Mr. Aswin Chandrasekaran
Mentor: Dr. Desmond Smith
Title: Identifying genetic circuits in cells
Aswin Chandrasekaran is a third year Molecular, Cell and Developmental Biology student conducting research in Dr. Desmond Smith's laboratory in the Molecular and Medical Pharmacology Department. Aswin is working on a project that is part of a recent trend in systems biology that involves the investigation of a methodical perturbation of biological systems. His project seeks to identify and map regulatory gene circuits in a mammalian system using microarrays. Identifying regulatory genes has vast implications because it allows for the detection of putative control genes in humans by homology. After completion of his bachelor's degree, Aswin plans to attend medical school and become a physician-scientist. He would like to thank Dr. Smith and Chris Park for their mentorship, as well as the Howard Hughes Undergraduate Research Program for fostering his research efforts.
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Mr. Thomas Clarke
Mentor: Dr. Rachelle Crosbie
Title: The Role of Phosphorylation in the Binding of Gas11 to Microtubules
Left to right: Dr. Janine Bekker, Tom Clarke, Dr. Rachelle Crosbie
Tom Clarke, a third year majoring in Molecular, Cell, and Development Biology, is currently conducting research under the guidance of Dr. Rachelle Crosbie. His research focuses on the microtubule binding protein Gas11. Dr. Crosbie’s lab identified a microtubule association domain within Gas11 called the “Gas11 Microtubule Association Domain” (GMAD). Tom is working to delineate the key regulatory features within the GMAD region. His preliminary results demonstrate that the GMAD is phosphorylated and that mutagenesis of predicted phosphorylation sites within the GMAD reduces Gas11 binding to microtubules. Tom is currently working to characterize the binding properties of these phosphorylation mutants in vivo and in vitro. He then plans to test whether these mutants affect cell motility. Additionally, Gas11 functions in the context of a larger protein complex, called the “Dynein Regulatory Complex” (DRC). Dr. Crosbie’s lab has recently designed two methods to biochemically purify the DRC from mouse tissue. It is hypothesized that this complex contains kinases and phosphatases that modify Gas11. Tom proposes to isolate and characterize these Gas11-regulatory proteins. After graduation, Tom plans to continue his education in graduate school.
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Title: Identification and characterization of constitutively active mutants of Schizosacchromyces pombe Rheb
Left to right: Dr. Fuyuhiko Tamanoi, Roman Deniskin, Jun Urano
Roman is a fourth year Physiological Sciences major and is working in the laboratory of Dr. Fuyuhiko Tamanoi under the tutelage of Jun Urano, Ph.D. He is currently working to characterize the protein Rheb. Rheb, or Ras homolog enriched in brain, is a member of the Ras superfamily of GTP-binding proteins. Rheb is highly conserved from yeast to mammalian cells. Rheb is a member of the TOR pathway which regulates protein synthesis as well as cell size. Rheb functions upstream and positively regulates TOR. Interestingly, Rheb is negatively regulated by the Tsc1/Tsc2 complex, which is mutated in Tuberous Sclerosis, a disease characterized by numerous benign tumors. This regulation is thought to be mediated by the GTPase activating protein (GAP) domain of Tsc2. Roman has chosen to further characterize this pathway in the fission yeast, Schizosaccharomyces pombe, where Tsc1/Tsc2, Rheb and Tor are conserved. His goal is to define other proteins and conduct biochemical assays on Rheb through identification of constitutively active mutants of S. pombe Rheb (SpRheb). The screen is based on the expected phenotypes of activated Rheb from TSC null mutants, namely resistance to canavanine (a toxic arginine analog) and thialysine (toxic lysine analog). Preliminary data published in Molecular Microbiology shows mutations in several G-boxes (domains involved in GTP-binding) of Rheb as well as one curious mutation at a residue that is conserved among Rheb proteins but outside the GTP-binding domain. Future biochemical assays will be pursued. Endogenous expression of the gene will also be explored. Upon graduation from UCLA, Roman has aspirations to attend medical school. He thanks all the members of the Tamanoi lab for their guidance and support.
Mr. Byran Harada
Mentor: Dr. James Bowie
Title: Structure and Function of the SAM Domain of Diacylglycerol Kinase δ
Bryan is a third-year biochemistry major, mathematics minor researching under the direction of Dr. James Bowie in the Department of Chemistry and Biochemistry. For his project, Bryan is studying the structure and function of diacylglycerol kinase δ (DGKδ). DGKδ is a member of a family of kinases which convert diacylglycerol to phosphatidic acid. Since both diacylglycerol and phosphatidic acid are important lipid second messengers, the DGK isozymes may play an important role in regulating the signaling pathways which involve these two molecules. DGKδ possesses a sterile alpha motif (SAM) domain, which is a conserved structural motif found in a variety of proteins. Most SAM domains mediate protein-protein interactions, and the SAM domain of DGKδ has been shown to mediate the homo-oligomerization of DGKδ. This oligomerization is thought to be involved in determining the intracellular localization of DGKδ. In order to study the role of this oligomerization, Bryan has developed a novel in vivo fusion-reporter screen to identify monomeric mutants of the SAM domain of DGKδ. Identification of these monomeric mutants will reveal which amino acid residues are critical for oligomer formation as well as provide mutant proteins for further biochemical, biophysical, and structural characterization. Ultimately, Bryan aims to map the oligomeric interface of the DGKδ and solve the structure of the SAM domain of DGKδ, so that he can use this structural information to study the function of DGKδ oligomerization in vivo. This information may give insight into the regulation of DGKδ’s activity and its role in intercellular signaling. After graduating from UCLA, Bryan plans to attend graduate school and pursue a Ph.D. in biochemistry or biophysics.
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Mr. Omid Hariri
Dr. Paul Micevych
A Membrane Estrogen Receptor Mediates Intracellular Calcium Release in Hypothalamic Neurons and Astrocytes
Omid is a fourth year Neurocience major and first year graduate student in the Department of Neurobiology. He is conducting his graduate studies in the laboratory of Dr. Paul Micevych. An essential question in the current field of neurobiology is how sex steroids affect the Central Nervous System (CNS). This question has recently gathered a great attention because of the global actions of estrogen on brain functions such as: neuroprotection, perception, response to pain and the regulation of food intake. The goal of Omid’s project is to understand estrogen induced signaling process in behaviorally relevant opioid circuits.
Membrane associated ERα appear to directly activate mGluR 1a (Boulware. Based on the activation of mGluR 1a by estrogen, receptor stimulates phospholipase C (PLC). Eventually two intracellular messenger, inositol1,4,5-trisphosphate (IP 3) and diacylglycerol (DAG) that are produced when PI 4,5-bisphosphate [PI(4,5)P 2] is hydrolyzed by the activated PLC. IP 3 diffuses through cytosol and release Ca 2+ from endoplasmic reticulum by binding to and opening IP 3-gated Ca 2+-release channels in the endoplasmic reticulum membrane. Diacylglycerol stays in the plasma membrane and together with phosphatidylserine and Ca 2+ help to activate the enzyme protein Kinase C (PKC), leading to phosphorylation of cAMP responsive element (CRE)-binding protein (CREB) and regulation of transcription. Omid’s goal is to define the proximal signaling cascade in NPY cells that initiate this behavioral cascade. Subsequent experiments will determine [Ca 2+] to study the hypothesis responsible for mediating activation of NPY neuron in the arcuate nucleus. Upon obtaining his Master Degree from UCLA, Omid plans to attend medical school. He thanks all the members of the Micevych lab for their guidance and support.
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Mr. Maziyar Kalani
Mentor: Dr. Owen Witte
Title: First Principle Predictions of the Structure of G-Protein Coupled Receptors
Membrane bound G-protein coupled receptors have profound affects on cellular signaling, homeostasis, and regeneration pathways. Although GPCRs comprise only a small segment of the genome, they represent over 55% of all pharmaceutical drug targets. The membrane bound nature of GPCRs has resulted in little high resolution crystal data for this family of proteins; with the exception of the high resolution crystal structure of Bovine Rhodopsin, there are no high resolution X-Ray crystal information available for the other GPCRs. At the current state of the filed, the advent of novel ab initio modeling methods have provided excellent insight into the structure function relationship for this class of receptors.
Utilizing a novel and powerful computational technique, MembStruk, developed at the California Institute of Technology, Maziyar is working on predicting the three-dimensional structure of the newly deorphaned GPR30 receptor, an estrogen-binding GPCR found in the Endoplasmic Reticulum. He will then utilize the structural information to identify the binding site of 17β-Estradiol, Tamoxifen and Faslodex and map critical residues within the binding site involved in differential binding. He also plans to engineer the protein to bind 17α-Estradiol and activate the signaling pathway as a means to validate the structure experimentally .
Maziyar is a fourth year, Biochemistry major here at UCLA. He aspires to begin an MD/PhD program in the upcoming year.
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Ms. Toni Lee
Mentor: Dr. Han Htun
Differential Compartmentalization by Nuclear Localization Sequences
Toni, a fourth year Biochemistry and Anthropology major, is continuing her research under the mentorship of Dr. Han Htun in the Departments of Obstetrics and Gynecology & Molecular and Medical Pharmacology at the David Geffen School of Medicine at UCLA. In the last year, Toni has examined the role of nuclear localization sequences (NLSs) in the differential compartmentalization of an RNA-binding protein within the nucleus and has found that all NLSs tested have a propensity to target the nucleolus. While the NLSs were examined within the context of an RNA-binding protein, which the Htun lab has developed for the purpose of monitoring transcription in living cells, RNA binding is shown to play a minor role in nucleolar localization. Toni is currently in the process of finalizing her analysis of the fluorescence microscopy data and in helping to prepare a manuscript for publication. During this time, Toni continues her laboratory research in addressing the utility of this RNA-binding protein for monitoring a site of transcription. After graduation, she plans to continue research either as environmental researcher or physician scientist.
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Ms. Yara Mikhaeil
Mentor: Dr. V. Reggie Edgerton and Dr. Niranjala Tillakaratne
Title: Spinal AMPA receptors in spinally transected mice after step training .
Yara Mikhaeil is a third year Neuroscience major working with Drs. Reggie Edgerton and Niranjala Tillakaratne examining the biochemical pathways underlying spinal learning. Edgerton lab has previously demonstrated that even in the absence of supraspinal input, mice are able to learn and retain specific motor tasks. Yara is focusing on comparing and contrasting the biochemical pathways that have been established in the hippocampus, to those that may be occurring in the spinal cord. More specifically, Yara examines the changes of AMPA receptors in adult mice whose spinal cord had been completely transected at a mid-thoracic level and had learned to step through a robotic assisted step training regimen. AMPA receptors are fast-acting ionotropic glutamate receptors that consist of a combination of four subunits: GluR1, GluR2, GluR3 and GluR4, and are critically involved in excitatory transmission. GluR1 is the subunit of interest in most research projects due to the fact that it is phosphorylated in two specific sites: ser-831 by CaMKII and PKC and ser-845 by PKA. Yara performs immunohistochemistry on the spinal cord tissue to determine the levels of phosphorylated and non-phosphorylated AMPAR in the muscle specific motoneurons using specific antibodies to these receptors. These changes will reflect both the phosphorylation of pre-existing AMPA receptors as well as the insertion of silent synapses, which is the addition of new AMPA receptors. The findings of this project will provide more insight in the molecular pathways involved in spinal learning. For example, the phosphorylation of serine 845 will tell us that the following pathway is involved in spinal cord learning: Adenyl Cyclase ® cAMP ® Protien Kinase A ® serine 845. The project relates the behavioral learning of motor tasks by spinally transected mice to the learning that occurs in the brain, especially in the hippocampus. In addition, it compares the pathways involved in cumulating long term memories in both the brain and the spinal cord. Eventually, these findings will be beneficial in development of therapeutic treatments to improve stepping in spinal injured patients. In the future, Yara plans to pursue a joint MD/PhD degree and continue her research in the field of neuroscience.
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Ms. Alissa Minkovsky
Mentor: Dr. Christopher Denny
Title: Establishing stable expression of EWS/FLI1 in primary cell lines in the search to find a Ewing's Sarcoma cell of origin
Left to right: Christopher Denny, Alissa Minkovsky, and Gary Potikyan
Ewing’s sarcoma is a poorly lethal pediatric cancer characterized by a chromosomal translocation that results in the juxtaposition of the EWS gene on chromosome 22 with one of five different ETS family transcription factors of chromosome 11, the most common of which is the Fli1 gene. Alissa Minkovsky, a Junior Microbiology, Immunology, and Molecular Genetics Major, is studying this EWS/Fli1 chimeric gene in the lab of Dr. Christopher Denny her first year here at UCLA. She is working, with the guidance of Gary Potikyan, towards attaining stable expression of the EWS/Fli1 protein in murine embryonic stem cells by altering tumor suppression pathways in order to come closer to finding the cell of origin in EFTs. Stable expression of EWS/Fli1 has been achieved in NIH3T3 murine cell lines which already possess numerous mutations in tumor suppression pathway genes but expression has been toxic to most primary cell lines. Strong evidence exists that alteration of the INK4a/ARF network, is necessary for oncogenesis in Ewing’s. The knockdown of p16, a protein that is upstream in the p53 and RB tumor suppressor pathways, will hopefully make expression of EWS/Fli1 stable in mouse embryonic cells so that the cell types that do tolerate expression of EWS/Fli1 when the ES cells are differentiated in an tetracycline-inducible system can be identified. Alissa is studying towards a MD/PhD and a career in biomedical research.
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Ms. Sarin Prakobwanakit
Mentor: Dr. Samson A. Chow
Title: Characterizing the Karyophilic Properties of Lentiviral Integrase in the Mechanism of HIV Nuclear Import
Sarin is a fourth year Molecular, Cell, and Developmental Biology major student who is currently conducting research under the guidance of Dr.
Samson Chow of the Molecular and Medical Pharmacology department. Dr.
Chow?s laboratory investigates various aspects of the multi-faceted step of an HIV infection. Sarin is currently working on a project that aims to better understand the mechanism in which the HIV complex is able to traverse an intact nuclear membrane, a crucial step for viral infection. HIV has very unique characteristics that continue to perplex scientists in the way that it behaves. As a member of the retrovirus family, it must first transcribe its RNA into a DNA form.
It will then form a large nucleoprotein complex called the pre-integration complex (PIC), which is largely responsible for HIV?s karyophilic properties. HIV is uniquely able to enter the nucleus without nuclear membrane disintegration; this indicates that there may be special interactions between certain components of the PIC and the host cell that allows for this passage to occur. Specifically, integrase, a component of the PIC, is a key suspect in this mechanism.
In her project, Sarin is working on mapping the karyophilic property of integrase and identifying other cellular factors that help to mediate the nuclear import of integrase. This project will give a clearer picture of the entire mechanism of HIV nuclear import. Sarin plans to enter a joint MD/PhD program that will allow her to pursue her ambitions for both research and medical practice. Sarin would sincerely like to thank Cora Woodward for her continual guidance and support, as well as the rest of the members of the Chow lab for their continual guidance in her research experience. Additionally, she would like to thank the supporters of the Howard Hughes Undergraduate Research Program for giving her their support during her duration in the program.
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Ms. Zalya Sanchez-Galvan
Mentor: Dr. Michael Sofroniew
Title: Reactive Astrocytosis after CNS insult
Zalya, a third year Neuroscience major student, is currently conducting research under the mentorship of Dr. Michael V. Sofroniew of the Neurobiology department. Dr. Sofroniew's laboratory investigates reactive astrocytosis in response to Central Nervous System (CNS) insult. Glial cells, including astrocytes and microglia are the primary responders to CNS injury. A question that our laboratory is trying to answer is on the signaling mechanisms through which reactive astrocytosis is regulated after CNS injury. Our laboratory has focused on ablation of dividing reactive astrocytes in GFAP-TK transgenic mice; using this model, we have studied the effects of astrocytes death in the immediate vicinity of spinal cord injuries. Another transgenic model consists on the deletion of genes of specific interest in defined populations of cells using Cre-loxP technology, in our case we focus on astrocytes. Zalya will conduct spinal cord crush injury surgeries on transgenic models to study reactive astrocytosis in vivo, and will subsequently conduct behavioral analysis. Understanding the multiple activities of reactive astrocytes may lead to novel therapeutic strategies to improve outcome after a wide variety of CNS insults by promoting certain astrocytes activities, while inhibiting others. Zalya plans to enter a Neurobiology graduate program that will allow her to pursue her scientific interests. Zalya is also an NIH Undergraduate Scholar and will conduct three years of postdoctoral research at NIH.
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Mr. Grant Sanders
Mentor: Dr. Michael Teitell
Title: Bacterial Production of GST-labeled Polynucleotide Phosphorylase (PNPase) for Use in Identifying Novel Interacting Proteins of PNPase by Far Western Analysis
Grant Sanders is a third year student majoring in Microbiology, Immunology, and Molecular Genetics who is conducting research under the guidance of Dr. Michael Teitell in the department of Pathology and Laboratory Medicine. His research focuses on the protein, Polynucleotide Phosphorylase (PNPase), which is a 3’-5’ exoribonuclease localized to the inner mitochondrial membrane. Immunoprecipitation experiments have shown a tight binding between PNPase and the oncoprotein, T Cell Leukemia-1 (TCL1). TCL1 is a 14-kDa protein that has been studied extensively by the Teitell Lab and its overexpression in TCL1 transgenic mice has been shown to result in B cell lymphomas of the germinal center. Mammalian PNPase, however, has only been recently identified and both its cellular function and the role it plays in conjunction with TCL1 remain unclear. Grant’s project will include elucidating the role PNPase plays is cancer formation by identifying any additional binding partners in the cell, something that has been troublesome in the past due to artifacts produced with immunoprecipitation experiments. Grant will perform a Far Western Analysis to identify any binding partners of PNPase in both the mitochondria and cytosol. He is currently optimizing a protocol to purify large quantities of Glutathione S-Transferase (GST) tagged PNPase to be used as a probe in his experiment. Being extremely insoluble, the GST-PNPase probe is difficult to purify yet Grant has successfully altered the protocol to efficiently create substantial amounts of the intact protein. The Far Western Analysis will hopefully shed light onto PNPase’s function in the cell and will help Grant further characterize the protein’s role in B cell lymphoma formation. After graduating from UCLA, Grant plans on attending medical school and continuing biomedical research in an academic setting. Grant would like to sincerely thank Dr. Michael Teitell and Cynthia Balatoni, as well as all of the other members of the Teitell Lab, for their guidance and unwavering support. He would also like to thank the Howard Hughes Undergraduate Research Program for the opportunity they have provided him.
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Ms. Sylvia Vong
Mentor: Dr. Harold G. Martinson
Transcription and 3’ End Processing
Sylvia Vong is a fourth year Biochemistry major, working in the lab of Dr. Harold G. Martinson. The Martinson group is interested in transcription termination and coupled 3'-end processing of eukaryotic pre-mRNAs. Her current project is interested in characterizing an little known alternative function of the cleavage and polyadenylation apparatus. Recent work in the lab has found that polyadenylation can take place at the 3'-end created by severing the RNA tether. Although proper poly(A) cleavage is inhibited by severing this tether, polyadenylation occurs robustly. This phenomenon is called Poly(A) signal-dependent polyadenyation at remote 3'ends (PPR). Previously, she has found constructs with the same poly(A) signal may be opposite in their ability to support PPR. Through mutational analysis, she hopes to identify the sequence dependence of PPR ability. Through these studies, she hopes to better understand PPR in and of itself, as well as its relatiion to proper 3'end cleavage and polyadenylation.
Sylvia is currently applying to graduate school. She plans to pursue a PhD. in neuropharmacology.
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Mr. Kevin Yackle
Mentor: Dr. Utpal Banerjee and Dr. Gerald Call
Title: Characterization of Two Nuclear-Encoded Genes Putatively Involved in Mitochondrial Protein Translation
Currently Kevin is conducting research in genetics to study developmental biology in Drosophila melanogaster under the mentorship of Dr. Utpal Banerjee and Dr. Gerald Call. He is working to characterize two nuclear-encoded genes that are putatively involved in mitochondrial protein translation. These genes were identified through a genetic screen on the 3R chromosome which utilized mitotic recombination technology in order to reveal interesting cell cycle phenotypes caused by lethal mutations. These mutations were mapped and homology searches classified as nuclear encoded genes whose proteins function within the mitochondrial protein synthesis. This mechanism of function within the mitochondria of higher organisms like Drosophila has not been shown thus far. Experiments will be conducted in an in vivo (adult fly) and in vitro system (cell culture). The in vivo research will involve the re-introduction of wild type genes in order to rescue the mutant phenotypes of mitochondrial staining which show respiration and protein phenotypic effects in mutant organisms. The in vitro experiments will use dsRNA to knockout these genes in cell culture. To study mitochondrial function ATP levels will be measured to show respiration and Western analysis will reveal effects on protein translation. Lastly, fluorescent fusion proteins of these genes show localization patterns within the cell. Through these assays these nuclear genes can be characterized as functioning within the mitochondria where they play important roles in protein translation. After graduation Kevin plans on joining an MD/Ph.D program with the goal of becoming a medical scientist. Kevin would like to thank and acknowledge Dr. Utpal Banerjee, Dr. Gerald Call, everyone in his lab, and the Howard Hughes Undergraduate Research Program.
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Ms. Yawei (Jenny) Yang
Mentor: Dr. Stephen Cederbaum
Title: Elucidating the roles of the Arginine and polyamine metabolism on neuronal stem cell proliferation and differentiation using Small Interference RNA
Jenny is a fourth year Molecular, Cell, and Developmental Biology major conducting research under the mentorship of Dr. Stephen Cederbaum at the David Geffen School of Medicine. The focus of the Cederbaum lab has been to characterize the roles of Arginase as the last enzyme in the Urea cycle as well as to study the physiological effects of the enzyme deficiency characterized by the human genetic disease Arginase Deficiency. Jenny’s research focused on elucidating the roles of Arginase in the brain using comparative studies in an Arginase I knockout mouse model. Preliminary studies done last year on primary neuronal stem cells isolated from both wildtype and Arginase I knockout mice have suggested that (1) a loss of arginase I expression affects both proliferation and differentiation of neuronal stem cells in culture and (2) that these arginase I null stem cells actually exhibit an increase in proliferation and differentiation when cultured under appropriate conditions. In order to conduct more comprehensive studies, Jenny created Short Interfering RNAs (siRNAs) which allow for the specific silencing of a target gene by inhibiting translation and inducing targeted RNA degradation within a cell. Several siRNAs targeted against various genes that play a role in arginine metabolism were constructed and verification of the ability of each siRNA to abrogate the targeted genes has been done with both RT-PCR and fluorescent microscopy to reveal a decrease in gene expression. The effects that siRNA perturbation has on proliferation and differentiation will be studied in two cell lines that are suitable models for neuronal stem cells, specifically SH-SY5Y, a human neuroblastoma cell line, and NTera2/D1, a human embryonal carcinoma cell line. Using both siRNA gene knockdown studies as well as differentiation induction studies, Jenny hopes to better understand the role of Arginase on neuronal stem cell proliferation and differentiation. For this work, Jenny received Highest Honors on her senior thesis this past Spring.
Jenny is currently also a Beckman Undergraduate Research Fellow as well as a Departmental Scholar and is currently working towards a joint Bachelors and Masters degree. Upon graduation, Jenny plans on pursuing a joint MD/PhD in order to become a research physician specializing in the field of neurobiology, and she hopes to make strides in the fight against neurodegenerative diseases. She would especially like to thank her mentors, Drs. Cederbaum, Iyer, and Grody, and the rest of the Cederbaum and Grody labs for their mentorship, guidance, and support. She would also like to thank the Arnold and Mabel Beckman Foundation and the Howard Hughes Undergraduate Research Program for their generous support.