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2019 Research Showcase
UR Abstracts
Abstract Title : Search for Potential Drug Leads against Giardia lamblia using a High-throughput Screening Assay
Abstract : The parasite, Giardia lamblia, is a common protozoan affecting 280 million people annually and giving rise to 500,000 new cases each year. According to the World Health Organization, giardiasis contributes to 846,000 deaths a year for diarrheal diseases. Currently, the main drugs used to treat giardiasis belong to nitroimidazoles. Despite the effectiveness of nitroimidazoles, up to 20% of giardiasis cases fail to be treated due to resistance of G. lamblia to these nitroimidazoles. As a result, my research question will attempt to find new drugs that are effective against G. lamblia. Primary screening of compounds will be performed at a single concentration in triplicate. Compounds showing more than 50% inhibition in the primary screen will be selected for secondary screening to determine EC50. The growth inhibition by compounds will be measured by determining the ATP content of the cells, using a bioluminescence-based cell viability assay. Our findings will indicate which compounds have the most potential to go into further studies to develop a drug lead for G. lamblia.
Abstract Title : The Elasticity of Chromosome Tethers is Dependent on Phosphorylation State
Abstract : The mitotic phase of anaphase is responsible for producing two cells with identical copies of the genome. During this phase, chromosomes are pulled away towards opposite poles of the cell. Tethers are postulated to be elastic connections that exist between the ends of chromosomes on sister chromatids. By investigating the function of these tethers, we may find their implication in the loss or over-expression of genetic information, as well as in unregulated cellular division. In these studies, cells are treated during early anaphase with Calyculin A (Cal A), to prevent dephosphorylation in order to determine how the phosphorylation state of tethers may affect chromosome movement. Moreover, Individual chromosomes are cut by laser microsurgery in order to monitor backward movement of a chromosome during Cal A treatment at anaphase’s most phosphorylated state (early anaphase) and least phosphorylated state (later anaphase). At its least phosphorylated state, the cell loses tether elasticity, but retains tension. Our results show that chromosomes move backward after Cal A treatment and at a faster rate than control cells.
Abstract Title : Discovery and Biosynthetic Studies of Oxazolone Natural Products
Abstract : In the last nine months I have been performing research in the Moore Lab at the Scripps Institution of Oceanography. I have isolated and determined the structure of some unique oxazolone natural products from a metabolic pathway in the marine bacterium Pseudoalteromonas rubra. I will discuss how I discovered and isolated these compounds, my studies of their biosynthetic pathway and the novel enzyme that produces them, and their hypothesized ecological role in the environment.
Abstract Title : Phenotypic Differences in Mouse Tissue Revealed Using Lectin Histochemistry
Abstract : All mammalian cell surfaces are covered with an array of Sialic acids which are monosaccharides with a nine-carbon backbone, and terminate cell-surface glycans. The biosynthesis of sialic acids are dictated by glycosyltransferases (such as ST6Gal-I or ST3Gal-I) which are enzymes that direct sialic acids to specific linkages to the underlying glycan. The presence of different branches terminating in Sias of different organs and the structural differences play roles in  interactions with pathogens or affect embryonic development or cancer progression. Plant derived lectins which are carbohydrate binding proteins have been used to detect cell associated glycoconjugates. The lectins used in this study, SNA, MAH (MAL2), MAL1 were used to perform comparative histochemistry in different species (mouse, human, and chimps) to detect and differentiate structures.  (SNA binds to ⍺2-6 sialic acids, MAH (MAL2) binds to ⍺2-3 O-glycans, and MAL1 binds to ⍺2-3 N-glycans).   Lectin immunohistochemistry was done on frozen sections or paraffin sections and it was noted that in many cases over fixation suppressed accurate detection of the glycoconjugates detected. Since sialic acid epitopes vary from organ to organ across species, identifying these differences in staining and assay methods (frozen versus paraffin) is vital in understanding & properly analyzing lectin histochemistry results.
Abstract Title : Analysis of Intercortical Protein Transport in the Developing Rodent Brain
Abstract : Learning, cognition, and memory are complex properties of the brain; the arrangement, and composition of neural circuits allows for cellular processes to conduct these behaviors. To understand the molecular basis of these behaviors it is important to analyze biochemical properties in vivo; however, the methodologies that we use to study the proteomic composition and spatial arrangement of these circuits must be developed. Therefore, we have established a strategy using succinimide esters of biotin for proteomic analysis of a large population of axonal transported proteins, that we named the “transportome”, in the visual system by tracing neuronal retina ganglion cell projections to the superior colliculus and the lateral geniculate nucleus in vivo. This was done by labeling retinal proteins with intravitreal injections of NHS-biotin, which covalently binds to lysine residues in proteins. The biotinylated proteins are then transported via the optic nerve to various retinal targets in the visual system. The biotinylated transported proteins were purified using DiDBiT, a technique developed by our lab, and identified using tandem mass spectrometric methods. In addition, transported proteins can be visualized using light and electron microscopy techniques using streptavidin conjugated to fluorophores or HRP. This strategy allowed for our lab to identify proteins transported through the visual system. NHS-Biotin could potentially be used to study the population of proteins transported in long range axonal projections other than retinal projections in the optic nerve. To test whether we could expand this strategy to study the axonal transportome in other neuronal circuits in the developing brain, we performed unilateral intracortical injections of NHS-biotin in postnatal day 3 rat pups. Using immunohistochemistry, we visualized the distribution of biotin-labeled proteins in axon commissures, including the corpus callosum and anterior commissure. The biotin labeled axonal bundles of the commissures indicate intracellular transport of biotinylated proteins. We examined the presence of biotinylated protein in ipsilateral and contralateral hemisphere samples, relative to the injection site with western blots, which revealed significant transport of specific biotinylated protein across commissures. Contralateral cortex tissue showed biotinylated proteins indicating that the developed strategy can be applied to investigate long-range axonal projections and proteomic conditions of neural circuits. In a western blot to investigate the presence of biotinylated Tau protein, the assay showed that consistent presence from ispilateral and contralateral corpus callosum samples exist due to the rapid axonal transport within commissures; these data are further supported by samples from the anterior commissure. In summary, we adapted the in vivo NHS-biotin protein labeling technique to study intercortical transportomes in the developing central nervous system. By tagging proteins with NHS-Biotin, we demonstrated that immunohistochemistry and immuno-purification can be utilized to analyze protein transport in brain circuits.
Abstract Title : A Comparison of Anxiety and Depression Screening Scores in Breastfeeding Women Exposed to Marijuana, Alcohol, Nicotine, and/or Prescription Medications
Abstract : Approximately 10-20% of women in the United States experience symptoms of postpartum depression or anxiety, respectively (CDC, 2017). Marijuana is one of the most widely used substances during pregnancy, with a reported 4.9% of women between the ages of 15 and 44 using it during pregnancy in 2016 (Ryan, Sheryl A. et al, 2018). With more states legalizing marijuana use for medicinal and recreational purposes, it is important to determine who is using marijuana while breastfeeding, and what co-exposures are associated with its use. Previous research has noted that individuals with depression or anxiety may be more likely to use marijuana. The objective of this research was to assess whether marijuana, with or without co-exposures to alcohol or nicotine, was associated with symptoms of anxiety or depression in breastfeeding moms. Edinburgh Postnatal Depression Scale (EPDS) and State Trait Anxiety Inventory (STAI) scores were collected from breastfeeding women enrolled in Mommy’s Milk, a cross-sectional study and Human Milk Biorepository at UC San Diego. Participants were categorized into one of four exposure groups: marijuana only (n= 24), marijuana and co-exposures (alcohol and/or nicotine, n= 64), alcohol and/or nicotine (n= 526), and unexposed (n= 297). We conducted multivariable logistic regression to estimate the association between exposure group on symptoms of depression or anxiety. Models were adjusted for race, maternal and child age, employment status, and use of antidepressants. Compared to women with no marijuana, alcohol or nicotine exposures, breastfeeding women exposed to marijuana only or marijuana plus alcohol or nicotine were at increased odds of experiencing clinical symptoms of postpartum depression [(aOR 4.6, 95% CI 1.8, 12.0), (aOR 2.4, 95% CI 1.2, 4.9) respectively]. Additionally, compared with women with only alcohol/nicotine, women with marijuana, alcohol and nicotine had higher odds of depressive symptoms (aOR2.5, 95% CI 1.2, 5.0). However, this did not hold true for anxiety symptoms; the only statistically significant difference between groups was between women with marijuana, alcohol and/or nicotine compared with women who used alcohol and/or nicotine with no reported marijuana (aOR 2.2, 95% CI 1.2, 4.0). In summary, women who reported marijuana use were more likely to also report symptoms of depression compared with women without marijuana use. These same findings were not seen with respect to symptoms of anxiety. While cross-sectional data cannot establish temporality, women who use marijuana while breastfeeding may be a group who would benefit from additional postpartum screening of depressive symptoms.
Abstract Title : The effects of antiretroviral drugs on microvascular cells: Tenofovir induces cell senescence in 3D multi-layer co-culture
Abstract : Antiretroviral (ARV) therapy is recommended for all adults with HIV infection, regardless of their CD4+ T-cell count. With the advent of ARV drugs, HIV infection has become manageable and plasma HIV-1 RNA load can be suppressed to undetectable levels, resulting in a drastic increase in life expectancy. With this success, clinical attention has shifted towards the improvement of treatment that optimizes long-term safety. A clinicopathological study of HIV-infected persons by our group revealed an association between the use of ARV drug emtricitabine (FTC) and occurrence of cerebral small vessel disease. FTC is commonly prescribed with tenofovir (TFV) in ARV regimens. Here we explored in vitro the cytotoxic effects of FTC, TFV, and FTC+TFV at clinically relevant concentrations (vs. vehicle) on primary human brain microvascular endothelial cells, smooth muscle cells, and pericytes grown in 3D multi-layer co-cultures. β-Galactosidase activity was used as a marker of cell senescence. Using two-way ANOVA, we found that there was no significant interaction effect of cell types and ARV drugs, whereas the main effect of ARV drugs was observed. ARV drug affected all cell types. Compared with vehicle, TFV exposure increased β-galactosidase activity, whereas exposure to FTC alone did not show any significant effect (Dunnett’s post hoc t-test). In conclusion, TFV exposure induced cellular senescence of primary human brain microvascular cells in 3D multi-layer co-cultures. The chronic effects of long-term exposure to TFV should be studied in animal models.
Abstract Title : Investigating the Relationship Between Sitting Time on DNA Methylation: A Predictive Marker of Aging and Disease
Abstract : Epigenetic regulation includes DNA methylation where a methyl group is added to cytosine residues of DNA, resulting in decreased gene transcription. While part of normal gene regulation processes, aberrant DNA methylation patterns can occur as a result of environmental stress and unhealthful lifestyle behaviors, potentially leading to increased aging rate and disease risk by decreasing expression of genes involved in oxidative stress protection, cell cycle control and DNA repair. We are studying whether aging-associated DNA methylation patterns are correlated with sitting time in overweight postmenopausal women. From a 518-woman sample, controlled for age (≥55 years), BMI (≥25kg/m2), and low physical activity, the women in the highest and lowest mean sitting bout duration quartiles were chosen for methylation analysis. Genomic DNA was extracted from participant buffy coat samples using a Qiagen DNA Kit. The DNA will be treated with bisulfate and analyzed using site-specific probes that measure intensity of methylated and unmethylated sites on DNA sequences known to be associated with aging. Average apparent methylomic aging rate (AMAR) will be calculated for the two groups (low and high quartiles of mean sitting bout duration) and compared using a two-sided t-test. Increased sitting duration is correlated with higher insulin resistance and triglycerides. Methylation analyses are on-going. We predict that longer sitting bout duration will be associated with higher AMAR. The mechanisms by which sitting time influences aging-related cardiometabolic disease are unclear and may include epigenetic DNA methylation.
Abstract Title : B-Arrestin-2 integrates cytoprotective signaling induced by APC/PAR-1 & S1PR1
Abstract : Our laboratory focuses on understanding the role of highly druggable G-coupled protein receptors (GPCRS) in endothelial barrier stability, which is implicated in sepsis. Sepsis is a life-threatening condition that occurs in patients that results from a massive immune response to a bacterial infection. The endothelial barrier dysfunction, vascular leakage and tissue edema are associated with sepsis morbidity and mortality. Endothelial cells line the lumen of blood vessels and form a semi-permeable barrier that is tightly regulated to control vascular leakage. Mortality is high in patients with severe sepsis and no drug treatments are currently available. The goal of our research is to identify new signaling pathways that can enhance endothelial cell function and viability. Endothelial barrier stability is induced by activated Protein C (APC) through protease activated receptor-1 (PAR1), a G protein coupled-receptor (GPCR). APC activation of PAR1 signals via the β-arrestin-2 scaffold to activate Rac1 to enhance stabilization of the endothelial barrier. In addition to PAR1, another GPCR, sphingosine 1-phosphate receptor-1 (S1PR1), appears to contribute to endothelial barrier stabilization. However, it is not known how signaling by these two receptors is coordinated to promote barrier stability and endothelial cell viabilty. Our work suggests that APC / PAR1 promotes activation of S1PR1 through β-arrestin-2. Utilizing pharmacological agents including the S1PR1 antagonist, Sphingoskine kinase (Sphk) inhibitor, and β-arrestin-2 siRNA knockdowns, we demonstrate that APC-stimulated phosphorylation of Akt, an indicator for anti-apoptosis, requires S1PR1, SphK1 and β-arrestin-2. The mechanisms that enhance endothelial barrier integrity is poorly understood, and the proposed studies provide important new insight into how PAR-1 and S1PR1 work together to enhance endothelial cell survival that is critical for maintaining endothelial cell function.
Advisor : DR. PATRICK D. HSU
Abstract Title : Discovery and Development of Novel Type VI CRISPR Enzymes for Programmable Transcriptome Engineering
Abstract : The discovery of microbial CRISPR-Cas systems has revolutionized our ability to control the genetic code at the heart of all life. By repurposing these ancient adaptive immune systems into precise tools for DNA editing, science is uncovering new possibilities in medicine, bioengineering, and synthetic biology. More recently, the elucidation of Type VI CRISPR ribonucleases has extended this potential beyond the genome, making targeted RNA editing a reality. Known as Cas13, this family of enzymes offers promising new tools for the specific, consistent, and robust mediation of RNA-knockdown in cells. However, currently known orthologs have varying levels of cytotoxicity, limiting their usefulness for experimental and therapeutic applications. Clearly, there is a pressing need to expand the Cas13 family tree and identify new protein variants that combine high enzymatic activity with low toxicity, making the next generation of basic and translational research possible. Here, I outline a practical framework for the discovery and development of these CRISPR enzymes, focused on a metagenomic extension of the Cas13d family of RNA-targeting effectors. This consists of two parts: a computational phase of bioinformatic data-mining to discover putative orthologs of Cas13d, and an experimental phase to assess their activity, toxicity, and specificity as RNA-editors. Results from this project have the potential to characterize previously-unidentified Type VI CRISPR enzymes that are better suited to transcriptome engineering than those currently known. This will improve our ability to regulate cellular activity, make bold new experiments possible, and pave the way for the eventual development of RNA-based in vivo therapies.
Abstract Title : Small G Protein RalA Regulates Inflammation in Adipose Tissue
Abstract : Type II diabetes is a major health concern. Inflammation contributes to diabetes pathogenesis. Inflammation is initiated by macrophages which are recruited into diabetic adipose tissue by the signals that are not well understood. Small g protein RalA increases protein trafficking towards plasma membrane in response to insulin. We found that in mice constitutive RalA activation (which mimics insulin signaling) dramatically increases inflammation in brown adipose tissue. By mass spectrometry, we identified several proinflammatory proteins with known function in macrophage recruitment, that were enriched in plasma membranes of brown fat in mice with activated RalA. Next, we will test whether adipocytes with activated RalA increasingly recruit macrophages and will test the role of the candidate proteins identified by mass spectrometry in this process. Our studies may reveal a mechanism for hyperinsulinemia-induced inflammation.
Advisor : DR. KANG ZHANG
Abstract Title : Using a computational and biochemical approach to develop graphene oxide-based platform for hepatocellular carcinoma detection
Abstract : Traditional cancer diagnostics primarily uses tissue biopsy and sequencing analysis in order to analyze morphological and alterations in the genetic code. However, there are a number of issues associated with this approach: (1) invasiveness of surgical intervention, (2) cost, and (3) limited technical accuracy of biopsy analysis. This stems from an incomplete understanding of the underlying mechanisms of cancer, and an incomplete catalog of the known mutations that can cause a particular disease further complicates efforts to develop diagnostic tools. In order to address key bottlenecks in liquid biopsy and noninvasive cancer detection techniques, our team focused on using epigenetic determinants for diagnostic purposes. Presented here is a novel workflow for diagnosing cancer by using promoter methylation as an indicator of interest. Key promoter regions of interest are first identified via a novel hybrid analysis of methylation signal data with Random Forest and LASSO linearization. After identification of the relevant markers, a novel fusion protein of MBD-HRP is used to detect the presence of hypermethylated regions of interest and provide a quantitative, fluorescent readout for clinical relevance. Further modifications using graphene oxide (GO) and signal amplification strategies were used to improve and validate the sensitivity of the assay. The workflow was validated as a proof of concept in hepatocellular carcinoma with a 2100 patient cohort with a sensitivity of 5 pM.
Abstract Title : Effect of Cryoablation on Nerve Structure and Function
Abstract : Clinical researchers are currently looking into ways to best address pain associated with surgery, yet postoperative pain remains a large issue. Peripheral nerve cryoablation, or freezing of nerves to mute sensation, is a method that seems promising for long term pain relief since it does not require additional device implantation. However, there is a question of whether damaged neurons are able to fully recover. This unknown is especially unknown for nerves that carry both motor and sensory nerves. For example, rotator cuff muscles are tenuous before surgery and have shown a lack of recovery post-surgery, possibly due to axonotmesis (axon breakage) of suprascapular (mixed motor and sensory) nerve. Although substantial nerve regeneration is expected after cryoablation, there remain concerns about residual and persistent motor weakness caused by either incomplete regeneration or motor unit clustering, like the effects of nerve crush, which also results in axonotmesis. Performing functional and structural tests on Male Lewis rats’ fibular nerves before and after cryoablation surgery, such as staining, imaging, and analyzing the nerve histologically as well as performing motor and sensory testing, we can test tissue and functional level recovery in mixed motor and sensory nerve cryoablation.
Abstract Title : The Regulation and Interplay between chromosomal axis protein Hop1 and AAA+ ATPase Pch2 in Budding-Yeast
Abstract : Hop1 serves an important role in the overall organization of the chromosomal axis and recombination aspect in Prophase I of meiosis. Its presence, along with several other proteins, signal for the promotion of double strand breaks (DSBs) and crossover (CO) events between homologous chromosomes. The main structural element of Hop1 that helps facilitate this function is known as the HORMA domain. The HORMA domain is a highly conserved interprotein regulator that can be observed in similar well-characterized proteins such as the spindle assembly checkpoint protein Mad2 and the meiotic recombination protein Rec7. HORMA domain containing proteins act in conjunction with ‘closure motif’ proteins by having them wrap their built-in C-terminal ‘safety belt’ region around the domain, rendering the domain topologically closed. Knowing this mechanistic property of Hop1, two conformations are categorized by the interaction of the closure motif protein, one with the closure motif interacting with the HORMA domain, and the other having the closure motif off or removed. This remodeling between these two conformations has been attributed to AAA+ ATPase Pch2 due to Hop1’s similarity to Mad2 and Pch2 being implicated with the disassembly of the closure motif from Mad2’s HORMA domain. Here, we take steps to explore Pch2’s remodeling capabilities with Hop1’s HORMA domain and the conditions for regulation of these conformations.
Abstract Title : Creating a Rubric to Assess Knowledge of How a Vaccine Works
Abstract : Vaccination is a major controversial public health issue. It is unclear how much people know about vaccines and how their knowledge influences what they believe about vaccines (Jacobson et al 2007). It is assumed that advanced biology majors have a more accurate understanding of how vaccines work when compared to non-biology majors and entering biology majors, but it is not clear if that is the case; in fact, many biology students use alternative ways of thinking in their explanations of biological phenomena (Tanner and Coley 2015). Therefore, this project asks: to what extent do students at various levels of biology education (non-biology majors, entering biology majors, and advanced biology majors) have an accurate and complete understanding of how vaccines work? We began to address this question by creating a rubric to assess knowledge in response to an open-ended prompt asking, “How does a vaccine work?” Participants were 263 students and 21 biology faculty at a large urban public comprehensive university. The rubric was created through reading descriptions of how vaccines work in commonly used immunology textbooks and analyzing faculty responses to the “How does a vaccine work?” prompt. In future, we plan to use this rubric to explore questions like whether a student’s knowledge of vaccines correlates with their belief in common vaccine misconceptions or their acceptance of vaccines.
Abstract Title : A Novel Method To Infer Horizontal Gene Transfer
Abstract : Horizontal gene transfer (HGT) is an evolutionary mechanism that involves one species stealing genes from another species. Although common in bacteria, a large knowledge gap exists in the occurrence of HGT outside the bacteria kingdom. Recent studies describe rare instances of gene flow between bacteria and metazoans (multicellular animals with a germline). However, no one has looked for inter-kingdom HGT at an extensive level. This project aims to establish a computational pipeline to comprehensively catalog instances of gene flow between bacteria and metazoans. This entails large scale comparisons of sequenced genomes of bacteria against metazoans for any similarities using paralleled BLAST searches and subsequent mapping of filtered BLAST hits above a statistical similarity threshold onto a metazoan phylogenetic tree to infer relationships between hits. The resulting phylogenetically-informed analyses are stored into a database that will catalog high-confidence instances of HGT. Additionally, phylogenetic trees representing hits capture functional information for better data visualization and prioritization. The outcome is to understand the frequency, direction and mechanisms of gene transfer, thereby develop and test functional hypotheses about the utility of newly co-opted genes.
Abstract Title : Effectiveness of Inquiry-Based Teaching In Undergraduate Lab Courses
Abstract : Undergraduate biology-related laboratory courses are designed to introduce students to various fields using hands-on learning methodologies. A key component of lab courses is the level of inquiry, which is the process of examining and explaining scientific phenomenon through experimentation. Inquiry can be defined at different levels based on the degree of guidance and information presented to the student, of which two major categories are low inquiry (e.g. guided inquiry with questions and experimental format provided by instructor) and high inquiry (e.g. open inquiry with questions and experimental format chosen by students). The effect of low inquiry versus high inquiry labs was studied in an introductory biochemistry lab course (BIBC 103) where students took pre-class and post-class assessments designed to measure scientific reasoning skills. The student outcomes for both classes were assessed by metrics such as average change in score as well as normalized learning gains for each student. Results were mixed; two previous high-inquiry labs yielded strong learning gains and score increases with small effect sizes (Cohen’s D), while one low-inquiry lab yielded very low learning gains and another low-inquiry lab had strong learning gains and score increases with medium effect size.
Advisor : DR. PARTHA RAY
Abstract Title : Characterization of the anti-KIT aptamer binding to Gastrointestinal Stromal Tumor cells and its effect on the cancer cell viability.
Abstract : Aptamers are single-stranded oligonucleotide-ligands (DNA or RNA) selected by an in vitro process called SELEX (Systematic Evolution of Ligands by Exponential Enrichment). They bind their target proteins with high affinity and specificity, analogous to monoclonal antibodies. However, compared to antibodies, aptamer production is cost-efficient and faster. Gastrointestinal Stromal Tumor (GIST) is the most common mesenchymal neoplasms of the gastrointestinal tract. GIST cells stain positive for the proto-oncogene c-KIT, a transmembrane receptor tyrosine kinase. We investigated the binding affinity and specificity of an anti-KIT aptamer. A stable transgenic cell line for expressing c-KIT was established. c-KIT gene expression was under Doxycycline (Dox) inducible promoter. Thus, c-KIT is expressed only when Dox is added to the cell culture media. The protein expression was assayed by staining with Fluorophore-conjugated anti-KIT aptamer and using Flow-cytometry. An anti-KIT monoclonal antibody was used as a positive control. The aptamer and antibody had comparable affinity of binding to the cell line expressing c-KIT. In the absence of Dox, there was no detectable aptamer binding, demonstrating the selectivity of the aptamer. Also, a scrambled aptamer, used as a negative control, did not bind the cells. The aptamer demonstrated similar affinity and selectivity of binding to GIST-T1 cells. Additionally, the aptamer binding to GIST-TI cells inhibited the binding of Stem Cell Factor (ligand of the c-KIT receptor). We are currently investigating if the aptamer binding can inhibit the growth of GIST-TI cells in vitro. The findings indicate a potential clinical use of the anti-KIT aptamer for GIST therapy.
Abstract Title : Transcriptional regulation of protooncogene myc by HPV-associated enhancer in HeLa cells and real-time imaging by MS2-stable integration at myc locus.
Abstract : Human papillomavirus(HPV) infection is one of the major causes of cervical cancer. Previous studies have revealed that HPV integration is at the 8q24 site of the HeLa cell line, which is near protooncogene MYC. Also, the integration at this site is known to have a high association with tumorigenesis in HeLa cells. However, the details of how MYC expression is regulated at the molecular level is poorly understood. Recent studies provided evidence for a long-range chromatin interaction between integrated HPV fragments, MYC, and its potential enhancer region (8q24.22) in HeLa cells. To aid in the understanding of this unknown molecular mechanism of the transcriptional regulation of MYC, we seek to obtain spatiotemporal information of the MYC gene interacting with its potential enhancer. To confirm the functional significance of this potential enhancer, we targeted the integrated HPV sequence at the MYC enhancer for knockdown using CRISPR-KRAB. Using PRO-seq and qPCR technologies, we quantified and validated that the enhancer’s knockdown significantly reduces the expression of MYC gene transcription. We then observed the interaction between MYC and the HPV-associated enhancer through 4C-seq technology, further confirming the enhancer’s regulation of MYC. Lastly, we will stably integrate MS2 and PP7 repeats into the enhancer and gene transcripts, respectively, to provide a 4D readout of the position and transcriptional activity of each element over time. This live-cell imaging system will be used to characterize the spatiotemporal dynamics of the interaction and regulation between MYC and the HPV-associated enhancer.
Abstract Title : Intracellular Calcium Increase caused by a Single Cell Death in Neuronal Cultures
Abstract : Traumatic brain injury (TBI) is caused by a strong impact to the head that results in immediate neuronal dysfunction and dysregulation as well as cell necrosis. Secondary effects of the injury lead to greater cell death as well as scarring. Calcium has been implicated in neuronal dysfunction as well as in the secondary response of neuronal cell death. We utilize the laser to selectively lyse a cell within a mixed cortical culture and monitor the calcium response of surrounding cells. Results show that the death of a single cell leads to cytoplasmic calcium spikes in surrounding cells. These spikes may be a result of an influx from both extracellular and intracellular calcium. The inhibition of NMDA receptor fails to reduce the size of the calcium spike significantly. The opening and inhibiting of the IP3 receptor located on the endoplasmic reticulum by 2-APB significantly reduce the intracellular contribution to the spike. These results suggest that the intracellular contribution of the calcium spike is from the ER.
Abstract Title : Fear of Fish: Consumptive and Non-Consumptive Effects of Fish on Plankton Communities
Abstract : Trophic cascades have shown that the presence of predators can have a great effect on the entire ecosystem. Previous studies have looked at the classic example of sea otters in the kelp forest where the removal of predatory otters increases the sea urchin population and in turn decimates the kelp population. We looked at the effects of fish predators on zooplankton prey and therefore on phytoplankton. We wanted to see whether those effects are due to direct consumptive or indirect non-consumptive effects. We asked the following questions: How do consumptive and non-consumptive effects of fish on plankton contribute to trophic cascades in lakes? Do the strengths of consumptive and non-consumptive effects of fish on plankton depend on the plankton’s evolutionary history? We used two zooplankton communities near Yosemite National Park -- one community from a fishless lake (naive) and the other from a fish lake (experienced) -- and placed them in mesocosms. They were exposed to one of three treatments: no fish (control), free fish (consumptive effects), or fish in a semipermeable cage (non-consumptive effects). We found that consumptive effects, not non-consumptive effects, drive trophic cascades in the lakes in the Eastern Sierras. Some individual species do respond to non-consumptive effects, but overall, zooplankton abundance does not. We also found that the trophic cascade we witnessed was stronger in zooplankton communities that have evolved alongside fish.
Advisor : ERIC ALLEN
Abstract Title : Analyzing the Effects of a Transversion Mutation in acpP gene in Escherichia coli AN62 on Lipid Membrane Composition and High Hydrostatic Pressure Tolerance
Abstract : Escherichia coli (E. coli) is a well-studied model organism that has been successfully used to study pressure effects on bacterial cell processes. A Previous study done by Marietou et al. demonstrated that gradually increasing the growth pressure over 62 generations in E. coli MG 1655 increases its pressure tolerance up to 60MPa. Our experiment aimed to address the hypothesis raised by the study stating that a transversion mutation (T to G) at position 131 resulting in V43G amino acid change was necessary for the piezoadaptation observed in the high-pressure adaptive strain named E. coli AN62. Furthermore, we focused on the dramatical increase in the ratio of cis-vaccenic acid (18:1) to palmitoleic acid (16:1) observed in E. coli strain AN62 comparing to its parental strain MG1655. The elongation of palmitoleic acid to cis-vaccenic acid is mediated by 3-oxoacyl-[acyl-carrier-protein] synthase II encoded by fabF. To evaluate whether this particular mutation was directly or indirectly related to FabF activity resulting in the observed phenotype and whether it is necessary for high pressure growth, the effects of the transversion mutation (T131G) in acpP gene on lipid membrane profile and growth ability at high pressure were investigated.
Advisor : JING WANG
Abstract Title : Exploring the Role of Sensory Information in Patterning Complex Social Behavior in Male Drosophila melanogaster Using Machine Learning Algorithms
Abstract : Flexible processing of a variety of sensory cues in the nervous system enables appropriate selection of behavioral programs essential for the survival of an animal. However, relatively little is known about how multisensory information is integrated to generate ethologically relevant complex behaviors. Here, we addressed this question by analyzing social behavior at high spatial and temporal resolution with a genetically tractable model organism, Drosophila melanogaster, also commonly known as fruit flies. Drosophila males use visual, olfactory, gustatory and mechanosensory cues to find potential mates, leading to courtship and eventual copulation. During courtship, Drosophila males exhibit a series of patterned behaviors including searching, chasing, tapping, licking and singing towards the female fly. We first applied machine-learning algorithms to classify each of these stereotyped behaviors from video recordings of a pair of interacting male and female flies. We then analyzed how the choice of specific courtship behaviors by male flies depended on the relative position and movement of female flies. Furthermore, we evaluated how the absence of single sensory cues altered the behavioral pattern of male flies by using sensory mutations and controlling light illumination. With this approach, we developed an understanding of how sensory information was constantly monitored under different contexts to pattern behavior for optimal outcomes.
Advisor : DR. STANLEY LO
Abstract Title : Transfer student experiences and science identity formation in biological sciences
Abstract : More than 40% of undergraduates begin their post-secondary education at community colleges, and these transfer students are disproportionately first-generation (FG) college students or under-represented minorities (URM). Studies have shown that transfer students tend to have a significant decrease in academic success compared to their peers who begin their higher education directly at universities. At UC San Diego, approximately 18.5% out of the total undergraduate population are Biological Science majors, and approximately 31.8% are transfer students. Studies suggest that the unique experiences of science students contribute to the development of their overall science identity, which is a critical element in student success and persistence in higher education. Science identity describes how experiences in science exert influence on how students think of themselves, and one’s science identity can influence student learning and persistence in science over time. Because of the large difference in the general graduation rates of transfer students vs. non-transfer students, it is critical to understand how transfer students’ experiences contribute to science identity formation and persistence in the biological sciences. Our research project explores three domains of science identity: competence, interest, and recognition. Competence is a characteristic which demonstrates that a student possesses the understanding and knowledge of meaningful science content and is motivated to understand their world scientifically. Interest is described in regard to student disciplinary interest and is also viewed as a fluctuating element that can be influenced by the intersections of gender, race, and ethnicity. Recognition is a characteristic defined to be a student’s own self-recognition as a science person and the recognition they receive as a science person from meaningful scientific others, such as faculty and other authority figures. Our research project aims to answer three related questions: (1) What do transfer students in biological sciences experience in regard to competence, interest, and recognition? (2) How are students interpreting these experiences to derive meaning that ultimately influences science identity formation? (3) What additional factors influence how and why do individual students may interpret similar experiences in different ways?
Abstract Title : Analysis of the Beuffert Genome
Abstract : Bacteriophages are viruses that infect bacteria, and these extremely diverse entities are common and found everywhere where bacteria exist. As they infect their host bacteria, the phage contribute to evolution and genetic diversity, and thus have been widely studied as an alternative treatment for bacterial infections. In the fall of 2018, a new streptomyces phage, Beuffert, was isolated. To investigate the genes that control the phage function, the genome of this phage was sequenced and compared to related phages. This genome was 129,935 nucleotides and contained 237 genes, 39 tRNAs, and 1tmRNA. Through the genome annotation software PECAAN, basic functions of the genes were determined, however 177 genes had no known function. When annotating this genome, we found a variety of genes that are predicted to encode endonucleases. Upon closer analysis, three of these genes have homology to known Cas genes, which are endonucleases used by the CRISPR system in bacteria to protect against phage infections. We are now analyzing these genes to determine whether their role in the phage genome can be predicted.
Abstract Title : Analysis of Cheese Bacteria Proteus hauseri for Antibiotic Production
Abstract : Cheese is home to a diverse collection of bacterial species that interact with each other and components of milk. To study and characterize these communities, we isolated bacteria from multiple types of cheese (Brie, Hartwell, Cambozola Black Label Champignon, Stilton, Morbier, Taleggio, Pleasant Ridge Reserve, Wagon Wheel, Comte). By streaking a sample of cheese onto plates and performing repeated purifications of the bacterial colonies present, we were able to isolate pure cultures of bacteria, and identify them through 16S rDNA sequencing. Ribosomal DNA is chosen for bioinformatic comparison due to its universally and functionally conserved properties. We tested all of the cheese species for antibiotic production against E.coli and found a single species with high levels of antibiotic production. This species was originally isolated from a Morbier cheese sample from Secret de Scey, France, and based on 16S rDNA sequencing, is classified as Proteus hauseri. To learn more about our strain, we prepared a DNA library for Proteus hauseri for Illumina sequencing. From the results, we can learn about potential antibiotic production and its mechanism of infection, proticine and type VI secretion systems. Lastly, we will test our strain against other cheese bacteria to test its ability to kill other species.
Abstract Title : The Role of Mitonuclear Interactions in ATP Production Rates of Hybrid Populations of Tigriopus californicus, an Intertidal Copepod
Abstract : Interactions between nuclear and mitochondrial genes play an essential role in the production of cellular metabolic energy because the enzyme complexes of the mitochondrial electron transport system (ETS) are comprised of subunits that are encoded in both the mitochondrial and nuclear genomes. These strong mitonuclear interactions lead to the coevolution of nuclear and mitochondrial genes. The importance of this coevolution on ETS performance was tested in the intertidal copepod Tigriopus californicus, a species with high levels of genetic divergence between populations. Using interpopulation paternal backcrosses, we were able to construct hybrid populations that had mitochondria from the maternal population and a preponderance of nuclear genes from the paternal population. Due to potential mitochondrial-nuclear incompatibilities, we predict that the hybrid crosses will display decreased mitochondrial ATP production compared to parentals. ATP assays were conducted in order to separately assess ETS function using substrates for Complex I, Complex II, and Complexes I+II combined. The results displayed an overall trend of decreased ATP production rates when Complex I (which has both nuclear and mitochondrial subunits) was involved. Additionally, there was an increased variance in ATP production rates of hybrids. These results suggest that the mitonuclear mismatch in hybrids can result in decreased ATP production rates and could potentially be responsible for hybrid breakdown.
Abstract Title : Domesticating New Marine Bacteria Chitosan-Alginate Micro-Orbs
Abstract : This project focuses on developing a new culturing method for marine bacteria that is both sterile and efficient. Using alginate and chitosan to encapsulate marine bacteria in nutrient-diffusing micro-orbs that mimic their natural environment could help to increase the percentage of culturable marine bacteria (3). Through this encapsulation method, we may be able to discover new secondary natural products produced by known or previously uncultured bacteria, which could lead to more knowledge of biosynthetic pathways and possibly new drug discoveries.
Abstract Title : A Metabolic Survey of Actinomycete Bacteria Responses to Low Oxygen Conditions
Abstract : The rich and diverse family of Actinomycete bacteria colonize the ocean sediment and are widely known for their impressive secondary metabolism. Secondary metabolites are known to have a wide array of functions, including the ability to harvest electrons. Some of these molecules have are thought to play an important role in Actinomycete respiration, especially when sediments become hypoxic. This study was designed as a survey of the secondary metabolites produced by specimens across 35 different genera in the family Actinomycetaceae, some of which have been newly discovered. The analysis of this project focuses on the production or upregulation of extracellular electron shuttles, which aid in anerobic respiration. By investigating what molecules bacteria produce when stressed by low oxygen conditions, we can gain greater insight into the complex world of microbial chemical ecology and the natural role of natural products.
Abstract Title : Liver heparan sulfate proteoglycans impact on FGF1 binding to adipose tissue
Abstract : Type-2 diabetes, a chronic condition in which blood glucose levels are deregulated due to cells becoming more insulin resistant, is a global epidemic that compromises an enormous health and economic burden. Our laboratory investigates how glycans, like heparan sulfate proteoglycans (HSPGs), impact developmental factors of metabolic diseases and chronic conditions such as type-2 diabetes. Adipose tissue plays a critical role in regulating glucose levels. Recently, fibroblast growth factor 1 (FGF1) has been implicated in increasing insulin sensitivity in adipose tissue. Therefore, FGF1 is investigated for therapeutic use in type-2 diabetes. The mechanism depicts HSPGs to be significant cofactors for FGF1 binding to the membrane’s receptor. Our preliminary results show that sulfation of HSPG is critical in adipose tissue for FGF1 mediated glucose lowering. The liver contains vast amounts of FGF receptors and HSPG; therefore, we hypothesize that the liver is a prime competitor for FGF1 binding when FGF1 is injected as a drug.
Abstract Title : Sex Differences in Cocaine-seeking Behaviors Using Escalation, Motivation, and Compulsivity of Intravenous Cocaine Self-Administration
Abstract : Not only there are no current FDA approved treatments for cocaine addiction, but also many of experiments so far have been performed mainly in males. However, studies performed have failed to address sex differences, previously seen in humans. Women tend to be more vulnerable to cocaine than men, but cocaine addiction and compulsive cocaine intake is more commonly reported in men. To assess the differences in cocaine intake between male and female rats, we performed a genome wide association study (GWAS) on Heterogenous Stock (HS) rats which mimics better the genetic diversity in the human population. In this study, both male and female rats self-administered cocaine intravenously (6h daily) that led to escalation. Compulsivity and motivation were also recorded using foot shock and a progressive ratio schedule of reinforcement. We found that females pressed more than males during all the duration of the experiment, suggesting that they tolerate or metabolize cocaine better than males. However, they show lower escalation, motivation and compulsivity related behaviors compared to males, suggesting that may be genetic traits responsible for these behaviors are dissociated from their effect on cocaine self-administration. The similar sex differences seen in these rats and in humans can be instrumental in developing treatments for cocaine addiction that are effective and gender-specific.
Advisor : JON SHURIN
Abstract Title : Fear of Fish: Consumptive and Non-Consumptive Effects of Fish on Plankton Communities
Abstract : Trophic cascades have shown that the presence of predators can have a great effect on the entire ecosystem. Previous studies have looked at the classic example of sea otters in the kelp forest where the removal of predatory otters increases the sea urchin population and in turn decimates the kelp population. We looked at the effects of fish predators on zooplankton prey and therefore on phytoplankton. We wanted to see whether those effects are due to direct consumptive or indirect non-consumptive effects. We asked the following questions: How do consumptive and non-consumptive effects of fish on plankton contribute to trophic cascades in lakes? Do the strengths of consumptive and non-consumptive effects of fish on plankton depend on the plankton’s evolutionary history? We used two zooplankton communities near Yosemite National Park -- one community from a fishless lake (naive) and the other from a fish lake (experienced) -- and placed them in mesocosms. They were exposed to one of three treatments: no fish (control), free fish (consumptive effects), or fish in a semipermeable cage (non-consumptive effects). We found that consumptive effects, not non-consumptive effects, drive trophic cascades in the lakes in the Eastern Sierras. Some individual species do respond to non-consumptive effects, but overall, zooplankton abundance does not. We also found that the trophic cascade we witnessed was stronger in zooplankton communities that have evolved alongside fish.
Abstract Title : Identifying and Validating the Presence of Specific Biomarker miRNA's for Placental Age During Pregnancy
Abstract : Chromosome 19 contains a large cluster of miRNA's known to be associated with placental formation and development. Previously in our lab, a model data set derived by using samples from UCSD patients was studied to discover miRNA biomarkers for placental age and to create a model for neonatal age prediction. Our research aims to validate these miRNA biomarkers for placental age by using a sample set obtained from a collaborator in Israel. Since the model and validation data sets have different variability, due to factors such as location, collection methods, patient demographics, etc., we are able to compare them and evaluate whether the miRNA biomarkers signals are stronger than the natural variance between the two sample sets. Our procedure entailed isolating RNA from human serum and plasma, converting the RNA to DNA, performing PCR, and indexing each sample for next generation sequencing. We performed various clean up and size selection procedures the desired miRNA fraction. We then created a serum pool and a plasma pool to send out for sequencing. We analyzed the sequencing data for two main factors across the longitudinal cohort: read depth, complexity, and the number of miRNA matches each sample had with an international miRNA database. Using this information, we were able to compare the two biofluids and filter the results to create a high quality data set for the subsequent bioinformatic data analysis pipeline.
Abstract Title : The fungal pathogen C. albicans promotes increased GBS colonization during urinary tract infection
Abstract : Urinary tract infections (UTIs), considered one of the most common microbial infections, also rank among the top nosocomial infections and are associated with increased morbidity and cost of care. Certain population groups, including pregnant women, elderly, and patients with catheters, are especially at risk for developing UTIs. While uropathogenic E. coli (UPEC) is the cause of at least 70% of urinary tract infections, other etiologic uropathogens include gram-positive bacteria such as Group B Streptococcus (GBS) and fungal agents including Candida albicans. While the pathogenesis of these organisms has been extensively studied in single-organism UTI models, there is an increasing interest in how the presence of multiple distinct organisms during infection can influence disease progression and symptoms. In fact, polymicrobial UTIs are particularly prevalent among the elderly, immunocompromised, and those with indwelling catheters, diabetes, and HIV. Upwards of 20% of women with symptoms of UTI potentially have polymicrobial growth within their urine. For example, coinfection with GBS promotes persistent UPEC infection of bladder and kidneys during UTI. GBS urinary tract infection during pregnancy is particularly concerning, as it can lead to increased risk of intrapartum fever, chorioamnionitis, and preterm delivery. Synergistic interaction between GBS and the fungal urogenital pathogen C. albicans has been described during vaginal colonization, and the carriage of Candida is an independent risk factor for GBS colonization. Thus, we investigated whether co-inoculation of C. albicans with GBS during UTI would also promote GBS colonization. Co-culture with C. albicans increased bacterial adherence to human bladder epithelial cells and promoted GBS colonization in an in vivo murine model of uncomplicated UTI. This increased colonization was dependent on the fungal adhesin protein Als3, as co-inoculation with fungal strains lacking this protein failed to promote increased colonization both in vitro and in vivo. Further investigation of pathogen interaction during polymicrobial UTI will lead to better understanding of patient susceptibility and disease progression, potentially reducing the incidence of recurring UTI and other urologic issues.
Abstract Title : The activation of specific FZD receptors using a Wnt mimetic
Abstract : Wnt signaling is an important pathway for developmental processes such as cell proliferation, cell fate determination, and cell polarity. The Wnt pathway is comprised of 19 secreted Wnt ligands that interact with a family of 10 transmembrane proteins called Frizzled (FZD) receptors. Wnts can activate several different FZD receptors, and lack specificity. As these FZD receptors are not necessarily biologically redundant, it would be beneficial to field to study the role of individual FZDs. This can be done through the specific activation of FZD receptors. Previous work in the lab has shown that it is possible to activate the Wnt signaling pathway specifically through FZD7 through the use of a bispecific antibody that mimics the action of Wnt by heterodimerizing FZD7 and its co-receptor LRP6. The FZD7 epitope recognized by the antibody was mapped to generate a tag. This then allowed for the sensitization of the Wnt mimetic to other FZDs. FZD2 was tagged and it was specifically activated by the Wnt mimetic. Our results demonstrated that the combination of the FZD7 tag and the bispecific antibody can be used to activate a specific FZD, allowing for the investigation of that receptors unique role in the Wnt signaling.
Abstract Title : Into the Unknown: Analysis of Previously Unsequenced Hafnia Phages
Abstract : Bacteriophages, also known as phages, are viruses that infect bacteria and are some of the most common entities on the planet; they keep bacterial growth in check, which allows microbial ecosystems to prosper. Phage are also known to infect the bacterial species associated with fermented foods, like cheese and yogurt, leading to failures in their fermentation processes. One of these bacterial species is Hafnia alvei, a cheese bacterium that intensifies the flavor of cheese. It has been observed that H. alvei is frequently infected by phage, but not much else is known about the phages that target this bacterium. Understanding how these phages infect the cheese bacterium can allow us to prevent phage infections. In order to understand the how closely related the bacteriophage infecting H. alvei are, we are using Illumina sequencing to sequence the genomes of two phages that infect H. alvei. With these sequences we may better understand the reason why bacteriophage infections of H. alvei occur so frequently. In addition, we will test the host range by trying to infect additional sequenced isolates of H. alvei. By exploring the relationship between these two phages, we will be able to learn more about the balance between bacteria and bacteriophage in both cheese and other microscopic biomes.
Abstract Title : Pre-clinical model of nicotine vapor self-administration shows dependence in rats
Abstract : In the last decade, the number of individuals that electronically vaporize nicotine and other substances of abuse has raised significantly. However, epidemiological evidences for the beneficial or risky effects of electronic cigarettes are very contradictory. Performing these studies in humans requires longitudinal observation for each individual, a large number of subjects in a specific age range and consistent self-reports or nicotine blood level analysis. In this study, translational research in both male and female rats was performed in order to mimic electronic cigarette use. A clinically relevant dose of nicotine (0.5 mg/ml) was selected to produce blood levels similar to those observed in chronic smokers and IV self-administration in rats. Behavioral observations were then compared with rats administering nicotine-free vapor as a control group. Findings revealed that daily access to nicotine vapor produced long-term symptoms of nicotine dependence including anxiety-like behavior, hyperalgesia, and relapse. These data provide pre-clinical evidence that electronic vaporization of nicotine leads to addiction-like behaviors in rats.
Advisor : PAUL R. JENSEN
Abstract Title : Micro-scale distribution and diversity of the marine bacterial genus Salinispora in marine sediments
Abstract : The ocean harbors astonishing levels of microbial diversity, much of which has not been characterized. Among the bacteria that reside in ocean sediments is the marine-obligate actinobacterial genus Salinispora. Members of this genus have been recovered from tropical and subtropical marine sediments collected around the world and produce potent bioactive compounds, making them a useful model organism for natural products discovery. Previously, the phylogenomic diversity and biosynthetic potential of the three Salinispora species were characterized by sequencing the genomes of 118 strains isolated from global locations. However, the fine-scale spatial distributions and genomic diversity of three Salinispora species remain unknown. Here, by plating the sediments we were able to isolate over 400 bacterial strains from 16 one-meter square marine sediment quadrants in Fiji, and we used molecular tools and growth assays to identify 176 as members of the genus Salinispora. We used PCR to amplify the 16S rRNA genes of each strain and determined that 172 strains were Salinispora arenicola and four were S. pacifica, all with different morphology. Our results demonstrate the potential of an incredible micro-scale diversity of Salinispora arenicola perhaps beyond what is typically considered a bacterial species or that Salinispora grow in a large hyphal network throughout the marine sediment. Genome sequencing of the new Salinispora strains is underway which will allow us to fully understand the extent of micro-scale genomic diversity and chemical potential of Salinispora bacteria from a 1-meter square quadrant.
Abstract Title : Investigation of the Demographics and Beliefs of University Students Who Reject Vaccinations
Abstract : Despite the fact that vaccines are generally safe and effective, many people choose not to vaccinate (Jacobsen et al 2007). Even students in our biology classes can have alternate conceptions regarding core concepts in biology, that can complicate their’ ability to learn. (Tanner and Coley 2015). Therefore, our project seeks to understand the ways in which students who reject vaccines are different from students who accept vaccines. To answer this question, we asked 96 non-biology majors and 101 entering biology majors at a large diverse urban public comprehensive university whether they agreed with common misconceptions about vaccines as well as whether they would vaccinate their children. Of the non-biology majors, 9.4% (9/96) would not vaccinate their children, while 9.9% (10/101) of the entering biology majors would not vaccinate. We then analyzed whether their acceptance of vaccines correlated with their belief in these misconceptions. We also looked at how demographic characteristics, such as gender, ethnicity, first-generation status, and years in college, differed in people who rejected or accepted vaccines. In the future, we plan to analyze their reasoning around vaccine misconceptions to see how that affects their acceptance of vaccines.
Abstract Title : Analysis of the King2 Phage
Abstract : Bacteriophages are viruses that infect bacteria (called the host) and reproduce inside it. Bacteriophages can be found almost everywhere and are constantly evolving through mutations. As a result, there are more species of bacteriophage than any other form of life. Phages have several uses in biology. They can be used as vectors to inject new DNA into bacterial cells or they can be used as models to help us understand evolution. In addition, phages can be used to treat deadly bacterial infections in phage therapy. Our goal was to discover and characterize a novel bacteriophage on the University of California San Diego campus. Though varied, bacteriophage can be identified by their life cycle, shape, host range, and cluster. We discovered a bacteriophage that was found to be a lytic, myoviridae phage that can be characterized into the subcluster AO1 that infects Arthrobacter sp. ATCC 21022, and named it King2. We analyzed the sequenced genome of King2 to determine the functions of genes and compare to the genes of related bacteriophage. We discovered a large portion to be genes that coded for proteins of unknown function. Due to the rapidly evolving nature of bacteriophages, this result can be expected and indicates that bacteriophage sequencing and annotating can be a promising path for discovering novel proteins and biological interactions that can have implications for applications in various fields of scientific study.
Advisor : DR. BETTY SHIH
Abstract Title : Elevated Erucic Acid Concentration in Women with Anorexia Nervosa
Abstract : Background: Anorexia nervosa is the deadliest eating disorder and psychiatric illness whose etiology remains unclear. Anorexia nervosa patients have shown an aversion toward foods high in fats and calories, which may have hindered treatment success as suggested by lower fat intake in anorexia nervosa patients with poor treatment outcomes when compared to patients with successful treatment outcomes. The high-fat foods that anorexia patients avoid are one of the major sources of omega fatty acids, which play important roles in healthy brain development and are implicated in cardiovascular, inflammatory, and psychiatric disorders. Additionally, altered levels of omega fatty acids have been found in patients with eating disorders, suggesting possible involvement in the pathophysiology of eating disorders. Thus, this study aims to explore the role of common omega fatty acids in the risk and clinical phenotypes of anorexia nervosa. Methods: Blood samples were collected from women with anorexia nervosa and age- and sex-matched healthy controls before and two hours after the subjects ate a breakfast sandwich (fasting N= 6 anorexia nervosa patients and 16 controls; postprandial N=6 anorexia nervosa patients and 12 controls). Questionnaires were administered to all study subjects to assess anxiety (BAI), depression (BDI), and food aversion (FAQ). Additionally, anorexia nervosa patients completed two eating disorder questionnaires (EDE-Q and EDI). Mass-spectrometry based lipid analysis was used to quantify both fasting and postprandial plasma levels of seven common fatty acids, which included two omega-6, four omega-3, and one omega-9 fatty acid. Results: Of the 7 fatty acids, most of them showed no statistically significant difference between women with anorexia nervosa and healthy controls, both at fasting and postprandial timepoints. Fasting levels of the monounsatured omega-9 erucic acid were significantly higher in anorexia nervosa patients than in healthy controls (136.95 ±75.29 pmol/ml and 77.16 ±67.68 pmol/ml, respectively; p=0.01). Similarly, in the fed state, women with anorexia nervosa had higher plasma levels of erucic acid than controls (124.18± 49.61 pmol/ml and 67.05±33.51pmol/ml, respectively; p=0.02). At the fasting timepoint, women with anorexia nervosa showed a positive correlation between erucic acid and change in BAI (rho=0.90, p=0.04), whereas controls exhibited a negative correlation between erucic acid and change in BAI (rho= -0.83, p=0.04). Erucic acid was not significantly correlated with BMI, BDI, or specific food aversions in both patients and controls. Conclusion: Compared to healthy controls, women with anorexia nervosa showed significantly higher levels of erucic acid both before and after eating a sandwich enriched with omega-6 arachidonic acid, suggesting that erucic acid may be involved in the pathophysiology of anorexia nervosa. However, further studies with larger sample sizes are needed to confirm the association between erucic acid and anorexia nervosa status and the (lack of) correlations between erucic acid levels and comorbid phenotypes in anorexia nervosa.
Abstract Title : HVCx Neuron Modeling with Variational Annealing Method
Abstract : HVC neurons are an important components in songbird’s forebrain that allows them to precisely learn a vocalization of a specific melody. However, the electrophysiological characterization of component HVC neurons is currently poorly understood. Consequently, a complete biophysical model of HVC neurons is difficult to develop to understand the functionality of them. Thus, inspired by the data annealing method, we are interested in building an accurate model to characterize the HVC neurons. Using the conductance-based model developed by colleagues, we performed the variational annealing method to estimate the biophysical parameters. The model, along with the optimized parameters, was then used to generate predictions of HVC neuron’s response to specific stimulus. These predictions were later verified with data collected in vitro experiment under similar condition. Our model produced the characterization of the HVC neurons responsible for song production in the songbird. This method can be applied in medical researches of neurodegenerative diseases to understand the biophysical changes in neuron.
Advisor : LISA A. LEVIN
Abstract Title : Novel Deep-Sea Communities in Los Angeles' Backyard
Abstract : Methane seeps are chemosynthetic ecosystems that arise along continental margins supporting heterogenous faunal assemblages. The Point Dume methane seep (PDMS), found 7 km off the coast of Malibu, CA at ~730 m water depth in the oxygen minimum zone (OMZ), is characterized by a submarine river channel with methane seepage. Microhabitats associated with these methane seeps include bacterial mats and clam beds with a high affinity to sulfide, as well as transitional habitats such as ampeliscid amphipod beds and arborescent foraminifera beds, with an intermediate affinity to sulfide. The goal of our research was to understand the effect that methane seepage has on the sediment-dwelling macrofaunal invertebrate community at PDMS. To do this, we wanted to compare community characteristics at each of the different habitats within the PDMS as well as non-seep affected (inactive) background sediments, to understand if each habitat exhibits distinct invertebrate density, vertical distribution, composition, and diversity. We also compared this methane seep with other seeps found along the Eastern Pacific Ocean. Sediment core samples were collected from each of the five habitats and sectioned vertically. Each vertical fraction was sieved, sorted for macrofaunal, and identified down to family level. We found the active habitats supported lower diversity, fewer polychaete species, and a higher rank 1 dominance, which is the proportional representation of the most abundant family, when compared to transitional and inactive seep habitats. Variations between habitats occurred for a few species. The ampeliscid amphipod bed had a significantly greater density of ampelescid amphipods than the other four habitats. Species composition varied across habitat and seepage activity. Specific habitat comparisons showed that the bacterial mat was composed of significantly fewer species than the background habitat. All of the habitats had the majority of animals concentrated in the top two cm, and vertical distribution was not significantly different across the five habitats. Neither habitat nor seepage had an effect on density. The discovery of new seeps is occurring rapidly with each seep exhibiting a distinct community of invertebrates. Understanding the unique communities found at each seep lends itself to understanding their function in the global chemical cycling, ecosystem services such as fisheries, and the effects these communities might face in light of warming oceans and decreasing oxygen concentrations (Levin et al. 2016). Levin, L. A., A. R. Baco, D. A. Bowden, A. Colaco, E. E. Cordes, M. R. Cunha, A. W. J. Demopoulos, J. Gobin, B. M. Grupe, J. Le, A. Metaxas, A. N. Netburn, G. W. Rouse, A. R. Thurber, V. Tunnicliffe, C. L. Van Dover, A. Vanreusel, and L. Watling. 2016. Hydrothermal Vents and Methane Seeps: Rethinking the Sphere of Influence. Frontiers in Marine Science 3:72.
Abstract Title : Time-Restricted Feeding Differentially Affects Atherosclerotic Lesion Development in ApoE-/- and Ldlr-/- Mouse Models
Abstract : Cardiovascular disease (CVD) is the worldwide leading cause of death in both men and women. Atherosclerosis, or the narrowing and hardening of arteries, is the leading cause of cardiovascular disease. Time-restricted feeding (TRF) has been shown to be protective against dietary and genetic determinants of CVD, such as insulin resistance, hyperlipidemia, and inflamed/hypertrophied adipose tissue. We hypothesized that TRF would also protect against genetic causes of atherosclerosis. To test this hypothesis, we placed ApoE-/- and Ldlr-/- mice on a high fat, high cholesterol (i.e. atherogenic) diet. The mice were either fed ad libitum or had time-restricted access to food for only 8 hours during the nocturnal (i.e. active) period (n=6 per condition, n=24 total). Their weight and food intake were monitored for a 23-week treatment period. Lipoprotein profile, metabolic phenotype (food intake, physical activity, RER) and atherosclerotic lesion development were assessed. Despite cumulatively consuming the same number of calories, TRF mice in both genetic conditions weighed more than their ad libitum counterparts. Total serum triglycerides and cholesterol levels were assessed after 17 weeks of treatment. While there was no significant difference in total serum triglyceride and cholesterol levels in ApoE-/- mice, Ldlr-/- TRF mice had significantly lowered cholesterol levels (p=0.0039). Moreover, Ldlr-/- mice on a TRF regimen had significantly reduced atherosclerotic lesion development (aortic arches p=0.011, aorta p=0.039) and an increased RER during their inactive phase, while TRF in ApoE-/- mice failed to alleviate atherosclerotic lesion severity (aortic arches p=0.372, aorta p=0.707) and no difference in inactive RER was observed. Overall, TRF requires ApoE to produce its cardiovascular benefits.
Abstract Title : Phages of UCSD
Abstract : The 246 phages that have been isolated at the University of California, San Diego display immense diversity in terms of their host range, genes, morphology, etc. This diversity can be attributed to evolutionary mechanisms, such as gene mutations and horizontal gene transfers, that can occur during host infection and phage replication. Using two phages isolated in UC San Diego, Edgyboi and Ungolyant, we intend to observe those evolutionary mechanisms in action to demonstrate how phages have come to be so diverse. More specifically, we will observe whether or not the propagation of phages on different bacteria will expand their host range, as gene mutations and/or horizontal gene transfer can occur during their life cycles. In order to do so, Arthrobacter sp. will be used as a measure of wild-type plaquing efficiency, as both phages are known to infect Arthrobacter sp. with high EOP. In addition, two bacterial species closely related to Arthrobacter sp. will be used to first see whether or not the two phages can infect those bacteria and how efficiently they are able to do so. If a phage is shown to have a very low EOP for one of the two species, we will try to propagate the phage in varying concentrations of that bacterial species. Therefore, the phage can have a chance to improve its EOP and fully include the species into its host range, which if successful, would demonstrate evolutionary mechanisms in action and the continuous growth of diversity in the phage world.
Abstract Title : Is increased Vitamin A to the brain related to more severe symptoms of Multiple Sclerosis?
Abstract : The blood-brain barrier (BBB) is a term used to describe the unique properties expressed by brain endothelial cells. For patients with the disease Multiple Sclerosis (MS), BBB dysfunction is an important factor in the formation of autoimmune neuroinflammatory lesions in the spinal cord. Therefore, understanding the mechanisms that lead to BBB dysfunction and lesion formation may provide vital therapeutic targets that limit the pathology of MS. Our lab used experimental autoimmune encephalomyelitis (EAE), the most commonly used MS mice model to identify molecular changes at the BBB during the course of EAE. We identified that the Stimulated by Retinoic Acid 6 (Stra6) gene, a transporter that brings Vitamin A into the brain, is upregulated at the BBB during the peak of EAE, and thus our aim is to identify the role of Stra6 at the BBB during MS. In order to do this, we induced EAE in mice that were wild type, heterozygous, or knockout (KO) for the Stra6 gene. We examined and scored the clinical symptoms of the mice along a thirty-day period after inducing EAE and analyzed symptom progression over time. We also characterized the hallmarks of EAE by measuring the size of the lesions and immune cell infiltration. We observed that Stra6 KO mice showed less severe clinical symptoms compared to mice heterozygous for Stra6 or wild-type mice. Through immunofluorescent staining, we also observed in Stra6 KO mice fewer infiltrating lymphocytes that crossed the blood-brain barrier to attack the myelin sheath covering the neuron axons. With these results, we concluded that Stra6 is detrimental during EAE, as worse clinical symptoms and higher numbers of lymphocytes were observed in Stra6 wild-type and homozygous mice compared to Stra6 KO mice.
Advisor : YISHI JIN
Abstract Title : Determining Functionality of Single Amino Acid Mutations in C/EBP bZIP Transcription Factor
Abstract : In humans, innate immunodeficiency is a disorder where the immune system is not able to fight against infections. Key components of the innate immune system such as toll-like receptors (TLRs), which recognize an infection, and mitogen-activated protein (MAP) kinase pathways, which regulate signal transduction are defective. The innate immune system of Caenorhabditis elegans (C. elegans) also utilize a set of TLRs and highly conserved MAP kinase pathways, similar to humans. C. elegans is the perfect model organism for studying the innate immune system because they have a smaller proteome that is highly homologous to humans. Additionally, their genome is easily manipulated by genetic tools such as Ethyl methanesulfonate (EMS) and CRIPSR to create mutant models to study. EMS is a chemical used to create random mutations throughout the genome, usually creating single base pair changes. CRISPR on the other-hand is used to create a specific mutation at a desired location in the gene, and is generally used to make insertion or deletion mutations. The Jin lab has recently determined a gene, nipi-3, that is part of the innate immune system of C. elegans, is also required for normal animal development. Without a functional protein, animals will have arrested development in the larval stage and will not reach adulthood. However, mutations in genes downstream of nipi-3, such as a particular MAP kinase pathway and the associated transcription factor are found to suppress the lethality caused by the loss of functional NIPI-3. My work in the lab has mostly focused on the downstream transcription factor, CEBP-1. C/EBPs are a family of transcription of factors that regulate cell proliferation and other cellular processes. The Jin lab created a point mutation in cebp-1(ju1521) using EMS, and a single base pair deletion in cebp-1(ju1590) using CRISPR. These mutations are of interest because they cause subtle changes to the amino acid sequence of CEBP-1, but can suppress the lethality of loss of nipi-3 as effectively as total loss of CEBP-1 function. The Jin lab has shown that CEBP-1 is required for repair of damaged neurons. Associating this with that fact that single amino acid mutations in cebp-1 suppress nipi-3 null lethality, we wonder if these mutations can also affect neuronal repair. To determine this, I performed a variety of crosses with a neuronal marker and cebp-1(ju1521) or cebp-1(ju1590). Our results show that ju1521 has a partial effect on suppressing neuronal repair. Currently, we are looking into another gene found in an EMS screen that can suppress nipi-3 null lethality.
Abstract Title : Patterning of the extracellular matrix: formation of cuticle struts by BLI collagens in Caenorhabditis elegans
Abstract : The Caenorhabditis elegans cuticle is a complex extracellular matrix composed primarily of secreted collagen proteins. Collagen proteins form the outer (cortical) and inner (basal) layers of the cuticle, which are connected by a medial layer of column-like “struts” oriented orthogonally to the cuticle plane. Under electron microscopy, struts appear in a hexagonal array with evenly distributed lateral spacing; however, the mechanism underlying this patterning has not yet been identified. We are studying strut biogenesis and patterning by investigating a classical set of mutants that display cuticle blistering (Bli) phenotypes. Mutations in bli genes disrupt strut formation and lead to a separation of the cortical and basal cuticle layers; we have chosen three cuticle collagens (bli-1, bli-2, and bli-6) for further molecular genetic analysis. Loss of function in either bli-1 or bli-2 results in blister formation, while gain of function mutations in bli-6 leads to blistering. By examining genetic interactions that affect Bli phenotypes, we hope to identify other factors involved in strut biogenesis and construct a more complete model for strut formation and patterning.
Abstract Title : A Computational Approach for Analysis and Interpretation of the Transcriptional Profiles Generated by Next-generation Single Cell Functional Genetic Screening
Abstract : As a consequence of modern sequencing technology, we now a complete catalog of somatic mutations in the cancer genome. The next challenge is how to obtain a complete functional annotation of genetic variants at the cellular and organism levels. The use of the CRISPR/Cas9 system has enabled us to perform precise and rapid genome editing and generate genetic perturbation to probe cellular circuits. Through filtering low expression genes and low expression cell samples within each perturbation, we obtained top gene sets enriched in each perturbation. We then performed NMF (Non-Negative Factorization) to detect potential genetic variants across individual cells as well as across different cells types. Further more, we performed clustering and partition to discover novel interactions between modified genes and the controls.
Abstract Title : Studying the impact of Protein X signaling on gene expression modulation in epithelial cells
Abstract : Inflammatory Bowel Disease (IBD), most commonly categorized as Crohn's Disease (CD) and Ulcerative Colitis (UC), is a disease characterized by chronic inflammation of the gut. One aspect in the treatment of IBD is the regulation of immune cells to dampen the immune response causing the chronic inflammation. Another aspect of treatment is repairing the barrier function of the gut epithelium through mucosal healing. This is equally important to prevent leakage of the intestinal microbiome into the gut walls which causes inflammation. Data from genome-wide association studies (GWAS) indicate that patients with specific single nucleotide polymorphisms (SNP) in the gene sequence of Protein X have been identified as higher risk for IBD, and Protein X has been suspected to be a potential therapeutic target that can address both of the aspects of IBD treatment discussed above. Our research attempts to investigate the pathway that Protein X signals through, in order to discover downstream biomarkers as well as validate Protein X as a drug target. The following tools were generated to help understand its mechanisms of action: Protein X knockout (KO) mice and highly specific antibodies against Protein X; which were selected by FACS analysis of antibody-binding to WT and KO cells. RNAseq analysis was performed to compare gene expression of cells treated with agonist compound to Protein X versus treatment with vehicle. qPCR techniques are then employed to confirm the differential expression of these genes. Our research also explored whether Protein X signaling activates ERK, Src, or Akt by phosphorylation, using bone marrow-derived macrophages (BMDMs) from KO and WT mice.