2009-10 Levinson Scholar
Daniel Kashima entered the University of Washington as a music major and one who enjoyed learning about Biology. Over his first two years as an undergraduate, he developed a heightened passion for learning more about the brain and thus applied and also was accepted into the Neurobiology program. The idea of conducting research in this area intrigued him and he feels fortunate to be accepted as an undergraduate researcher in the laboratory of Dr. Ed Rubel in his third year.
Dr. Rubel’s lab attracted Daniel as the research there focuses on auditory neuroscience. This effectively allows Daniel to bridge his two passions. His research uses techniques in molecular biology and physiology to uncover more information on the chicken sound localization circuit. Currently, he is using immunohistochemistry to look for an expression gradient of Eph receptors/ephrins along the midline of the chicken auditory system through development.
In his fifth year, the research component of his education continues to fascinate Daniel. He is currently trying to decide on what post-undergraduate path to pursue. Whether as an M.D., PhD, or both, research will undoubtedly be a major part of his future.
Daniel will graduate from the College Honors Program this Spring with degrees in Neurobiology and Music (classical guitar).
Mentor: Edwin Rubel, Otolaryngology-HNS
Project Title: The Role of Eph/ephrins in Axon Sorting in the Avian Sound Localization Circuit
Abstract: Auditory brain nuclei are organized in a tonotopic manner: the sound frequency to which neurons respond best, progressively and predictably shifts with its anatomical position, preserving the neighbor relationships of the auditory sensory epithelium, the cochlea. In birds, the bilateral nuclei n. magnocellularis (NM) and n. laminaris (NL). both part of a circuit responsible for sound localization, are arranged tonotopically. In each nucleus. neurons in the caudolateral region respond best to low frequencies (LF) and those in the rostromedial region respond best to high frequencies (HF). Our results show that axons projecting from NM to NL, thereby crossing the midline and forming the crossed dorsal cochlear tract (XDCT), are also arranged tonotopically. The mechanism governing this arrangement during development, however, is unknown. Studies done on the visual and olfactory systems implicate an expression gradient of Eph receptors and ephrins to form topographic axonal pathways. Although these molecules have been shown to be expressed in the chick auditory brainstem, no effort has been made to look for a gradient in expression in the XDCT. In order to address this, we will build on our results and look for a molecular gradient in XDCT. We will prepare parasagittal slices of chicken brainstem containing XDCT of 4 different developmental stages and use immunohistochemical methods to look for gradients in EphB2, EphB5, and ephrin-Bl expression. The slices will be imaged using widefield and confocal microscopy. Parasagittal sectioning incorporates the entire tonotopic extent of the XDCT into single slices such that protein expression levels will be compared within single slices. The presence of a gradient in Eph/ephrin expression will provide further insight into a possible mechanism underlying the tonotopic arrangement of the XDCT during development and can serve as a model to study the influence of molecular markers on axon sorting.
Encouraged by her lab’s strong support, Sherry Lee wants to learn more about cancer etiology in order to better understand how it ravages the human body. Thanks to her mentor, Dr. Paul Nghiem, Sherry has realized that a career in medicine is truly gratifying not when a physician simply prescribes readily available drugs, but when a physician can propose and test a more effective therapeutic treatment. Currently, Sherry is investigating the functional relevance and role of microRNA gene regulation in a deadly form of skin cancer called Merkel cell carcinoma. As a Mary Gates Research Scholar and a Levinson Scholar, Sherry hopes to rely on her family’s, her lab’s, and the foundations’ support to achieve beyond their expectations. Overall, her undergraduate research experience has led her on a promising path to pursue a career in translational medicine. She plans to attend a Medical Scientist Training Program after graduation to continue biomedical research.
Mentor: Paul Nghiem, Medicine-Dermatology and Pathology
Project Title: A Merkel cell polyomavirus-encoded microRNA expressed in human Merkel cell carcinomar
Abstract: Merkel cell carcinoma (MCC) is a rare but lethal skin cancer that is associated with immune suppression. People immunosuppressed with AIDS are twenty times more likely to get MCC. Merkel cell polyomavirus (MCPyV), an immunogenic virus, causes more than 70% of MCC. However, 90% of MCC patients have no identifiable immune suppression. In this population, MCC tumors proliferate by escaping the immune system, perhaps with the help of MCPyV. One potential mechanism the virus employs to escape the immune system is through microRNAs (miRNAs), small noncoding RNAs that downregulate the expression of genes that might be recognized by host immune system. During the early stage of my research project, I helped to discover a Merkel polyomavirus-encoded miRNA and to quantify expression of the Merkel-miRNA in MCC tumors. The Merkel-miRNA was expressed in strongly virus positive tumors (n= 11) and not expressed in virus negative tumors (n=4). Also, I detected MCPyV DNA in MCC patients’ blood samples. Because the Merkel-miRNA was predicted to downregulate lymphocyte genes, I hypothesize that it will be present in the blood. For aim 1, I will test blood samples for Merkel miRNA expression. Furthermore, I used a validated in silico algorithm called TargclScan to predict the Merkel-miRNA’s human target genes, which are mostly important for immune functions. For example, the gene PSME3 activates the immunoproteasome, which triggers immune responses. I hypothesize that these target genes will be downregulated in virus positive cells. I will quantify expression of Merkel-miRNA target genes in miRNA-positive cells in aim 2. These two aims are essential components to understanding viral genetic control in MCC pathogenesis.
Specific aims:
- Detect Merkel-miRNA in blood samples from MCC patients
- Quantify gene and protein expressions of Merkel-miRNA target genes in cell line after transfection with Merkel-miRNA
Catherine Louw’s interest in performing scientific research was inspired by a summer session at the Seattle Biomedical Research Institute. This experience soon led her to embark on her laboratory experience in the Baker lab, after being intrigued by the idea of computational protein design. Catherine began working with graduate student Justin Siegel on a novel project, aiming to enhance the efficiency of a computationally designed enzyme that could one day be used in a pathway for carbon dioxide fixation. She has developed a high-throughput screening protocol to grow and assay potential hits in a fraction of the time that it would normally take. Catherine is currently a senior at UW, pursuing a dual degree in Biology and Biochemistry. After graduating she hopes to attend medical school to become a doctor in family medicine were she plans to utilize her experiences with research methodologies in order to better understand and incorporate new medical discoveries into her practice.
Mentor: David Baker, Biochemistry
Project Title: Enhancement of a Computationally Designed Enzyme: A Contribution to the End of Global Warming
Abstract: Global warming is a pressing issue worldwide and finding new ways to control excess carbon dioxide is becoming increasingly important. This study aims to develop a novel biological system which converts carbon dioxide into small sugars. To create this system, a new metabolic pathway is being constructed which employs six naturally-occurring enzymes and one computationally-designed enzyme called Formolase. Formolase is derived from a naturally-occurring enzyme, Benzaldehyde Lyase, which ligates two benzaldehyde molecules together, while the desired reaction of Formolase is to ligate three formaldehyde molecules together to create dihydroxyacetone. A computer program called Rosetta was used to create Formolase, identifying four mutations to make within the active site in order to drastically alter substrate specificity. The enzyme would now catalyze the polymerization of formaldehyde over that of benzaldehyde, and when tested, these mutations altered specificity as predicted. In order to further understand this change in specificity, I determined the contribution of each mutated residue to the activity on formaldehyde. This study illuminated the key mutations necessary to maintain desired activity, as well as mutations which could be changed in the future to improve the activity of the enzyme. Although Formolase demonstrates enhanced activity on formaldehyde, the activity must be increased further before it can be used in the metabolic pathway. Therefore, I am now working on mutating additional residues in order to enhance activity. We also plan to alter the structure of the protein by adding new loops and tails to form more contacts with the formaldehyde ligand, which we hypothesize, will further improve our enzyme. Once sufficiently active, Formolase will complete the novel metabolic pathway and provide an alternative method for converting carbon dioxide into a more useful material. In this way, global warming can be controlled and the amount of carbon dioxide in the atmosphere decreased.
As a student in the Bioengineering department, Alyssa Sheih’s research interests involve addressing current medical problems through new diagnostic methods and therapeutics. Her passion for this area led her to Professor Hong Shen’s lab in the Chemical Engineering department, as a 2008 UW Amgen Scholar. Since then, Alyssa has been studying the design of drug delivery systems and their interactions with the immune system. Currently, she is excited for the opportunity to study cell-based therapy for neurodegenerative disease, with the funding of the Levinson scholarship. After graduation, Alyssa plans on pursuing a Ph.D. degree in Bioengineering with a research focus on immunology.
Mentor: Hong Shen, Chemical Engineering
Project Title: Engineering T Cells for Brain Imaging and Therapeutics
Abstract: According to the Alzheimer’s Association, every 70 seconds, someone in America develops Alzheimer’s disease. Beta-amyloid peptides are believed to be the main cause of this disease, forming plaques between nerve cells and triggering inflammation. By destroying brain cells, Alzheimer’s causes severe problems with memory, thinking, and behavior. Currently, diagnostic capabilities for this disease are limited with physicians utilizing neurological exams and brain imaging. Because early diagnosis of Alzheimer’s disease and clinical evaluation of therapeutics is important, there is a need for an accurate diagnostic tool for Alzheimer’s disease. Here we propose using a contrast agent for brain imaging that can bind to beta-amyloid peptides, allowing for the detection of Alzheimer’s disease. Previously, non-cellular vehicles such as nanoparticles have been used to deliver contrast agents to the brain, by targeting receptors on the blood brain barrier to achieve transport. However, other types of cells also express these receptors, resulting in nonspecific targeting of the brain. These particles can be immunogenic and are vulnerable to clearance by our immune system. Here we propose another solution by using T lymphocytes, which are immune cells capable of crossing the blood brain barrier and localizing near sites of inflammation in the brain. These cells will serve as vehicles modified to carry a contrast agent along with antibodies specific for beta-amyloid peptides. Initially, a proof of concept study will be completed to show that the surface of T cells can be modified to carry a fluorescent label and an antibody for ovalbumin, a more available protein that will serve as a substitute for beta-amyloid peptides in these experiments. This surface modification procedure will be optimized to maximize the binding capability of T cells without compromising its functionality. This project presents one of the first steps towards developing an accurate diagnostic tool for Alzheimer’s disease.
When Mark Shi first came to the University of Washington, he was unsure of what he wanted to study and pursue as a career. That all changed while he was taking introductory biology, where he became fascinated with the intricate, behind-the-scene mechanisms that mediate our daily lives. Mark pursued this interest by joining the neurobiology program, where he had the opportunity to learn about one of the most complex and least understood systems of the human body. After his first year in the program, Mark began working in Dr. Bosma’s developmental neurobiology lab. This research has allowed Mark to expand on his knowledge of the nervous system by investigating new questions in the field. After graduating this year, Mark plans on continuing his research in Dr. Bosma’s lab while applying for medical school. He hopes to further develop his investigative research skills with the support of the Levinson Scholarship as he pursues a career as a physician.
Mentor: Martha Bosma, Biology
Project Title: Activity-Dependent Regulation of 5HT-Postitive Raphe Neurons in Developing Mouse Hindbrain
Abstract: In many regions of the developing nervous system, spontaneous synchronous activity (SSA) plays a role in synaptogenesis, cell positioning, ion channel development, and neuronal migration. SSA has been observed in the hindbrain, the most caudal of the three primary divisions of the developing vertebrate brain that develops into the cerebellum, pons, and medulla. These regions coordinate complex muscular movements, equilibrium, and autonomic functions. In previous studies, our lab has identified an internal pacemaker, which drives SSA during a discrete window of time during embryonic development; this pacemaker region is a cluster of serotonergic (5-HT) neurons located between rhombomeres (r) 2 and 3. We are currently examining the mechanism by which SSA develops in the hindbrain. Using intracellular calcium imaging to visualize electrical events, we have observed SSA primarily propagating rostro-caudally along the midline of hindbrains at embryonic day (E) 11.5. Culturing hindbrain tissue from E10.5 to E11.5 has allowed us to pinpoint requirements for SSA in vitro. We have shown that hindbrains cultured in the presence of ketanserin, a blocker of the 5-HT2 receptor, have SSA with reduced frequency at E11.5, and fewer 5-HT-positive neurons, as shown with immunocytochemistry. Tissues cultured in high concentrations of extracellular potassium or Valproic Acid (VPA), the latter of which is a model for autism in mice, exhibit an increased frequency in SSA. Furthermore, tissues cultured with VPA have SSA with greater amplitude that extends beyond the midline to lateral regions. We therefore postulate in this model of cultured hindbrain that activity itself is able to regulate the appearance and excitability of the cluster of 5HT-positive pacemakers that drive SSA in the hindbrain.