Daniel is a senior at the University of Washington majoring in biochemistry and molecular, cellular, and developmental biology, with a minor in chemistry. An internship at the Fred Hutchinson Cancer Research Center during his sophomore year got him interested in research. Daniel joined the Brockerhoff lab in the department of biochemistry during the summer of 2017. His first project involved classifying and quantifying a series of proteins responsible for Calcium transport into the retinal mitochondria. His current project focusses on studying how the mitochondria in the retina respond to circadian rhythm and cellular stress. His research aims to support advancements in understanding blindness associated with mitochondrial damage, like Age-Related Macular Degeneration. Daniel is also an Undergraduate Research Leader who enjoys educating other undergraduates about research. When Daniel is not glued to a computer screen, he usually enjoys hiking, reading books, and playing guitar. After graduation, Daniel plans on taking a gap year, conducting clinical research at the NIH’s National Cancer Institute. He eventually plans on pursuing an MD/PhD degree and researching Leukemia. He would like to thank Dr. Susan Brockerhoff, Rachel Hutto, and the URP for their support. Lastly, he would like to thank Dr. and Mrs. Levinson for their generosity in supporting undergraduate research.
Mentors:
Susan Brockerhoff, Biochemistry; Rachel Hutto, Biochemistry
Project Title:
Determining the Effect of Circadian Rhythm and Cellular Stress on Mitochondrial Biogenesis in Zebrafish Retina
Abstract:
Circadian rhythms, or biological clocks, are molecular changes in cells that occur with daily periodicity, independent of light-dark cycles. Mitochondrial stress from Ca2+ and reactive oxygen species (ROS) can disrupt circadian cycles and cause cellular damage. Retina cells, which have a huge energy demand, especially in darkness, are hypothesized to regulate their mitochondria numbers in response to circadian cycles and cellular stress. My project aims to determine the rates of mitochondrial biogenesis in the retina in response to both the circadian clock and mitochondrial stress. Zebrafish retinas, collected from a light/dark cohort and a dark-only cohort, will be collected at different timepoints throughout the day to determine circadian effects on mitochondrial biogenesis. Additionally, I will compare healthy retinas to those experiencing mitochondrial stress due to Ca2+ overload or inhibition of the electron transport chain. The expression of several genes controlling mitochondrial biogenesis, such as mitochondrial DNA polymerases, promotors, and growth factors, will all be determined via qPCR. I will also use a fluorescent sensor, named MitoTimer, to measure rates of mitochondrial biogenesis. If mitochondrial biogenesis follows circadian rhythm, I expect the light/dark and the dark-only adapted cohorts to have the same expression of mitochondrial biogenesis genes and the same fluorescence patterns of MitoTimer. In the stress models, I expect the mitochondrial biogenesis genes to be downregulated to minimize stress arising from the mitochondria. Knowing how mitochondrial biogenesis is regulated and how it changes in response to chronic stress will help guide future research relating to mitochondrial diseases affecting the retina. The relevance of discovering how retinal mitochondria respond to stress and circadian rhythm exists in applying this research to help prevent or cure blindness associated with mitochondrial damage.
Irene is currently a senior at the University of Washington working on her double degree in Microbiology (B.S.) and Medical Anthropology (B.A.). Since high school, she has been passionate about global health and infectious disease research that helps the world on a global scale. As an undergraduate researcher in Dr. Murphy’s laboratory, Irene is beginning to realize that dream. The Murphy laboratory is focused on developing a novel and effective DNA vaccine for malaria, a disease that claims nearly a half-million young lives each year. Irene’s current research project is to use an “Acute Challenge” model to test Plasmodium yoelli candidate antigens, of unknown protective potential, that are putatively exported or secreted from the parasitophorous vacuole (PV) into the infected host cell cytoplasm. The candidate antigens that prove to be protective will then be assessed for localization in sporozoites and infected hepatocytes and will have their immunogenic MHC class I epitopes defined. The results of her research can then be used to create poly-specific vaccines for malaria. After finishing her undergraduate studies at UW, she plans to attend graduate school and pursue a Ph.D. program in either immunology or pathobiology. Irene is honored to be a Levinson Emerging Scholar and is incredibly grateful to Dr. Sean Murphy and Brad Stone for their support and mentorship. She extends her gratitude to Dr. and Mrs. Levinson for their generous contribution to her research and strong support of undergraduate research.
Mentors:
Sean Murphy, Laboratory Science; Brad Stone, Laboratory Science
Project Title:
The Acute Challenge Model: Assessing Pre-Erythrocytic Plasmodium T Cell Antigens for Malaria Vaccine Development
Abstract:
For many years, concerted efforts to combat malaria through the use of antimalarial drugs, bed nets, and other public health measures led to marked reductions in morbidity and mortality. Unfortunately, progress has stalled. Reductions in malaria have leveled off and even reversed in certain areas (WHO, 2017). As of 2016, there were 216 million cases and 445,000 deaths annually due to Plasmodium infections (WHO, 2017). To regain momentum and accelerate malaria eradication efforts, an effective and durable vaccine is needed. The Murphy Laboratory focuses on developing novel pre-erythrocytic (PE) malaria vaccines that can effectively stop the Plasmodium sporozoite (spz) before the clinically symptomatic blood stage begins. Identification and inclusion of multiple different protective Plasmodium antigens is thought to be crucial to developing a broad immune response and durable protection against this intracellular parasite. To test and define protective antigens, the Laboratory developed an “Acute Challenge” (AC) model in order to sensitively measure T-cell responses that are completely or partially protective. In this model, DNA vaccines encoding Plasmodium yoelii proteins are delivered by gene gun to induce CD8+ T-cell responses in BALB/c mice. At the peak of the immune response, we challenge the mice with luciferase-expressing P. yoelli sporozoites and measure the parasite burden and protection using IVIS imaging. A known protective epitope derived from P. yoelii circumsporozoite protein (CSP) induces a potent and protective response in this system. My project is to utilize the AC model to assess P. yoelli candidate antigens, of unknown protective potential, that are putatively exported or secreted from the parasite-containing vacuole into the host cell cytoplasm. Confirmed protective antigens will then be assessed for their localization and defined T-cell epitopes. The results will be used to create vaccines designed to maximize such responses and target the responding T-cells to the liver.
Andrea is currently a senior at the University of Washington studying chemical engineering. She became interested in biomimetics during the end of freshman year after taking Professor Sarikaya’s Modern Materials Seminar, and joined Professor Sarikaya’s lab the following year. Her interests include how biological principles from nature can be used to build and tune constructs at the molecular scale, and the implications of this for biomedical applications. She is currently investigating the biological mechanisms for mineral formation in nature, specifically, how peptides are able to control the synthesis and crystallography of enamel mineral. Her plan for the future is to continue researching for the biomedical field as a PhD candidate, and to conduct research in the biotechnology industry afterwards. She would like to thank her mentors, Professor Sarikaya and PhD candidate Deniz Yucesoy, for their guidance, as well as Dr. and Mrs. Levinson for their support for her undergraduate research.
Mentor:
Mehmet Sarikaya, Materials Science & Engineering
Project Title:
The Foundation of Biomineralization via Peptide-Ion Interactions
Abstract:
Biological mineralization is the formation of minerals in hard tissues guided by proteins. Unique aspects of these minerals include the control of hierarchical structure at the nanometer scale, unparalleled intricate architectures, and multifunctional properties that have bio-nanotechnology applications. To produce biological hard tissues for replacement therapies in tissue-related diseases, numerous biomineralization strategies have been developed. However, there is currently no in-depth understanding of how proteins regulate the synthesis of these inorganics or the physiological nature of the minerals. Thus, the ability to control mineral formation for biomedical applications is still limited to the use of a few mineral directing proteins. Biomineralization can also be controlled using short peptides domains that are derived from natural proteins known to have a regulatory role in mineralization. Our laboratory has designed peptides derived from protein amelogenin (ADPs) using combinatorial selection and computational design, whose utility in rebuilding hydroxyapatite mineral on tooth surfaces has been demonstrated in a variety of case studies. The aim here is to understand the fundamental mechanisms of biomineralization guided by ADP5 and develop a methodology to form hydroxyapatite with strict control on its growth kinetics and mineral crystallography. The research approach involves the mutation of ADP5 to investigate the effect of charge on mineralization kinetics, mineral morphology, and peptide-mineral binding kinetics. Insight into the correlation between peptide sequence domains and biomineralization outcomes can be used to optimize peptide sequences for hard tissue repair. The developed method has high potential to develop non-invasive oral health care products by restoring mineral loss, which is the root cause of all dental ailments. This research will help bring both clinical and over-the-counter dental products into the market that repair and prevent tooth decay.
Tonio is a senior majoring in Biology (Molecular, Cellular, Developmental) and Environmental Science & Resource Management. He joined the Torii Lab during fall quarter of his junior year. The Torii Lab’s focus on studying the stomatal differentiation pathway fit perfectly in line with Tonio’s interest in understanding plant biological systems at the molecular scale, while applying these concepts and information to ecosystems as a whole. His research involves examining and analyzing the phenotypic effects of manipulating the auxin-dependent developmental pathways via a synthetically engineered, orthogonal auxin-receptor pair in Solanum lycopersicum, or tomatoes. He is optimistic that this study will yield exciting agricultural implications, as well as a tool to study auxin-specific pathways in plant development. After completing his undergraduate education, he hopes to attend graduate school to continue studying plant growth and development and develop mechanisms to more sustainably live within our environment. Outside of academics and research, Tonio seeks to acknowledge and address issues of injustice and inequities within academia and society as a whole. He also enjoys backpacking and basketball. Tonio would like to thank his mentors, Dr. Keiko Torii and Dr. Aarthi Putarjunan for their time, knowledge, and support, as well as the entire Torii lab for their efforts caring for the tomatoes. Lastly, Tonio wants to thank Dr. and Mrs. Levinson for their generosity and support which allows him to focus more time and energy on exciting, important research.
Mentors:
Keiko Torii, Biology; Aarthi Putarjunan, Biology
Project Title:
Extrapolating A Synthetic, Orthogonal Auxin-TIR1 Receptor Pair in Solanum Lycopersicum to Assess Phenotypic Differences
Abstract:
Auxin/IAA is a bioregulatory plant hormone that impacts virtually all aspects of growth and development. Due to its seemingly universal effects, IAA is a prime candidate for agricultural use, but is held back by its unknown involvement in other signaling pathways. The TIR1 protein acts as a receptor for IAA and is active in its degradation, thereby releasing auxin-response factors (ARFs) to reprogram gene expression. Using a synthetically engineered, orthogonal auxin-TIR1 receptor pair, I plan to study and quantify the phenotypic impact of IAA treatment at various developmental stages in Solanum lycopersicum, or tomatoes. Introducing this synthetic, manipulatable auxin-receptor pair to a crop plant species will provide the groundwork for functionally extrapolating this system into other model fruit organisms and provide exciting insights into the developmental process of tomato fruits. My project aims to convey that this engineered auxin-TIR1 receptor pair can be used by biologists and farmers as a mechanism to understand precisely IAA’s involvement at specific cellular development stages, as well as an agricultural tool to control plant growth during precise maturation stages.
Ethan is a senior at the University of Washington majoring in biochemistry with a minor in mathematics. After his freshman year, he began working in Dr. Stan Fields’s lab studying genetics and molecular biology. In his current research, Ethan is investigating the biosynthesis and evolvability of lasso peptide antibiotics and how they respond to genetic mutation. Through this study, he hopes to gain insight on how manipulating the microbial pathways in which these peptides are synthesized allows for the discovery of novel antibiotics and their ability to overcome bacterial resistance. After graduating from UW, Ethan hopes to attend a graduate program in biochemistry studying drugs and medicine. Apart from science, Ethan enjoys skiing, running, hiking, and playing the piano. He would like to thank his mentors Stan Fields and Ben Brandsen for their continued guidance with his work as well as Dr. and Mrs. Levinson for their generous support of undergraduate research at UW.
Mentor:
Stanley Fields, Genome Sciences
Project Title:
Designing Novel Peptide-Based Antibiotics through Analysis of Lasso Peptide Bioacivity
Abstract:
Antibiotic resistance is a growing threat to public health. This resistance, coupled with a dearth of new antibiotics, makes development of new antibiotics of critical importance. Many antibiotics derive from microbial pathways that synthesize complex natural products, and engineering these pathways to produce new antibiotics is an exciting prospect. I aim to use such a strategy to produce variants of the antibacterial lasso peptides Microcin J25, Klebsidin, and Acinetodin in E. coli, named for their distinct lariat knot structure. Each peptide is produced by three biosynthetic enzymes that modify and export a ribosomally-synthesized precursor peptide. I hope to use an assay that relies on cellular growth to (1) measure the activity of many lasso peptide variants in parallel, and to (2) identify lasso peptide variants that overcome a known resistance mutation. When expressed in E. coli, wild type lasso peptides inhibit growth of the host cell. By expressing a library of lasso peptide variants, each within a single cell, and using deep sequencing to count the frequency of each variant before and after selection, I will obtain functional data for thousands of lasso peptides in one experiment. Using this assay, I hope to generate functional scores of each single amino acid mutation in Microcin J25, Klebsidin, and Acinetodin. This data will provide us an understanding of each lasso peptide’s tolerance to mutation and help us characterize the sequence of each peptide based on antibacterial activity. In addition, I hope to study how mutagenesis of the lasso peptides can combat antibiotic resistance. By performing the same selections in an E. coli strain resistant to the wild type lasso peptides, I hope to identify variants that overcome the resistance that wild type peptides cannot. Together, these studies will provide a framework for understanding lasso peptide engineering and identifying novel peptide-based antibiotics.
Jonathan is currently a senior at the University of Washington studying Aquatic and Fishery Sciences with a minor in Marine Biology. He has spent much of his earlier undergraduate years at the UW Friday Harbor Labs developing his fascination with fishes. Since then, he has recently joined Dr. Luke Tornabene’s lab to conduct a comparative study on gobies using museum specimens from several natural history collections. In this study, he will investigate the interaction between form and function with ecology, by using phylogenetics and morphology to identify the traits that have allowed some species of gobies to clean the ectoparasites off other fishes. Jonathan is also a Mary Gates Research Scholar and a former research intern at the Smithsonian National Museum of Natural History. These research opportunities have motivated him to pursue a PhD program after graduation to continue researching functional morphology in an evolutionary context. Jonathan would like to thank Dr. Luke Tornabene for providing mentorship and support throughout this project. Jonathan is also beyond grateful for the Levinson Emerging Scholars Program and would like to thank Dr. and Mrs. Arthur Levinson for allowing him to invest more time into his research
Mentor:
Luke Tornabene, Aquatic and Fishery Sciences
Project Title:
Evolution of Morphological Diversity in Caribbean Cleaner Gobies
Abstract:
Cleaning is a mutually beneficial interaction where a cleaner organism forages by removing the ectoparasites off a client. Among fishes, the cleaning behavior is present in 19 families and many of these groups are well adapted for cleaning. Cleaner gobies (Teleostei: Gobiidae) belong to one of the most speciose and diverse group of fishes, and make up the second largest group of cleaners. Most cleaner gobies belong to a single genus (Elacatinus) and are known as obligates, or mandatory cleaners, while some species of occasional cleaner gobies belong to a closely related genus (Tigrigobius). In general, specialized feeding behaviors like cleaning, can be linked to a specialized feeding morphology that enhances feeding performance. Until now, no studies have ever examined how the morphology of cleaner gobies differs from that of non-cleaners; nor have they determined if all cleaner gobies are converging on a similar morphology. The primary objective of this study is to take a comparative and phylogenetic approach to characterize morphological diversity in cleaner gobies. I will combine micro-CT scanning with clearing and staining to examine museum collection specimens and characterize several biomechanical predictors of feeding performance. The data will be used to compare cleaners and non-cleaners from both genera as well as analyzed in a phylogenetic context to assess patterns in cleaning evolution. This research will provide insight on the functional diversity of cleaner gobies and how they interact with their environment. Determining how similar or distinct different species of cleaners are, will also lay down to the groundwork for understanding the evolutionary processes and mechanisms that produce such specialized behaviors.
Anna is currently a senior in the Department of Bioengineering. She joined the Murry lab her freshman year and developed a strong interest in tissue engineering and regenerative medicine. Her current research focuses on improving stem cell-derived cardiomyocyte therapies to increase regeneration of the heart after myocardial infarction. Her approach focuses on providing a source of oxygen for cells by incorporating photosynthetic plant cell machinery into mammalian cells. After graduation, Anna plans to pursue a Ph.D. in bioengineering with a focus in tissue engineering and later a career in industry to translate research to medicine. Anna would like to thank her mentor, Christine Yoo, for her guidance and Dr. Charles Murry for his continuous support. She would also like to thank Dr. and Mrs. Levinson for their generous support and encouragement of undergraduate research.
Mentor:
Charles Murry, Pathology
Project Title:
Photosynthetic Stromal Cells to Improve the Survival of Human Stem Cell-Derived Cardiomyocytes in Hypoxic Environments
Abstract:
Myocardial infarction following ischemic heart disease is the leading cause of death worldwide. Therapies using direct injection of both human embryonic and human induced pluripotent stem cell-derived cardiomyocytes (hESC-CMs, hiPSC-CMs) into the infarct have been developed to repair damaged tissue, but low survival rates due to a lack of oxygen have prevented optimal remuscularization of myocardium. To increase survival of stem-cell derived cardiomyocytes in the ischemic conditions and lack of vasculature in the infarct, we propose the development of photosynthetic stromal cells to be injected alongside hiPSC-CMs. This will be accomplished by extracting intact chloroplasts, the photosynthetic machinery of plant cells, and inserting them into a stromal cell line through stimulation of an endocytotic pathway. Once the oxygen production level of the generated cells is quantified and optimized in hypoxic conditions under light exposure, they will be co-cultured with hiPSC-CMs in the same hypoxic environment under light exposure to demonstrate increased hiPSC-CM survival. Viability of the hiPSC-CMs will also be determined in co-culture with generated photosynthetic stromal cells in 3D tissue constructs to not only show increased survival in more physiological relevant conditions, but to demonstrate the ability of light to penetrate tissue and induce photosynthesis in the developed cells to generate oxygen for increased hiPSC-CM survival. If we can optimize the uptake of chloroplast into stromal cells and produce sufficient oxygen to improve the survival of injected cardiomyocytes, regeneration of the heart could be increased and improve the survival outcome of individuals who experience myocardial infarctions. These photosynthetic cells could then be applied similarly to other organs and the possibilities for regeneration would be endless.
Katie is currently a senior at the University of Washington majoring in neurobiology and minoring in political science. She has been engaged in undergraduate research for the entirety of her freshman-senior years at the University. She is currently working at Dr. Juliane Gust’s research lab investigating the causes of neurotoxicity in patients that are treated with CAR-T cell cancer therapy. In her free time, Katie enjoys traveling, dancing, and editing for Grey Matters- the undergraduate neuroscience research journal. After graduating this year, Katie hopes to take a gap year before applying to medical school. During this time, she would love to backpack through South America and Europe. Later, she also plans on obtaining a MPH to assist her in conducting clinical research. Katie would like to thank her mentor Dr. Gust, as well as Dr. and Mrs. Levinson for their generous support for her research.
Mentor:
Juliane Gust, Neurology
Project Title:
Investigating Neurotoxicity and Endothelial Activation after Immunotherapy with CAR T-Cell Cancer Treatment
Abstract:
Chimeric antigen receptor (CAR) T-cell therapy is the latest, highly promising treatment option available for those suffering from certain forms of cancer such as lymphoma and leukemia. Recent clinical trials have shown bone marrow remission rates as high as 83%. These engineered cells are able to recognize specific proteins found in tumors, and subsequently activate the body’s innate immune system to help destroy the malignancies. Despite its promise, a percentage of patients who receive this treatment develop a broad range of neurotoxic symptoms. It is still unclear whether the cause of this neurotoxicity is related to the CAR T-cells themselves, or the elevated cytokine release that often follows treatment. My research project will test the hypothesis that endothelial activation of vascular tissue in the brain, which would allow for increased permeability of immune cells through the blood-brain barrier, is contributing to the development of these clinical symptoms. Using a powerful laboratory technique called immunohistochemistry, I will use the antibodies claudin-5, zo-1, and cd31 to fluorescently label endothelial cell components on brain tissue harvested from a developed mouse model with similar neurologic dysfunction. I will then use 3D imaging technology and confocal microscopy skills to visualize and analyze the labeling of transmembrane and gap-junction proteins. If my hypothesis is correct, these proteins will display functional damage near the blood-brain barrier, therefore allowing the central nervous system to be penetrated with foreign cellular matter. Understanding the cause of CAR T-cell related neurotoxicity will be first step in promoting prevention and increasing the effectiveness of this encouraging, new cancer immunotherapy.
Meena Meyyappan is currently a senior at the University of Washington studying neurobiology. In the summer following her sophomore year, Meena participated in the Scan Design Innovations in Pain Research Program under Dr. Jennifer Rabbits. Through this program she had the opportunity to conduct clinical research and evaluate the psychometric properties of the Widespread Pain Index as an indicator of chronic pain in the pediatric population. She also got to develop a post-surgical chronic pain psychosocial intervention alongside her mentor and see the process of intervention development from conception to testing. Throughout her junior year, Meena was funded by the Mary Gates Endowment and was able to co-author a paper about the Widespread Pain Index and travel to the American Pain Society Scientific Summit to present her evaluation of the feasibility of the post-surgical psychosocial intervention. Following graduation, Meena plans to pursue a career in medicine and continue to foster her interest in research throughout her medical career. She would like to extend gratitude to her mentor, Dr. Jennifer Rabbitts, and Dr. and Mrs. Levinson for their generous support of undergraduate research.
Mentor:
Jennifer Rabbitts, Anesthesiology and Pain Medicine
Project Title:
LEAP: An Online Peri-Operative Cognitive-Behavioral Intervention for Adolescents and Parents
Abstract:
Modifiable psychosocial and behavioral factors place youth at risk for severe and persistent pain after major surgery. Opportunity exists prior to surgery to intervene with youth and families to prepare them for surgery including helping to manage distressing cognitions and teaching non-pharmacologic coping strategies, to reduce acute pain and prevent chronic post-surgical pain. This study aims to develop and evaluate a cognitive behavioral program in reducing acute and chronic pain in youth undergoing major surgery. In the pilot study, fourteen children ages 10-18 years (M= 14.5), 71.4% female, scheduled for major spine surgery, and their parents, were enrolled into the study. Enrollment rates were excellent with 88% (14 of 16) of families approached agreeing to participate in the study, with a range of 4 to 16 weeks (M = 6.3, SD = 1.3) until surgery at the time of enrollment. Teens and their parents completed three pre- surgical modules containing surgical preparation and relaxation tips during the month preceding surgery. Participants completed three post-surgical modules during the 6 weeks following surgery containing tips for managing pain and returning back to school and activities. Each module was accessed online and followed by a coaching phone call with a trained study team member to ensure comprehension of module content. All of the participants completed all pre-surgery and post-surgery modules within the prescribed time frame. Parents and teens completed assessments at four time points: 1 month pre-surgery, 1 week pre-surgery, 6 weeks after surgery, 3 months after surgery. The majority of parents and children (79%) completed the study phone calls within the prescribed time period. Preliminary results indicate excellent program feasibility. Further study must be done on patient outcomes to assess efficacy of the program in a larger population.
Alice is a junior at the University of Washington studying Microbiology and Molecular/Cellular/Developmental Biology. Her interests in oncology, immunology, and virology led her to join the Lagunoff Lab in in her freshman year. Since then, she has studied the interplay between Kaposi’s sarcoma-associated herpesvirus (KSHV) and the innate immune system. KSHV alters endothelial cells to cause Kaposi’s sarcoma (KS), the most common tumor in certain parts of Africa and in AIDS patients worldwide. While KSHV infects both blood and lymphatic endothelial cells (BECs and LECs, respectively), LECs become infected more easily and express fewer antiviral genes compared to BECs. Recent experiments have shown that LECs, but not BECs, have a defect in a critical antiviral pathway that is activated during herpesvirus infections. The focus of Alice’s project is to elucidate the mechanism behind this defect and to determine whether the defect plays a role in increasing susceptibility to KSHV infection in LECs. She hopes that results from her experiments will aid in future efforts to develop antiviral treatments. Aside from research, Alice has volunteered at Swedish Medical Center, served on the board of the ASUW Student Health Consortium, and is currently the Editor-in-chief of Capillaries, a journal for students to write and create works of art about their experiences with illness and healing (ranging from personal stories to reflections on public/global health issues). After graduation, she would like to engage in translational cancer research, and in the future, she plans to become a physician and pursue both clinical work and research. Alice is grateful for the mentorship of Dr. Michael Lagunoff and PhD candidate Daniel Vogt and for the generous support of Dr. and Mrs. Levinson.
Mentor:
Michael Lagunoff, Microbiology
Project Title:
Role of STING in Latent and Lytic KSHV Infection of Lymphatic Endothelial Cells
Abstract:
Kaposi’s sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi’s Sarcoma (KS), a highly vascularized tumor composed of cells of endothelial origin. KSHV, while possessing both lytic and latent replication programs, predominantly exists in the latent form during infection. While KSHV infects both blood (BECs) and lymphatic (LECs) endothelial cells, LECs are more susceptible to infection and express fewer antiviral genes during infection compared to BECs. Recent experiments have shown that LECs, but not BECs, have a defect in STING, a critical signaling protein that is activated during herpesvirus infections and results in the production of antiviral signaling molecules such as IFN-β. It remains unknown whether the defect in STING plays a direct role in increasing susceptibility to KSHV infection and if the defect impacts the ability of STING to suppress lytic reactivation in LECs. Accordingly, I propose to construct a constitutively active (CA)-STING and express it in LECs. Because CA-STING results in the continuous induction of IFN-β, I hypothesize that CA-STING-LECS will show decreased susceptibility to the establishment of latency by KSHV and have increased ability to suppress lytic reactivation compared to empty vector-expressing (EV)-LECs. First, I will infect EV-LECs and CA-STING-LECs with KSHV and measure the infection rates 48 hours post infection (hpi). I expect the number of infected cells in the CA-STING-LECs to be decreased relative to the EV-LECs. Next, I will infect EV-LECs and CA-STING-LECs with KSHV, and at 48 hpi, I will induce lytic reactivation in the two cell types and quantify the virus produced. If CA-STING suppresses lytic reactivation in LECs, I expect less virion production from CA-STING-LECs than from EV-LECs. The results from these experiments will further elucidate how KSHV exploits defects in innate-immunity to infect and transform host cells.
Luis is a senior at the University of Washington studying Molecular & Cellular Biology. He is particularly interested in how molecular processes in the brain orchestrate animal behavior, and how behavior can be modulated by changes in an animal’s environment. He is currently working on a project that aims to understand the neural processes involved in entraining circadian rhythms to a cyclic fear stimulus, by looking at the rhythm of expression of clock genes in the SCN master clock, and also in the amygdala, which is responsible for processing fear information. The experiment also serves as a way to understand the mechanisms that interlink anxiety disorders and fear with the circadian system. By understanding the neural circuitry and relationship between fear-coding centers and the circadian system, a basic understanding of how these systems interact with each other can be obtained for further extrapolation across other animal models, including humans. After graduation, his plan is to continue to graduate school to pursue a Ph.D. in Neuroscience, with his goal being to focus as a researcher on neurodegeneration and neurodegenerative diseases. He would like to thank his PI/mentor Dr. Horacio de la Iglesia for his continual support and guidance and Dr. and Mrs. Levinson for supporting his current and future work.
Mentor:
Horacio de la Iglesia, Biology
Project Title:
A Fear-Entrained Oscillator in the Mouse
Abstract:
Most organisms show a roughly 24-h cycle in their physiological and behavioral processes, called circadian rhythms, generated endogenously through the ~24h cyclic expression of genes known as clock genes. Expression of these genes oscillates in the master circadian clock of mammals – the suprachiasmatic nucleus (SCN) – and in nearly every cell of the body. Typically, circadian clocks and the rhythms they sustain are ‘entrained’ by the 24-h light-dark (LD) cycle. Our lab has found that fear can also behave as an entraining factor. We observed that when mice or rats need to leave a safe nesting area to access a foraging area, they forage and feed during the dark phase of the LD cycle. If the foraging area is rendered dangerous with random uncued footshocks during the active dark phase, the animals’ foraging and feeding activity shifts to the light phase. My goal is to understand the neural circuits and molecular processes involved in fear entrainment. I have analyzed the expression of clock genes in animals exposed to nighttime fear and control animals exposed to daytime fear; this allowed me to assess the circadian rhythm of expression of clock genes of interest (Per1 and Bmal1) in the SCN and amygdala, and I found that the amygdala entrains to fear but the SCN does not. As a next step, the goal is to validate my preliminary results and demonstrate that an intact molecular circadian clock is necessary for mice to entrain to cyclic fear, and that Bmal1 is a necessary component, by exposing mice with regionally knocked out expression of Bmal1 in the SCN or amygdala to this fear entrainment paradigm. These experiments serve to unmask the molecular mechanism of fear entrainment and could also help understand the mechanisms linking fear and anxiety disorders to problems with circadian rhythms and sleep.
Vidhi is a junior undergraduate in the department of bioengineering. She joined the Yager lab her freshman year because it brought together her interests in bioengineering, global health, and medicine. Her work has focused on incorporating smartphones as low-cost image readers for various diagnostic assays. Currently, Vidhi is developing a smartphone spectrometer to image paper-based fluorescent assays. Her project addresses the need for device independent imaging that is applicable to various lab-on-chip systems and brings mobile health technologies globally. In her free time, Vidhi enjoys working with her Bioengineers without Borders team and writing articles for The Daily. She thanks her mentors, Dr. Paul Yager and Kamal Shah, for their continued support on her research, as well as Dr. and Mrs. Levinson for their generous gift.
Mentors:
Paul Yager, Bioengineering; Kamal Shah, Bioengineering
Project Title:
Smartphone Spectrometer to Measure Fluorescence from a Paper-Based Membrane
Abstract:
Methicillin-resistant Staphylococcus aureus (S. aureus, MRSA) is a bacterium found on skin and in moist areas that accounts for 60% of S. aureus infections. MRSA is resistant to antibiotics that contain penicillin and cephalosporins, which are commonly used for clinical and hospital patients. In the US, MRSA yearly impacts 126,000 hospitalized patients and kills 5,000. Unfortunately, MRSA is difficult to treat, and diagnosis takes 48-72 hours. To speed-up treatment and prevent MRSA from spreading, there is a need for a rapid and sensitive MRSA screening system that is accessible in diverse healthcare systems. Such a need can be met with paper-based microfluidic devices (i.e. pregnancy tests) that are disposable, portable, and inexpensive diagnostic platforms. The Yager lab designs such devices for point-of-care (POC), or bedside testing, and recently developed “MAD-NAAT,” a sample-to-result nucleic acid amplification test, that detects MRSA DNA with colored-particles, or chromophores (i.e. gold nanoparticles). However, chromophore-based diagnostic tests have poor limits-of-detection, take about 1 hour, and are not quantitative. The latter problems can be addressed with a fluorophore-labeled diagnostic test, but it is imaged with expensive and non-portable readers. To address the previously stated needs and problems, I am working on our lab’s fluorescent “MAD-NAAT” device and developing an inexpensive smartphone spectrometer to measure fluorescence emissions. Smartphones are ubiquitous devices, used by 36% of the world population, and relatively low-cost. The proposed project will be the first demonstration of a smartphone spectrometer that measures fluorescence from biomarkers immobilized on a porous-membrane. The spectrometer will be USB-powered and innovatively apply optical components to gather, disperse, and quantify fluorescence emissions on a highly scattering paper surface, which is a challenging sample to image accurately. This project addresses the need for device-independent imaging platforms, which are applicable for various lab-on-chip systems and bring mobile health technologies globally.
Riley Stockard is a senior majoring in bioengineering with a research interest in synthetic biology. She is also a mathematics minor and completing departmental honors in bioengineering. Riley is developing a peptide binder enrichment screen by engineering yeast mating and sporulation to isolate strong peptide binders in large libraries. This technology aims to provide a cheap, simple, and versatile method of screening protein therapeutics to reduce the time required to bring these drugs to the market. Post-graduation, she hopes to work in industry for some time before returning for a PhD in bioengineering. Riley is honored to be a Levinson Emerging Scholar and would like to express her gratitude to her mentors in the Klavins Lab and to Dr. and Mrs. Levinson for their generosity in encouraging undergraduate research.
Mentor:
Eric Klavins, Electrical Engineering
Project Title:
Engineering S. cerevisiae Mating and Sporulation to Isolate High-Affinity Peptide Binders from Large Libraries
Abstract:
Since 1982, with the introduction of insulin as the first recombinant protein therapeutic, peptide and protein drugs have grown to encompass 10% of the pharmaceutical market and are the fastest expanding class of drugs. To search for strong binders for a therapeutic target, combinatorial peptide libraries of up to billions of different sequences are synthesized and screened against promising targets. Due to the enormous library size, screening for high affinity binders is a laborious and potentially bottlenecking step in developing protein drugs. Current approaches are also limited to screening a library of binders against one target, instead of multiple targets (library-on-library). Here, we aim to develop a peptide binder screen utilizing a simple workflow of repeated mating and sporulation of genetically engineered S. cerevisiae, or baker’s yeast. This technology improves the throughput of established screening methods through a library-on-library format that efficiently isolates high-affinity peptide binding interactions.
Eric is a junior studying bioengineering and applied mathematics at the University of Washington. Ever since he started his undergraduate studies, Eric has been fascinated by the field of biotechnology and has been exploring his interests through research. Specifically, he has worked on kidney organoid differentiation in the Institute for Stem Cell and Regenerative Medicine and capillary engineering in the DeForest Research Group. In his current research, he works with Dr. Cole DeForest to deploy small molecule chemotherapeutics to tumors in vivo. In addition, Eric is involved with programs within Bioengineers Without Borders, Undergraduate Research Program, and the College of Engineering. In the future, Eric hope to make an impact in the world by contributing to the ever-growing field of biotechnology through industry roles and graduate studies. Lastly, he would like to thank his mentor Dr. DeForest as well as the Levinson family for their generous support for undergraduate research.
Mentors:
Cole DeFores, Chemical Engineering; Christopher Arakawa, Pathology
Project Title:
Logic-Degradable Nanogels for Environmentally Triggered Chemotherapeutic Delivery
Abstract:
The delivery of cell and drug-based chemotherapeutics to tumors have presented major challenges in effective cancer treatment. Opportunities to improve current small molecule drug delivery systems exist by increasing overall delivery specificity and decreasing harmful off-target effects. Towards this, we have recently developed a chemical framework for creating user-programmable hydrogels that undergo programmed degradation in response to multiple environmental cues following Boolean logic. Exploiting this methodology, user-specified combinations of environmental inputs (e.g., tumor-presented enzymes, reducing conditions) yield material breakdown and accompanying therapeutic release. To translate these materials for chemotherapeutic delivery in vivo, we aim to establish strategies to formulate these stimuli-sensitive materials into nanogels that circulate in the bloodstream before acting on the desired target site. We will establish techniques to formulate gels on the 50-250 nanometers size scale, one which should enable circulation in the blood and uptake within tumors based on the enhanced permeability and retention effect. We hypothesize that different ultrasonication conditions will allow us to tune nanogel size and dispersity. We will characterize the therapeutic efficacy of these drug-containing gels in vitro through studies involving cervical cancer-derived HeLa cells This system is scalable, translational, and simple to recreate. In the future, these materials can effectively hone and selectively deploy small molecule chemotherapeutics to tumors in patients.