein-coupled receptors (GPCRs) that couple Gαs and increase the intercellular accumulation of this second messenger, are potential targets for treatment of PAH. We are wondering if previously unrecognized GPCRs, in particular ones that regulate cellular cAMP concentration or that are uniquely expressed, may be novel and innovative targets for treatment of PAH based on their regulation of muscle tone and proliferation in PAH-PASMC. Using a TaqMan® GPCR array and human PASMC we identified >130 GPCRs, at

least 50 of which regulate cAMP formation (Gαs/Gαi-coupled). We then used real-time PCR and GPCR agonists to investigate the relationship between GPCR expression and function by comparing the concentrations of cAMP (determined by radioimmunoassay) that are generated by the highest, intermediate and lower expressed Gαs-coupled GPCRs (determined by cycle threshold [ΔCt]) in PASMC. The balance between cAMP formation by adenylyl cyclases (ACs) and cAMP hydrolysis by phosphodiesterases (PDEs) controls the intracellular level of cAMP and the duration of its signaling.

As examples of the highest, intermediate, and lower expressed Gαs-coupled GPCR, we compared the adenosine 2B receptor (A2BR, ΔCt = 18), the vasoactive intestinal peptide receptor (VIPR1, ΔCt = 19), the prostaglandin I receptor 2 receptor (PGI2R, ΔCt =20), the prostaglandin E receptor 2 receptor (EP2R, ΔCT = 22) and the gastric inhibitory polypeptide receptor (GIPR, ΔCt = 24), respectively, cAMP accumulation (fmol / cell / 10 min treatment with agonist) in response to receptor agonists, CV1808 (A2BR, 1µM, 0.6 fmol / cell / 10 min), VIP

(VIPR1, 1µM, 0.2 fmol / cell / 10 min), epoprostenol (PGI2R, 10µM, 0.4 fmol / cell / 10 min), butaprost (EP2R, 1µM, 0.4 fmol / cell / 10 min) and GIP (GIPR, 1µM, 0.2 fmol / cell / 10 min) correlated with receptor mRNA expression (r2=0.31, meaning partial correlation with receptor expression levels and cAMP accumulation as a result of activation by agonist). Moreover, cAMP dose-dependently decreased PASMC proliferation (see Figure 1). Figure 1 shows that as we activate highly expressed Gαs – coupled GPCRs we get high cAMP levels and thus, lower proliferation. Receptors that show lower expression have lower cAMP levels and thus, higher proliferative effects.

The graph shows a strong correlation (r2=0.90) between cAMP accumulation and its effect on proliferation on PASMC. Overall these data show that the mRNA expression of Gαs-coupled GPCRs predicts functional response in PASMC, implying that this approach can identify GPCRs that may be therapeutic targets for PAH. Funded by NIH and the Doris A. Howell Foundation for Women’s Health Research.

Figure 1. Scatter plot of cAMP accumulation induced by GPCR-selective agonists and its correlation with their ability to decrease PASMC proliferation ([H3] Thymidine, cpm). sq.ucsd.edu

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SENIOR HONORS THESES

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SENIOR HONORS THESES

SENIOR HONORS THESES

The Senior Honors Theses section highlights the achievements of the accomplished undergraduate researchers in the graduating class of 2013.

Photo taken by Yasmeen Elsawaf on the shores of Melbourne, Florida. sq.ucsd.edu

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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 3.7 or higher, have senior standing, and commit to three consecutive quarters of research during their senior year. The goals of the program are to increase one-on-one interaction between students and faculty to encourage more biology majors to pursue independent research.

Each student in the program has a faculty mentor who provides guidance to the student during the year. In spring quarter each year, students in the honors thesis program participate in a poster session that showcases their research, and gives them the opportunity to discuss their research with faculty and their fellow students. Below are abstracts of all the outstanding research projects conducted by undergraduates in the program during the 2012-2013 academic year.

Role for AMPK-Activators in Treatment of Cryopyrin-Associated Periodic Syndromes (CAPS)

Akemi Brown, Eleanor Roosevelt College, Human Biology major, International Studies minor, Class of 2013 PI: Hal Hoffman, M.D., Professor, Department of Pediatrics, University of California San Diego Cryopyrin-associated periodic syndromes (CAPS) are rare inherited autoinflammatory disorders characterized by over activation of the cryopyrin inflammasome which results in a release of the proinflammatory cytokine interleukin-1ß (IL-1ß). Current therapeutic drugs for CAPS are costly, require frequent injections, and are ineffective in some patients; thus, more effective treatments for this study, fluorescence data was collected using the image analysis software CellProfiler. The results of this study were analyzed both independently and compared to data previously collected by less efficient methods. The data collected here strongly agree with the overall relative fluorescence results of previously collected data and in correlation of individual fluorescence values with those previously collected, indicating that the method used in this study can be an efficient alternative to previous methods in finding evidence for the above hypothesis. This study also indicates that the fluorescence data is robust in the face of measurement variability and thus suggests that any similar experiments can implement the methods used in this study for successful data collection.

Novel Karyopherin Regulation of Mitotic Assembly Events

Sarah L. Carmona, Sixth College, Human Biology major, Psychology minor, Class of 2013 PI: Douglass J. Forbes, Ph.D., Division of Biological Sciences, Section of Cell and Developmental Biology In eukaryotic cells, communication between the cytoplasm and the nucleoplasm is mediated by a large, multiprotein selective gateway (120MDa): the Nuclear Pore Complex (NPC). Macromolecules up to 40 kDa can diffuse passively through the nuclear pore, but cargoes greater than 40 kDa must be actively transported by a class of transport receptors known as karyopherins. During interphase, two classes of karyopherins are in charge of import (Importins) or export (Exportins) of nuclear cargoes. Unidirectional transport is made possible by the exclusive presence of the active form of the GTPase Ran (RanGTP) in the nuclear compartment creating a “RanGTP gradient” across the nuclear membrane. However, transporting cargoes through the NPC is not the only role of karyopherins.

Two major Importins - Importin Beta and Transportin - have also been shown to play a role in negatively regulating major mitotic assembly events. While Importin Beta’s regulatory mechanisms on mitosis have been studied, Transportin’s mechanism of action has been much less well studied. Using in vitro nuclear and mitotic reconstitution systems derived from Xenopus egg extract, we have examined the mechanism of binding of nuclear pore assembly targets to Transportin and have identified eight specific partners that bind to Transportin via its NLS cargo binding site during mitosis. In a second line of experiments, we have begun to study the potential action of a different cellular karyopherin on the multiple mitotic assembly events, which include mitotic spindle assembly, nuclear pore assembly, and nuclear membrane assembly. The results of these studies provide a better understanding of the mechanism of mitotic regulation by karyopherins.

Microbial Calcification: A New Method to Combat Microbial Infections at Wound Sites?

Pin-Wen Chen, Earl Warren College, General Biology, Class of 2013 PI: Paul A. Price, Ph.D., Division of Biological Sciences, Department of Molecular Biology The Mineralization by Inhibitor Exclusion (MIE) mechanism is the selective mineralization of collagen using a macromolecular inhibitor of mineral growth that is excluded from that matrix. Previous studies have shown that the cell wall of bacteria is also a target

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SENI0R HONORS THESES for calcification by the MIE mechanism, and that calcification kills the bacteria. Currently, we are able to consistently kill 90% of live Staphylococcus aureus bacteria via calcification at physiological pH and 37°C. Our goals are to improve the lethality of the MIE calcification procedure by determining the optimal conditions at which to conduct the procedure and to determine whether the cell wall of other pathogenic microorganisms is a target of calcification by this method. If successful, our calcification procedure could serve as an alternative treatment to combat pathogens that have become antibiotic resistant.

GCN5-Dependent H3 Histone Acetylation Influences the Fidelity of pre-mRNA Splicing in Yeast

Justin Cheng, Muir College, Biochemistry and Cell Biology, Class of 2013 PI: Tracy L. Johnson, Ph.D., Division of Biological Sciences Pre-messenger RNA contains noncoding sequences that are removed by the process of splicing, which is necessary for proper gene expression. In recent years, splicing has been shown to occur co-transcriptionally while RNA polymerase is actively engaged with the template. Chromatin modifications such as acetylation are an essential part of gene regulation. Recent studies suggest that histone acetylation dynamics are closely tied to spliceosome dynamics. Specifically, H3 histone acetylation by Gcn5 is necessary for proper splicing. Gcn5 is a histone acetyltransferase that has been found to genetically interact with the Msl1 and Lea1 components of the U2 snRNP to allow recruitment of the U2 snRNP to the intron branchpoint. We hypothesize that Gcn5 may be necessary for ensuring splicing fidelity at the intron branchpoint. Maximizing specificity of splicing machinery is important to prevent insertions, deletions, and frame shifts in mRNA. This study aims to better understand the role of Gcn5 in ensuring fidelity in yeast splicing.

The Physiological Role of Piezo1 in Mechanotransduction

Brian Chow, Thurgood Marshall, General Biology, Class of 2013 PI: Ardem Patapoutian, Ph.D, The Scripps Research Institute, Molecular and Cellular Neuroscience Other Advisors: Michael David, Ph.D, UC San Diego, Division of Biology, Molecular Biology Mechanotransduction, the conversion of mechanical forces into cellular signals, is essential for not only the senses of hearing, touch, and pain but also nonsensory physiology, including cardiovascular regulation, respiratory, and cell volume regulation. Although mechanotransduction is involved many essential physiology, the molecular identities of the mechanotransducers in vertebrate system has been elusive. Recently, Piezo1 and Piezo2 were identified as mechanically-activated ion channels and promising candidates for the mechanosensors in mammals. The Piezo family are evolutionary conserved throughout organisms. Here, we examine the physiological role of Piezo1 in mechanotransduction using mouse genetics. Piezo1 constitutive knockout mice are embryonically lethal. To circumvent the lethality and study the pleiotropic consequences in loss of Piezo1 function, we use the inducible, tamoxifen-activated conditional knockout (CKO) Cre/LoxP system. ROSA26 CreErt2, floxed Piezo1 mice was treated with tamoxifen to activate the ubiquitouslyexpressed Cre to excise critical exons of Piezo1, theoretically resulting in null Piezo1 alleles in all cells. QPCR analysis shows that there is indeed a loss of Piezo1 transcripts in various tissues after tamoxifen treatment. Calcium imaging on primary cell cultures isolated from the inducible CKO mice show there is a loss of Piezo1 function as observed by the decrease in calcium influx in the presence of the Piezo1 agonist. Preliminary metabolic behavior analysis reveals that tamoxifen-treated inducible CKO mice have decreased motor activity and increase in feeding. These results suggest that the ROS26 CreErt2 mice are useful genetic tools to dissect the physiological role of Piezo1 in mechantransduction.

Research Title: Assessing the Effect of PTEN/Nogo Deletions on Axon Regeneration and Sprouting After Spinal Cord Injury in Mice

Omeed Ghassemi, Warren College, Human Biology major, Psychology minor, Class of 2013 PI: Binhai Zheng, Ph.D., Department of Neuroscience Spinal cord injury (SCI) is mainly characterized by damage to axonal tracts in the central nervous system (CNS), which often leads to a loss of locomotor function and permanent paralysis from the site of injury downward. The CNS, unlike the peripheral nervous system, does not show regeneration after injury. This lack of regeneration is believed to be the result of complex neuronal extrinsic and intrinsic mechanisms. Nogo, a myelin-derived axon growth inhibitor (extrinsic factor), has been previously shown to inhibit axon growth in vitro, and the deletion of Nogo has been shown to promote axon sprouting (growth of uninjured axons) in the mice spinal cord. PTEN is a neuronal intrinsic molecule that negatively regulates the mTOR pathway. It has been demonstrated that deletion of PTEN in cortical neurons leads to the upregulation of the mTOR pathway and promotes significant sprouting and regeneration of corticospinal axons in mice. We examined the possibility that the removal of these two inhibitors of axon regeneration, Nogo and PTEN, in the CNS could lead to a further increase of axon regeneration and ultimately to functional recovery after SCI in mice.

In order to examine axon regeneration in the corticospinal tract (CST), we used the dorsal hemisection injury model; whereas for determining the amount of sprouting in the CST, the pyramidotomy injury model was used. The behavioral assays used to quantify the amount of functional improvement after dorsal hemisection were the ladder rung, open field test, catwalk, and rotarod. The behavioral assays used to quantify the amount of the functional recovery after pyramidotomy were the tape removal, catwalk, rearing, and ladder rung. Apart from functional improvement, we also assessed anatomical axonal growth by using a specific neuronal tracer to see if we sq.ucsd.edu

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SENI0R HONORS THESES could find correlations in functional recovery and anatomical axonal growth among the different genotypes. A correlation between this data would lead us to conclude whether functional recovery was a result of axon regeneration or axon sprouting.

Effects of Streptolysin O in Drosophila Development and Roles of HIF1 in Toxin-Mediated Apoptosis

Aaron Louie, Earl Warren College, Microbiology major, Global Health minor, Class of 2013 PI: Victor Nizet, MD, Department of Pediatrics, School of Medicine Other advisors: Annabel Guichard, Ethan Bier Group A Streptococcus (GAS) is a leading human bacterial pathogen responsible for a wide spectrum of local and systemic infections. Our laboratory has shown that the potent GAS pore-forming cytolysin called streptolysin O (SLO) accelerates apoptosis of host macrophages and neutrophils. However, the mechanisms involved in mediating these effects of SLO on host tissues are still incompletely understood. The fruitfly model (Drosophila melanogaster) offers a powerful genetic system for dissecting potential signal pathways and developmental consequences of endogenous SLO in the host. Using germline transformation and organ specific drivers we are engineering expression of SLO in different Drosophila tissues during development, discovering that expression of SLO in the fly midgut epithelium results in a high frequency of lethality. In contrast, SLO expression in other epithelial cell types does not seem to have much effect suggesting that gut epithelial cells are particularly susceptible to the action of this toxin. Morphological analysis of the intestinal tissues of SLO-expressing flies and controls is ongoing. In parallel studies, we are examining potential approaches to limit SLO damage to host cells, by exploring the role of a transcriptional activator hypoxia-inducible factor-1 (HIF-1) that serves as a master regulator of the inflammatory and innate immune function of white blood cells. Genetic and pharmacological approaches to modulate HIF-1 are being analyzed to define their contribution to the apoptosis phenotype in response to the bacterial pore-forming toxin. Since GAS can potentially produce severe, life-threatening infections, even in previously healthy individuals, our findings may contribute to our understanding on the mechanisms through which bacterial virulence factors like SLO interact with the multifaceted constituents of the host’s innate immunity.

Kainate Impairs Pattern Separation and Affects Dentate Gyrus Function in Long Evans Rats

Anelah McGinness, Revelle College, Physiology and Neuroscience major, Spanish Literature minor, Class of 2013 PI: Jill Leutgeb Ph.D., Division of Biological Sciences, Section of Neurobiology The hippocampus is the brain region responsible for encoding episodic memory (the “where, what, and when” of memories). Within the hippocampus, the dentate gyrus is the sub-region that specifically encodes pattern separation – the ability to distinguish or “separate” very similar memories from one another. Previous research has shown that during epilepsy, one finds neuronal reorganization in the dentate gyrus that may impair pattern separation in epileptic patients. In this set of experiments, we attempt to induce epilepsy and dentate gyrus reorganization through subcutaneous injections of kainite in male Long Evans rats until they presented class IV or class V seizures for one hour. Rats were subsequently monitored for seizures in the months after the initial induction, and no class IV or class V seizures were recorded in most rats. Control and induced rats up to 6 months of age were trained on an 8 arm radial arm maze for up to 2 weeks in a pattern separation task. The task required rats to remember the location of a food reward and choose the correct arm when presented with a choice between adjacent, very similar arms. Rats induced with kainite injections showed a significant impairment compared to control rats. Histological analysis was then performed to measure corresponding neuronal reorganization in the groups and compared to the results of the behavior.

CellProfiler as an Efficient Means of Collecting Data Related to the Study of Bacterial Aging

Rohan Mehta, Revelle College, General Biology/Mathematics, Class of 2013 PI: Lin Chao, PhD, Division of Biological Sciences, Section of Ecology, Behavior, and Evolution Current research on aging processes in E. coli has led to the hypothesis that mother cells partition the non-genetic damage they acquire over their lifetime unequally among their daughter cells, with the new pole daughter inheriting less damage than the old pole daughter.

This asymmetric partitioning is thought to improve the fitness of certain bacterial lineages by shifting the damage load elsewhere. An attempt to collect data on this partitioning process has been made in which GFP production is induced in E. coli and followed through the growth of a colony. The relative amounts of fluorescence between new and old pole daughter cells in a colony should provide information to support or reject the above hypothesis by reflecting the broader partitioning process. In this study, fluorescence data was collected using the image analysis software CellProfiler. The results of this study were analyzed both in themselves and compared to data previously collected by less efficient methods. The data collected here strongly agree with the overall relative fluorescence results of previously collected data and in correlation of individual fluorescence values with those previously collected, indicating that the method used in this study can be an efficient alternative to previous methods in finding evidence for the above hypothesis.

This study also indicates that the fluorescence data is robust in the face of measurement variability and thus suggests that any similar experiment can implement the method used in this study for successful data collection.

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SENI0R HONORS THESES Stretch Response of microRNA-148a: Modulation of Inflammatory and Calcification Pathways

Vishal Patel, Warren College, Human Biology major, Economics minor, Class of 2013 PI: Vishal Nigam, MD., Assistant Professor, Department of Pediatrics Aortic valve calcification is the third leading cause of adult heart disease and the most common form of acquired valvular disease in developed countries. However, the molecular mechanisms leading to calcification are poorly understood. In this study, we looked at the role that increased biomechanical strain plays in inflammatory and calcification pathways, specifically looking at the role of stretchresponsive microRNAs (miRNA). Using miRNA target prediction programs, we identified miRNA-148a as a regulator of IKK-ß, a known activator of inflammation. In 14% cyclic stretched AVICs, we found miRNA-148a to be down regulated while subsequently finding IKK-ß to be up regulated. AVICs transfected with a miRNA-148a mimic also showed decreased expression of IKK-ß and downstream interleukins (ILs) and matrix metalloproteinases (MMPs). Finally, transfection with a miRNA-148a inhibitor showed increased expression of IKK-ß, ILs, and MMPs. These findings suggest that increased stretch down-regulates miRNA-148a in AVICs, leading to increased expression of IKK-ß, which activates inflammatory pathways.

The Role of Tmem2 During Muscle Morphogenesis

Jenny Phan, Revelle College, Biochemisty & Cell Biology, Class 2013 PI: Deborah Yelon, Ph.D., Department of Biology, Genetics The transmembrane protein, Tmem2, has been shown to play a critical role during heart development in zebrafish. Mutant embryos lacking tmem2 exhibit severe atrioventricular valve defects as well as defects in cardiac contractility. Furthermore, maternal-zygotic tmem2 mutants exhibit additional somite defects, leading us to believe that Tmem2 plays a significant role not just in the development of cardiac muscle but in the development of skeletal muscle as well. Though muscular dystrophies often accompany cardiac defects, not much is known about the link between the two. We therefore chose to investigate the role of Tmem2 during muscle formation in further detail. By utilizing immunofluorescence techniques to visualize muscle fibers as well as different components of the extracellular matrix (laminin and fibronectin) that link the fibers to the myotendinous junction, we were able to visualize the influence of Tmem2 on the degree of extracellular matrix deposition and muscle fiber organization. Analysis of somite angles, length, and organization show that that Tmem2 does not play a significant role in myogenesis during the early stages of somite formation of zygotic mutants.

However, it may be necessary for muscle development during later stages of muscle fiber elongation and attachment. In tmem2 mutants, laminin deposition is altered and muscle fibers are less organized, exhibiting bent or wavy structures. Future extensions of these studies have the potential to reveal a significant mechanistic link between cardiac and skeletal muscle.

Probiotics Can Normalize the Gut-Brain Axis in Immunodeficient Mice

Carli Jordan Smith, Eleanor Roosevelt College, Physiology & Neuroscience, Class of 2013 PI: Dr. Kim Barrett, PhD, Department of Medicine, Division of Gastroenterology Other Advisors: Dr. Melanie Gareau, PhD, Department of Medicine, Division of Gastroenterology The brain-gut axis is increasingly recognized as an important regulator of intestinal physiology. Exposure to psychological stress causes activation of the hypothalamus-pituitary-adrenal (HPA)-axis and is linked to altered gut barrier function, intestinal microbiota, and changes in behavior. The primary aim of this study was to determine if the effects of psychological stress on intestinal physiology and behavior, including anxiety and memory, are mediated by the adaptive immune system. Furthermore, we wanted to determine whether treatment with probiotics could normalize these effects. The impact of psychological stress was assessed in adult Rag1 knockout mice (Rag1-/-) compared to wild-type (WT) C57BL/6 controls. Rag1-/- mice lack mature B and T cells, thus their adaptive immune system is compromised due to a lack of plasticity. Behavior was determined in Rag1-/- mice ± exposure to 1h of water avoidance stress (WAS) using the novel object and light/dark box tests. A subset of mice was treated with either probiotics (109 colony forming units [CFU]) or placebo from weaning (4 weeks) until sacrifice (7-8 weeks of age) via the drinking water. Corticosterone levels were determined by enzyme immunoassay (EIA). We demonstrated that behavior was altered in Rag1-/- mice compared to WT controls. Light/dark box testing revealed reduced exploratory behavior coupled with anxiety-like behavior in Rag1-/- mice, compared to WT controls. Nonspatial memory was also reduced, as demonstrated by the novel object test. Although WAS impaired behavioral measures in WT mice, it did not cause further dysfunction in Rag1-/-mice. Elevated serum corticosterone levels, indicative of activation of the HPA-axis, were observed at baseline in Rag1-/- mice and were not further enhanced by WAS. In addition, our group has shown that Rag1-/-mice displayed altered colonic physiology. Treatment of Rag1-/-mice with probiotics from weaning was accompanied by normalization of both behavior (non-spatial memory) and colonic secretory state compared to placebo-treated controls. In conclusion, immunodeficient mice display altered baseline behaviors, accompanied by an overactive HPA-axis and increased intestinal secretory state. Both the local (intestinal) and central (behavioral) changes were normalized by treatment with probiotics suggesting an overall benefit in health conferred by changes in the microbiota, independently of lymphocytes. Taken together, these findings indicate a role for T and B cells in maintaining intestinal and brain health in mice and show that the adaptive immune system mediates signaling between the gut and the central nervous system. sq.ucsd.edu

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SENI0R HONORS THESES Attenuation of Mitogenic EGFR Signaling by Phosphorylation of GIV, a Non-Receptor GEF for Heterotrimeric G-Protein

Andrew To, Warren College, Biochemistry and Cell Biology major, Music minor, Class of 2013 PI: Marilyn G. Farquhar, Ph.D., Department of Cellular and Molecular Medicine and Pathology Changes in signaling via epidermal growth factor receptors (EGFR) are some of the many aberrations characteristic of cancer cells.

EGFR signaling can be fine-tuned by the Ga-interacting vesicle-associated protein (GIV/Girdin), a multidomain molecular rheostat.

GIV modifies EGFR signal transduction pathways via interactions with heterotrimeric G proteins by assembling a complex that can lead to prolongation of EGFR signaling from the plasma membrane, resulting in amplification of migratory signaling. Trafficking of activated EGFR through early endosomes is regulated by interactions between the heterotrimeric G protein Gas and GIV. Disruption of the GIV/Gas complex by knockdown of Gas or overexpression of a GIV (F1685A) mutant defective in G-protein binding led to the accumulation of EGFR on early endosomes. This resulted in the amplification of mitogenic signaling via the ERK1/2 pathway and increased cell proliferation. Recent work has provided evidence that phosphorylation of GIV’s GEF domain altered its interaction with G-proteins. Specifically, phosphorylation of GIV at S1689 reduced its interaction with Gai3 decreasing motogenic AKT signaling while increasing mitogenic ERK signaling pathways. We have also identified another residue near GIV’s GEF domain that alters interaction with Gas upon phosphorylation. In vitro assays showed that a phosphomimetic mutation of a specific residue, increased interaction between GIV and Gas. Based on our previous findings, we hypothesized that increased binding of GIV to Gas may cause accelerated trafficking of EGFR through early endosomes, thereby attenuating mitogenic signaling through the ERK pathway. We investigated this hypothesis by creating stable HeLa cell lines expressing full-length GIV proteins bearing phosphomimetic mutations in its GEF domain. We established the kinetics of EGFR signaling by analyzing activation of several downstream kinases (ERK1/2 and AKT) over time. Preliminary results indicate that phosphorylation of GIV at a specific residue reduces motogenic signaling (AKT) and accelerates mitogenic signaling compared to cells expressing wild type GIV. These findings suggest that phosphorylation at this site may specifically attenuate cell proliferation induced by EGF. Future experiments are aimed at analyzing the cell cycle profile of the stable HeLa cell lines to establish that phosphorylation of GIV at this site results in reduced cell proliferation. The results of this study indicate that GIV can be phosphorylated at different residues that regulate G-protein binding to modify downstream signaling of EGFR. We hope that these findings may ultimately be useful in the design of therapeutic agents capable of modifying the abnormal cell growth that occurs in various cancers.

The Role of BMP Signaling in the Development of the Zebrafish Inflow Tract

Tina Vajdi, Marshall College, Biochemistry & Cell Biology Major, Class of 2013 PI: Deborah Yelon, Ph.D., Division of Biology, Section of Cell and Developmental Biology The cardiac inflow tract plays an important role in mediating cardiac function because it serves as a passageway for blood to enter the heart and acts as the heart’s pacemaker. Very little is known about the specification and differentiation of inflow tract cells; therefore, understanding the genetic pathways that regulate inflow tract development will be valuable for our comprehension of the causes of certain types of congenital heart disease. One pathway of interest is the bone morphogenetic protein (BMP) signal transduction pathway. Previous work has shown that zebrafish mutations that inhibit BMP signal transduction cause a reduction of cardiomyocyte number in the atrial chamber of the heart. By extending these studies, we have investigated whether inhibition of BMP signaling also affects the development of the inflow tract. Our findings show that there are fewer inflow tract cells when BMP signaling is reduced in zebrafish between 24 to 48 hours post fertilization. Therefore, BMP signaling is required to facilitate the specification and/ or differentiation of inflow tract cells.

Using Next-Generation Mapping Methods to Identify Genes that Mediate Responses to Water Limitation in Plants

Eric Yang, Muir College, Biochemistry & Cell Biology, Class of 2013 PI: Dr. Julian Schroeder, Distinguished Professor, Division of Biological Sciences, Section of Cell & Developmental Biology Plants respond to water limitation in a number of different ways. However, the mechanism(s) by which plants sense the initial osmotic stress signal remain unknown. The Schroeder lab has identified several Arabidopsis mutant lines displaying abnormal osmotic stressinduced calcium responses. Additionally, these mutants exhibit other osmotic stress-related phenotypes. Identification of the precise mutations causing these phenotypes could reveal genes necessary to sense water limitation. The rapid growth of sequencing technology now enables us to use whole-genome sequencing to map mutations. Previously, I have established an analysis pipeline for identification of causative mutations using whole-genome sequencing, which I verified using simulated and previously published data sets. My simulated data allowed me to determine the optimal experimental conditions necessary to yield positive results using this analysis method. Experimental data gathered from candidate calcium response mutants was determined as suboptimal, leading us to begin collection of data from osmotic root skew mutants to more effectively identify a potential osmotic sensor in Arabidopsis.

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SENI0R HONORS THESES Involvement of the Circadian Clock Gene Rev-Erb in the Protective Effect of Time Restricted Feeding Against Metabolic Disease

An Qi Yao, Revelle College, Human Biology major, Studio Art minor, Class of 2013 PI: Satchidananda Panda, Ph.D., Regulatory Biology Laboratory, Salk Institute For Biological Studies From a previous study, it was discovered that mice placed on a time restricted feeding schedule were protected against obesity and metabolic diseases. Two groups of mice were fed the same high fat content food containing 60% fat. One group was given unlimited food access 24 hours a day (ad libitum), while the other group was only allowed access to food for 8 to 9 hours per day (time restricted).

Despite the difference in access time, the two groups consumed the same amount of calories. However, even though the total food consumption for the two groups was isocaloric, only the ad libitum group became obese, as expected, while the time restricted feeding group did not, and were additionally protected against developing diabetes. The current research addresses the question of whether circadian clock genes are involved in the protective effect of time restricted feeding. In order to test the individual circadian clock genes, mouse models deficient in those particular genes are used. The model used in this project is a Rev-Erb a/ß knock out in which both isoforms of Rev-Erb are knocked out. These animals were then subjected to the same ad libitum versus time restricted feeding paradigm. Through regular monitoring of the animals’ weight and diabetic status as determined by insulin and glucose tolerance tests, it was shown that the Rev-Erb knockout mice placed on time restricted feeding still exhibited the protective effects demonstrated in the original study, and did not become obese or diabetic. These findings show that the Rev-Erb gene is not implicated in the protective effect of time restricted feeding. Further studies of mouse models deficient in other circadian clock genes may help elucidate the involvement of the circadian clock in time restricted feeding protection against metabolic disease. Identification of the specific circadian clock gene(s) involved would help us understand the mechanism behind this effect and provide vital information regarding whether this protective effect, as shown in mice, can exist in other organisms. This potentially includes humans, which if further studied may alter our understanding of how simple changes in consumption timing may protect us from obesity and diabetes.

Activating Honey Bee Immunity Against the Widespread Pathogen, Nosema ceranae

Adam Yen, Revelle College, Human Biology, Class of 2013 PI: James Nieh, PhD, Division of Biological Sciences, Section of Ecology, Behavior, and Evolution Honey bees (Apis mellifera) serve as pollinators for $15-20 billion of agricultural crops each year in the U.S., most importantly almonds, apples, and melons. The recent decline in honey bee health has placed great stress on agricultural production and can be at least partially attributed to infection by the fungal pathogen Nosema ceranae. Previous studies suggested that N. ceranae could only infect adult honey bees. However, by using controlled in vitro rearing conditions, we show that N. ceranae can infect honey bee larvae. In this study, we fed three-day-old honey bee larvae a single dose of 0, 10,000, or 40,000 N. ceranae spores per bee. We recorded mortality of adult bees that were kept in cages, and after their deaths, we counted N. ceranae spores in their midguts. We found significantly elevated spore counts in bees that were fed N. ceranae. Additionally, bees fed 40,000 spores had significantly decreased lifespans compared to uninfected control bees. We have now begun to investigate ways to protect honey bees from N. ceranae infection by activating honey bee immunity. Specifically, this paper focuses on exposing bees to probiotic bacteria, which can activate immune genes and lead to increased abaecin production. We test the hypothesis that probiotic bacteria can increase the ability of adult honey bees to fight off N. ceranae infection.

Telemetry Studies of Caveolin-3 Overexpressing Mice Reveal Characteristics of a Trained Athlete Heart

Judith K. Yu, Muir College, Biochemistry/Cell Biology, Class of 2013 PI: David M. Roth, Ph.D., M.D., Dept. of Anesthesiology UCSD and VASDHS Cardiac myocytes are rich in Caveolin-3 (Cav-3), a scaffolding protein essential for regulating signal transduction pathways and cellular processes. Cav-3 overexpression in the mouse heart (Cav-3 OE) has been shown to be protective against ischemia and heart failure.

However, characterization of the electrocardiogram (ECG) in these mice has yet to be collected. Utilizing telemetry, ECG data from Cav-3 OE and controls were collected. Both the Cav-3 OE and the transgene negative mice were implanted with transmitters, positioned in the abdominal cavity to transmit continuous lead II ECG signals and measure both activity and temperature. The mice were then monitored for three 24-hour light-dark cycles. Diurnal temperature variations were similar between the groups, and peak activity in both groups occurred at approximately 12 am each night. Analysis of the heart rates revealed that Cav-3 OE mice had a lower mean heart rate than control mice (516.1 ± 6.331 bpm vs. 535.8 ± 4.178 bpm; P = 0.0105, respectively). PR intervals were significantly prolonged for the Cav-3 OE (Cav-3 OE: 38.08 ± 0.16 msec, vs. Control: 34.61 ± 0.083 msec; P < 0.0001), and QRS complexes were slightly elongated for the Cav-3 OE as well (Cav-3 OE: 13.43 ± 0.032 msec vs. Control: 13.32 ± 0.042 msec; P < 0.05).

Interestingly, prolonged QT intervals were not observed in the Cav-3 OE mice (Cav-3 OE 152.8 ± 0.6236 msec vs. Control 170.8 ± 1.072 msec; P < 0.0001). Our data suggest that Cav-3 OE results in bradycardia with prolonged PR and increased QRS durations, which are findings similar to those seen in trained athletes. Cav-3 OE also results in a shortened QT interval, which has been shown to cause cardiac arrhythmias and sudden death. However, our mice are resistant to these arrhythmias suggesting that Cav-3OE may produce the ideal trained athlete heart. sq.ucsd.edu

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INTERVIEW

An Interview with

Nobel Laureate

Dr. Bruce Beutler

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INTERVIEW The Royal Swedish Academy of Sciences gives the Nobel Prize to individuals who, according to Alfred Nobel’s will, “have conferred the greatest benefit on mankind.” Dr. Bruce Beutler, UC San Diego alumnus, was awarded the Nobel Prize in Physiology or Medicine in 2011 for his work in innate immunology. The prize committee stated that the work of Dr. Beutler and his fellow award recipients in discovering the key principles of immune system activation “has opened up new avenues for the development of prevention and therapy against infections, cancer, and inflammatory diseases.”

Dr. Beutler graduated from UC San Diego after two years and a quarter at the age of 18 in 1976. He then attended medical school at the University of Chicago. Dr. Beutler worked at UT Southwestern, Rockefeller University, and back to UT Southwestern where he did his Nobel prize winning work. Dr. Beutler then worked at The Scripps Research Institute in La Jolla for over a decade and moved back to UT Southwestern in 2011. He is currently a regental professor and director of the Center for Genetics of Host Defense at the University of Texas Southwestern Medical Center, and he holds the Raymond and Ellen Willie Distinguished Chair in Cancer Research.

In November of 2012, UC San Diego was honored to welcome Dr. Beutler back to campus for a talk entitled “Reflections from a Nobel Laureate: An Evening with Bruce Beutler ’76.” At a session during this visit where students from the Division of Biological Sciences had the chance to meet Dr. Beutler in person, Dr. Beutler saw copies of Saltman Quarterly and Under the Scope journals and was impressed by how they were put together. Milli Desai had the opportunity to interview Dr. Beutler after his visit to discuss his undergraduate career at UC San Diego and the importance of scientific communication.

Q: How did you complete your undergraduate career at A: No I didn’t, but I did meet him. He was a friend of UCSD in two years? Was this difficult or did you always my father’s. I first saw him before I went to UCSD, when plan on achieving your degree in a short time? my older brother Earl was graduating. There were a lot of faculty and parents milling around together, and I A: I planned on moving quite quickly from the start. I happened to meet him then. I did not take a class with planned on being a him, which I regret. biologist and I planned on going the route of Q: Did the biology medicine to do that. It program at UCSD impact was somewhat difficult the rest of your career in as I had to take a lot of terms of research? classes but I had a lot of Advanced Placement A: In some ways, yes. credit from high school.

In those days there was With a certain amount very little in the way of of stress I was able to molecular biology, the do it. usual way of approaching biological questions was to isolate the proteins that Q: Did you have any favorite courses at UCSD? mediated certain functions. I did a lot of that early on in my career. That was the way I approached tumor necrosis A: I remember taking biochemistry with Dr. Paul Price. factor to have a feeling for what proteins could do— their He was a wonderful teacher. That class influenced me flexibility, their ability to change conformation, how they quite a bit. It’s been about 37 years or so and I have many responded to different conditions in whatever solvent memories of classes. The whole experience was very one placed them in. This was all very important for me pleasant for me. and it did influence me to take certain classes to begin to understand those things.

Q: Did you have a class with Dr. Paul Saltman at UCSD?

“It is unknowable how things will turn out; it is better not to agonize but to just do something.”

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Q: Did you live on campus when you attended UCSD? Were your roommates aware of your interest in biology?

Did your roommates always know they were living with a future Nobel Laureate?

A: I always made a point of being attached to a laboratory.

I did that in order to learn new techniques to try to understand the thinking that went on in certain disciplines.

I was attached to the Lindsley lab for much of the time I was at UCSD, and that was where I learned quite a bit A: I did live on campus for my first year in Argo Hall, about genetics. I think that’s a good idea even now; there facing Revelle Plaza. My roommate was David Kaplan, are many professors who would be very happy to have a and whether he knew I would go on in biology I don’t bright hardworking undergraduate working in their lab. really know. I certainly didn’t make a secret of it, but then It doesn’t matter so much what you concentrate on and one never knows how things will turn out at a relatively in what order at this stage of undergraduate careers. It is young age. unknowable how things will turn out; it is better not to agonize but to just do something.

Q: Did you always set the curve in your classes?

Q: Were there any challenges that you had to overcome A: Oh, no. I would say I was a good student, but not a when you were conducting research? great one. I could have done better in most of my classes if I had gone at a more measured pace. I don’t believe I A: Not many challenges. Things don’t always work, but was at the top of most of my classes, maybe just a few I tend to be patient with experimental research. I don’t of them. usually set out to prove something, but to understand how a system operates, and that takes a lot of time. One Q: Do you have any advice for undergraduates starting to has to be aware of that. conduct research?

Q: A lot of UCSD students aspire to have careers in healthcare professions.

Why is biological research important in those fields?

Dr. Bruce Beutler giving a talk to students and faculty at UCSD.

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A: Things go both ways. Biology has been tremendously important to medicine, it has made medicine and healthcare what it is. Without it we’d have nothing. There is a time within living memory— not within my memory, but within the memory of some— when medicine really had little to offer and doctors were mostly passive witnesses to the progress of disease. It’s no longer that way. Over the span of my own life, the lifespan of the average American has increased by about 30%. All of this is because of science. All of it. And I think people should be aware of that. The converse is also true—medicine helps science.

It certainly helped me to understand biology better. Someone who goes into medicine, who becomes closely familiar with disease and what can go wrong with organisms like humans will understand what has to go right for humans to be healthy and survive. sq.ucsd.edu

Dr. Bruce Beutler taking a picture with students during his visit to UCSD.

This is the reason that medicine has been so effective in elucidating biological concepts. It’s the same principal that I apply in genetics. When I mutagenize mice, for example, I do so in order to disrupt certain biological systems and then to figure out what went wrong.

do have to capture the attention of the reader early on in an article or grant proposal or research proposal. It often helps to very concisely lay out the big picture or question you are approaching in hopes that you achieve that, that you get their attention, that you interest them.

If you don’t do those things, then certainly they are not Q: The Saltman Quarterly program encourages students going to look at your paper the same way as they would if to publish their own research to disseminate scientific they understood the question right from the front. knowledge to UCSD and the greater community. What is the importance of scientific communication in terms of Q: That concludes our short interview, thank you again writing about your work and conveying it to others? for speaking to us!

A: It’s extremely important to be able to communicate effectively, to make people understand what you’re doing, to persuade them that you have the correct idea about how things work in whatever problem you are studying.

In some ways I think that is the single greatest predictor of success—how well one can write, and how well one can communicate orally. It is undoubtedly very important for people to get practice in that early on and to keep exercising it.

A: Thank you very much, it was nice speaking to you.

WRITTEN BY MILLI DESAI. Milli Desai is the Editor-inChief of Saltman Quarterly. She is a Human Biology major from Revelle College and is graduating in 2014.

Q: Do you have any tips about how to start writing about biology in a way that is interesting?

A: That is a very good question. I think everyone has to find his or her own way. In my own perspective, you sq.ucsd.edu

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SALTMAN DEDICATION

f o fe

i L e h t n a g n m i t r l u a t S c l i P Pau

A wise man once surmised, “the university is an organism and an organization which must create an internal environment to optimize three activities: teaching, learning, and service. How trivial these three words appear, yet how profound and complicated it is to carry out those processes effectively… there must be semipermeable membranes that define and limit the environment within the institution.” This man was none other than Dr. Paul Saltman, a figure who left an ever-lasting impact not only on the students and faculty of UC San Diego, but also on the world. Reading through stacks of papers written by Dr. Saltman himself that are available at the Mandeville Special Collections, I was captivated by the sheer genius and wit that Saltman’s voice held in each sentence. The pages were in their

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original form and were thus quite fragile, yet the words resonated with immense power.

Although there were many brilliant quotes to choose from, the quote written above stood out to me as one that encompassed not only Dr. Saltman’s passion for education, but also his love of science.

Clearly, Dr. Saltman did not just talk the talk, but truly walked the walk. Dr. Saltman dedicated his own life to those three things: teaching, learning, and service, and encouraged others to do the same on their own journeys to reach their unique human potential. In 1967 Dr. Saltman joined UC San Diego, taking the school to new heights and allowing it to evolve into the world-renown institution that it is today. Over a period of 32 years, Dr. Saltman took on the role of provost of Revelle College from 1967 to 1972, Vice Chancellor for Academic Affairs from 1972 to 1980, and continuously served as a beloved teacher, mentor, and researcher. As a researcher, Dr. Saltman engaged in the study of nutrition, specifically looking to make sense of the chemistry, biochemistry, and nutritional role of trace metals such as iron, copper, zinc, manganese, and others. Dr. Saltman combined common sense with scientific exploration and believed in the importance of sq.ucsd.edu

SALTMAN DEDICATION

the

amazing ability to inspire generations.

Following in the footsteps of Dr. Saltman, the Saltman Quarterly strives to communicate scientific research to the general public and encourage learning and education. With this being the now 10th edition of the Saltman Quarterly we hope to carry on Dr. Saltman’s legacy and remember a man who saw the value in human interaction and had an incomparable positive impact on the people who knew him. Dr. Saltman’s selfless dedication to humanity will never be forgotten. Dr. Saltman’s legacy has sparked the drive to keep teaching, learning, serving, and engaging with the world around us so that we too can challenge ourselves, inspire others, and effect change.

responsibility of a scientist to communicate science to the public. As a professor and leader, Dr. Saltman encouraged open discussion and showed concern for students’ education and overall well-being. Dr. Saltman was a beacon of light on the UC San Diego campus; whoever came into contact with him including students and teachers alike regarded him as a WRITTEN BY SAMEEHA KHALID. Sameeha Khalid is a role model and friend. With his bubbly personality and Human Biology major from Revelle College. She will be contagious smile, as well as his enthusiasm for learning, graduating in 2014. education, science, communication, and living life to the fullest, Dr. Saltman was a legendary individual with the

Left: Paul Saltman having an informal discussion with his students. Right: Paul Saltman playing basketball. He was an avid sportsman all his life.

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Staff Editor-in-Chief Milli Desai

Technical Editor of Graphics Sabrina Trinh & Vanessa Salcido

Executive Editor Vicky Hwang

Research Design Editor Anastasia Lam

Special Sections Editor Sameeha Khalid

Features Design Editor Shruti Shrivastav

Editor at Large Lawrence Ku

Review Board Managers Joseph Aleshaki & Tina Vajdi

Senior Research Editor Payal Desai

Web Editor Brianna Egan

Junior Research Editor Zac Hodosevich

Communications/Publicity Chair Namrata Vora

Features Editor Kevin Perez

Communications/Publicity Committee Sushmitha Divakar & Goonjan Agarwal

Production Editor Erica Couzens & Kriti Gupta

BS/MS Program Liaison Weston Davini

Senior Technical Editor for Content Amelia Wong Junior Technical Editor for Content Arjun Patel

Faculty Advisory Board

Staff Writers: Areana Park Chris Probert Michaela Go Andrew Lum Mandeep Bajwa

Staff Advisors

Ecology, Behavior, & Evolution David Holway & Heather Henter

Associate Dean for Education Gabriele Wienhausen

Molecular Biology Eric Allen & James Golden

Webmaster, Biology Computer Services Katie Lee

Cell & Developmental Biology James Wilhelm & Gabriele Wienhausen

Undergraduate Advisor, Student Affairs Hermila Torres

Neurobiology Jill Leutgeb

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Review Board Cell/ Developmental Biology: Alan Nguyen Alice Zalan Alyssa Nelson Andre Lum Andrew Lum Benjamin Yang Brianna Egan Camellia Lee Cris Molina Demiana Sidrak Diljot Kahlon Jeffrey Umotoy Jennifer Kuang Jonjei Ku Katherine Binning Murad Khan Myanna Olsen Priya Patkar Ragavi Sethurajan Ruby Kuang Safwanul Haque Sean Huynh Sora Chee Viktoriya Megrabova Yasmin Aghajan Adriana Ochoa Ecology, Behavior, Evolution: Andrew Alvarez Carolyn Kim Florence Lambert Haebih Liew Hannah Youngwirth Jacky Lu Lisa Robison

Misha Yalavarthy Priscilla Vu Richard Loi Rohan Mehta Sierra Donaldson Yaamini Venkataraman Neuroscience: Nicole Zawada Karen Fernandes Dimple Patel Brian Chow David Linderman Nimish Pratha Grace Gu Joseph Stacy Katherine Moran Billy Nguyen Shruti Shrivastav JungYeon Kim Julie Shen Joanna Coker Jasmine Chau Vedang Uttarwar Taruc Alvarez Yasaman Pirahanchi HyungJoo Yoon Shilpa Sharma Sarah Sypris Tricia Ngo Torrey Czech Sora Chee Shauheen Ladjevardi Neil Srinivas Maxwell Ruckstuhl Susie Dang

Presantha Vemu Jennifer Park Saumya Kumar Rannah Dabiri Stephen Howell Molecular Biology: Chirag Krishna Joseph Stacy Jennifer Kuang Lina Tsvirkunova Benjamin Yang Qianxue Lu Alisha Jain Brian Junho Chang Erinn Kuehne Jonathan Chan Christian Garcia Emma Jackson Linda Budd Kathleen McGuire Justin Sun Sher Khan Katherine Melville Sarah Kang Marie Lea Rayo JungYeon Kim Alexandra Chen Alan Du An Qi (Angela) Yao Victoria Cheung James Wang Jillian Freese Nareh Eshgian

Pictured: (bottom to top and left to right) Lawrence Ku, Kriti Gupta, Zac Hodosevich, Sameeha Khalid, Vicky Hwang, Milli Desai, Joe Aleshaki, Kevin Perez, Erica Couzens, Tina Vajdi, Goonjan Agrawal, Hermila Torres, Brianna Egan, Mandeep Bajwa, Dr. Gabriele Wienhausen, Areana Park, Arjun Patel, Amelia Wong, Michaela Go, Andrew Lum, Anastasia Lam sq.ucsd.edu

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