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2018 Research Showcase
UR Abstracts
Abstract Title : Generating the First Inducible Mouse Model for the Study of Sturge-Weber Syndrome
Abstract : Sturge-Weber Syndrome (SWS) is a congenitally acquired neurocutaneous disorder caused by a somatic activating mutation in the GNAQ gene, which encodes the Gαq heterotrimeric G protein. SWS is characterized by the development of facial port-wine stains, as well as ocular and cerebral vascular malformations. Approximately 3 of every 1000 newborns are affected by port-wine stains, where SWS risk in patients with facial port-wine stains increases to 25%. The recent discovery of the R183Q (p.Arg183Gln) mutation in Gαq as the single underling mechanism for SWS and port-wine stains has uncovered a path for us to explore potential mechanistic pathways and identify new therapies. By the use of genetic engineering techniques, we aim to generate an inducible mouse model expressing the GαqR183Q mutation, which will serve in the study and possible discovery of treatment for Sturge-Weber syndrome and similar congenital disorders. To explore such pathways and potential targets for treatments, a disease model expressing the GαqR183Q mutation must be generated. Early experiments in the generation of a mouse model included the confirmation of expression of GαqR183Q via western blot analysis, followed by the use of various DNA recombination techniques to prepare a plasmid for pronuclear injection. It is expected that doxycycline inducible expression of GαqR183Q in the endothelial cells (by the use of conditional and tissue specific systems) will lead to tumorous capillary malformations in the skin similar to port-wine stains, a characterizing feature of the Sturge-Weber syndrome. Thus, our efforts will result in a disease model that will allow us to target signaling pathways of interest and lead to potential new precision treatment options.
Abstract Title : PIRC-NET: Twitter-based on demand public health framework for HIV risk estimation
Abstract : As of 2016 the HIV/AIDS epidemics is still a key public health problem. Recent reports showed that alarmingly high numbers of people in vulnerable populations are not reached by preventative efforts. Despite technology improve-ment, we are not yet able to identify populations that are most susceptible to HIV infections. In order to enable evidence- based prevention, we are studying new methods to identify HIV at-risk populations, exploiting Twitter posts as possible indicators of HIV risk. Our research on social network analysis and machine learning outlined the feasibility of using tweets as monitoring tool for HIV-related risk at the demographic, geographical, and social network level. CO-PRESENTER: CLARK PHAN
Abstract Title : Medial entorhinal cortex inputs are not required for CA3 cells to retain trajectory information in a spatial memory task
Abstract : Spatial memory performance requires information about current and future locations. Recent studies have shown that hippocampal place cells contain information about both of these dimensions. The medial entorhinal cortex (mEC) projects directly to the hippocampus and is thought to be a key source of spatial and temporal information to hippocampal place cells. However, the specific contributions of the mEC to memory performance remain unclear, and we therefore used mEC-lesioned rats to understand the contribution of mEC to hippocampus- dependent memory and hippocampal physiology. MEC and sham lesioned rats were trained to perform a spatial alternation task in which the animals alternated between left and right sides of a figure-8- maze to receive a food reward. Both groups performed similarly during the trials without delay, but when a delay was imposed, the mEC lesion group made significantly more errors. After further training, the behavioral performance of the mEC lesion group improved for trials with a 10-second delay but showed a long-lasting impairment for trials with delays of 60 seconds. Recordings of hippocampal single units and local field potentials were performed during the spatial alternation task to investigate the circuit dysfunction that underlies the memory impairment as well as to identify which neuronal firing patterns could support the spared memory function in mEC-lesioned rats. CA1 cells in mEC lesioned rats discriminated less well between left and right maze segments than control animals. Unexpectedly, CA1 cells of control and lesioned rats distinguished equally well between left and right trajectories on the stem before the decision point. We therefore examined whether CA3 is a possible source for maintaining separate neural codes for left and right trajectories such that this information could be provided to CA1 on the stem. We found that a subpopulation of CA3 cells in mEC lesioned rats consistently distinguished between right and left sides of the maze. These data indicate that mEC inputs are critical for distinct CA1 neuronal activity patterns on the return arms of the maze, while CA3 inputs more effectively retain differences. By forwarding this information, CA3 inputs could contribute to the reemergence of distinct CA1 firing patterns on the center arm, such that left-turn and right-turn trajectories are successfully distinguished in mEC lesioned rats. Taken together, our results suggest that intrahippocampal processing of inputs from sources other than mEC can support spatial working memory and that spatial computations can thus partially recover in a circuit without mEC projections to the hippocampus.
Abstract Title : Analysis of Isolated Streptomyces Strains though Bacteriophage Host Range Experiments and 16s rRNA Sequencing
Abstract : Our research focuses on the phylogenetic relationships between Streptomyces bacteria, as well as the host range of Streptomyces phage isolated from the same soil samples. To understand the phylogenetic relationships between our bacteria, we took the approach of using 16s rRNA analysis because it is widely accepted as a biological measuring stick for inter-species relationships and provides a simplification over whole-genome sequencing. We collected and isolated Streptomyces from soil samples and sequenced the 16s rRNA genes. Using this data, we constructed a phylogenetic tree of our Streptomyces. To determine the host range of Streptomyces phage we isolated in our class, we tested our phage samples against several of the Streptomyces strains we isolated from the same soil samples. The phage were found to reliably kill Streptomyces platensis, but had lesser range in terms of other Streptomyces species isolated from the soil. Overall, we hope that this phylogenetic mapping may prove useful in later research in the study of antibiotics, as Streptomyces are common antibiotic producers. We also hope that understanding the host range of our phage will give an insight into some of the interactions in the soil microbiome. CO-PRESENTERS: Jeffery Sardina, Lauren Stuart, Laine West, and Savanna Senecal.
Abstract Title : Determining the Developmental Stages that Require Nascent Protein Polypeptide-Associated Complex -Alpha (NACA) for Drosophila Heart Development
Abstract : Congenital Heart Disease (CHD), malformations of the heart and great vessels present at birth, has significant impact on society and especially on the affected patients. Efforts are invested in identifying novel genetic components involved in pathogenesis in order to offer optimized treatments for patients. Nascent protein polypeptide-associated complex-alpha (NACA) is one candidate CHD gene that emerged from a GWAS study associating Single Nucleotide Polymorphisms (SNPs) with larger hearts in humans suggesting a possible role in heart morphology and development (van der Harst, et al. 2016). More recently, a missense mutation in NACA was associated with the CHD Tetralogy of Fallot (TOF) further implicating a role for NACA in cardiac development (Liu et al. 2017). While some studies have begun to study the role of NACA in the heart, its role in heart development remains unclear. This study focuses on determining the developmental stages that require NACA for proper heart development. In Drosophila melanogaster, constitutive knockdown of NACA in the heart throughout development leads to adult flies with no hearts. Using a temperature-dependent and heart-specific driver, we spatially and temporally controlled KD of NACA by expression of NACA-RNAi to different stages of heart development (embryo, larvae, pupae or adult) by raising ambient temperatures. NACA knockdown in adults only does not cause any changes in heart function or structure indicating that NACA is not necessary for cardiomyocyte maintenance but required during development for normal morphogenesis. Knockdown of NACA during embryo stages leads to a heart with a significantly smaller diameter but no change in fractional shortening. Interestingly, the posterior most segment of the heart was completely absent. Knockdown of NACA during mid larvae to pupae stages, increased fractional shortening and did not alter heart structure. We found that knockdown of NACA during both embryo and pupae stages led to an almost complete loss of the heart. Interestingly, NACA's effect during pupae stage is dependent on its knockdown during embryo stages as well, as knockdown during pupae alone had no significant effect on heart structure. Future research will be aimed at determining the mechanisms involved in NACA's regulation of heart development and we hypothesize that NACA may play a role in determining heart cell identity. By elucidating NACA's role in heart development, we could potentially uncover novel pathways and mechanisms involved in heart morphogenesis and pathogenesis of CHD.
Abstract Title : Benefits of Microfluidic Chambers for Neuroscience Research
Abstract : Neurons may have long axons (for some motor neurons extending >1 meter) and go through different microenvironments that can locally affect the neuron’s health inducing its degeneration. In order to mimic neuronal compartmentalization in vitro, microfluidic chambers represent a unique tool to enable distinguishing molecular mechanisms underlying neuronal dysfunction at the cell body and axon level. We utilize a two-chamber system that holds neurons in the proximal chamber, and axons that grow through microgrooves to reach a distal chamber in order to examine axon growth, anterograde and retrograde signaling, RNA localization and local protein synthesis, as well as axonal regeneration upon injury. To investigate the contribution of ALS-causing proteins to axonal stress, we model acute axonal injury by performing aspiration-based axotomy in the distal chamber and further analyze axonal recovery properties. Using iPSC-derived motor neurons we find that reducing expression of TAR DNA binding protein 43 (TDP-43) or stathmin 2 (STMN2) suppresses axonal regeneration, thus identifying a role of stathmin 2 in motor neuron vulnerability. Furthermore, the two-chamber system permits the incorporation of a second cell-type such as the muscle cells. In an initial effort, we have co-cultured motor neurons in the proximal compartment, with muscle cells in the distal chamber, to produce detectable neuromuscular junctions (NMJs) in the muscle compartment. Loss of NMJs which leads to muscle denervation is one of the earliest features of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease without an effective therapy. Therefore, this sophisticated compartmentalized motor neuron/muscle co-culture represents a valuable tool to validate compounds that promote muscle innervation or reduce/prevent muscle denervation induced by ALS-causing mutations, and further elucidate their cell target of action.
Abstract Title : Cellular Mechanisms of Neuroprotective Effects of TRiC Chaperonin in Huntington’s Disease
Abstract : Huntington’s disease (HD) is a “trinucleotide repeat” disorder caused by an increased number of CAG repeats in the HD gene that codes for Huntingtin protein (HTT) (Myers, 2004). It is characterized by a substantial loss of striatal medium spiny neurons (MSNs) and a loss of cortical pyramidal neurons (Zhao et al., 2016); cortical-striatal pathways are disrupted and degenerated in Huntington’s disease, resulting in atrophy and death of medium spiny neurons. MSNs are dependent on cortical neurons for brain-derived neurotrophic factors (BDNF) for survival. Thus, in this experiment, we used biochemistry to treat and live cell imaging to assay axonal dysfunction as well as cortical or striatal atrophy. We hypothesized that mutant huntingtin protein (mHtt) interferes with the axonal function for BDNF trafficking and signaling. Also, we hypothesized that TRiC chaperonin-based reagents mitigate mHtt toxicity to afford neuroprotection for MSNs in HD. We created an in vitro cortical-striatal system to study the cellular mechanisms atrophy and death of MSNs in HD and have shown that MSNs, not cortical neurons, are degenerated in BACHD transgenic mice. We have also demonstrated that axonal trafficking of BDNF in BACHD cortical neurons is deficient in BACHD mouse that likely contributes to atrophy of MSNs in HD. Finally, we showed that addition of exogenous BDNF or the apical domain of CCT1 is effective in preventing atrophy of MSNs in BACHD. CO-PRESENTER: ELIZABETH HO.
Abstract Title : Generation of an LRP6 deficient cell line via CRISPR-Cas9
Abstract : Wnt signaling is an important pathway for developmental processes such as cell proliferation, but when it is dysregulated, uncontrolled cell proliferation leads to cancer. At the core of this pathway are secreted Wnt ligands and transmembrane Frizzled (Fzd) receptors. In humans, there are 19 WNTs and 10 FZDs identified, each with different affinities for each other. In our lab, it has been shown that Wnt9a specifically interacts with Fzd9b in zebrafish. Wnt initiates signal transduction by binding to Fzd which oligomerizes with the transmembrane protein LRP6. To determine if this specific interaction is dependent on LRP6, we asked the following: Is LRP6 necessary for Wnt signaling to occur? To answer this question, we first needed to develop a cell line deficient for LRP6. My project uses the CRISPR-Cas9 system to generate an LRP6 deficient cell line, which we then verified by genotyping and characterized through protein analysis and activity assays.
Abstract Title : Antibiotics In Your Backyard
Abstract : Antibiotics have long been valued in medicine, research and the public for their usefulness in fighting harmful pathogens. However, pathogens evolve rapidly and quickly develop resistance to antibiotics. Science is unable to keep this pace because research and discovery in this area cannot innovate as quickly. Many people are continuing the search as there are many potential antibiotics that can be found in our very own backyards. A cure-all for antibiotic-resistant pathogens could be waiting right outside, ready to be discovered and it was with this goal in mind that we conducted our research. Our experiments revolved around Streptomyces, a bacteria found in almost any soil across the world and often used for antibiotic research. We collected soil samples from different areas across San Diego that were then plated in order to allow the bacteria in the soil to grow. From twenty-one plates, forty-two colonies of various strains of Streptomyces were found and subsequently transferred to new plates to be purified. The purification process was necessary to isolate Streptomyces from other types of bacteria and fungi that are found in soil along with Streptomyces. After approximately five weeks of purification, the Streptomyces samples were plated with common bacteria strains in order to observe the effect Streptomyces had on the growth of these bacteria and if the environment in which Streptomyces grows influenced its production of antibiotics. We obtained a variety of results from this experiment on the antibiotic production of the collected Streptomyces strains, as different strains produced differently sized killing zones for each bacteria. Furthermore, the strains had a different effect on the common bacteria when grown in a plate without antibiotics than when grown in a plate with antibiotics. These findings indicate the variability of Streptomyces antibiotic strength and type. Our efforts prove that there is much left to be discovered in the frontier of antibiotics and further analysis of these bacteria may yield effective antibiotics for the future. CO-PRESENTERS: Caroline Nguyen, Jeanna Sheen, Lily Wang, and James Medwid.
Abstract Title : A Comparison of Anxiety, Depression, and Stress Mood Scores in Breastfeeding Women Who Use Marijuana to Those Who Do Not Use Marijuana
Abstract : Background: Women are twice as likely as men to be diagnosed with anxiety and/or depression. The use of marijuana to treat anxiety and/or depression has become increasingly popular among breastfeeding women. Research Question: The aim of this study was to compare the difference in scores from standardized post-partum mood questionnaires between breastfeeding women who are either using or not using marijuana. Methods: Breastfeeding mothers ages 18 and older were enrolled into Mommy’s Milk between 2014 and 2017. Participants expressed a milk sample and completed an interview about their health and their child’s health, breastfeeding habits, and current exposures to medications and substances within the past 14 days. Participants completed the Edinburgh Postnatal Depression Scale (EPDS), Perceived Stress Scale-10 (PSS), and State-Trait Anxiety Inventory Y-1 Y-2 (STAI) shortly after milk collection via email. The 28 women who used marijuana and completed the mood questionnaires were matched to controls by maternal age and child age for a total of 56 controls (2:1 ratio). Results: There was a significant difference in the mean EPDS score between marijuana and non-marijuana breastfeeding women (p=0.05). The marijuana users had a higher EPDS score than non-marijuana users. There was no difference in the mean STAI and PSS-10 scores between marijuana and non-marijuana users. Conclusions: The screening results indicate that marijuana users may be at higher risk for postnatal depression than non-marijuana users.
Abstract Title : The parasitic plant dodder (Cuscuta californica) infection of buckwheat (Eriogonum fasciculatum) is higher in wetter soils
Abstract : Cuscuta californica, or California dodder, is a species of parasitic plant native to ecosystems throughout California, including coastal sage scrub. In the study area of the Scripps Coastal Reserve in La Jolla, California, C. californica is primarily sighted on California buckwheat, Eriogonum fasciculatum, despite the presence of several other known possible hosts. Detailed work analyzing the factors that affect host selection has been carried out with related parasitic plants, such as Cuscuta campestris, due to their roles as agricultural parasites, but the factors influencing the host selection of C. californica are not all known in detail. Additionally, the strength of the effects of C. californica on its hosts will need to be examined to look for any larger effects it may have on its ecosystems. We hypothesized that the soil moisture levels beneath E. fasciculatum plants infected with dodder would be higher than those under uninfected E. fasciculatum. This difference might be an influence affecting host selection of C. californica, or it might be an effect of C. californica on the host (if it allows moisture to condense on the plant or traps moisture). To test for causality, we removed C. californica from eight host plants and remeasured soil moisture. Finally, to consider host health effects, stem elongation of E. fasciculatum plants infected and uninfected by dodder was measured. Stem elongation was significantly lower in infected than uninfected plants.
Abstract Title : In Search of Novel Antibiotic Therapeutics
Abstract : Once hailed as panaceas for bacterial infections, antibiotics are declining in efficacy due to the rise of resistant pathogens. Therefore, it is imperative to find novel therapeutics. The objective of this ongoing project is to characterize the antibiotic properties of 113 natural products obtained from the National Cancer Institute’s Developmental Therapeutics Program. These compounds were tested against a variety of Gram-negative and Gram-positive bacterial species including Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Bacillus subtilis. Minimum inhibitory concentration (MIC) assays were done to determine potency and spectrum of activity. The fluorescent microscopy technique of bacterial cytological profiling (BCP) was used to evaluate the mechanism of action (MOA) of the natural products in a sensitized strain of E. coli. Since BCP only identifies MOA at the biosynthetic pathway level, it was necessary to then generate resistant mutants via serial passaging to determine the molecular target. Preliminary BCP results indicate hits on each of the five major biosynthetic pathways by at least one compound screened. This project has the potential to reveal a novel antibiotic with a unique MOA that could then be developed into a therapeutic agent.
Abstract Title : Comprehensive genomic and transcriptomic analysis of papillary thyroid carcinoma reveals common and unique mutations and immune-associated gene expression dysregulations among the various cancer subtypes
Abstract : Although papillary thyroid carcinoma (PTC) is the fastest growing cancer, little effort has been made to develop novel treatments for this disease. Additionally, little is known about the genetic and trascriptomic differences that cause largely different clinical outcomes among the three most prominent variants of PTC: the classical, follicular, and tall cell variants. To evaluate potential pathways useful for immunotherapy and for differentiation of unique PTC variants, this study computationally analyzes the dysregulation of immune-associated genes and associated aberrantly expressed microRNAs as well as mutations in PTC patients, identifying genomic and transcriptomic alterations common and unique to the three PTC subtypes. RNA sequencing data of 548 patients from The Cancer Genome Atlas (TCGA) was separated into six cohorts, each comparing data from patients with a variant of PTC to that of normal patients or to that of patients with a different PTC variant. Differential expression analysis of mRNA expression identified significantly dysregulated genes between patients of the two groups in each cohort. After filtering dysregulated genes by selecting those with immune system association, gene dysregulation was compared to relevant clinical variables. Gene expression was then correlated with miRNA expression using the Gene Set Enrichment Analysis (GSEA). Mutations associated with gene expression were identified using REVEALER. The analyses identified genes that were commonly dysregulated in all PTC subtypes in comparison to normal samples, including genes in the S100 family and ANGPTL1. In addition, dysregulation of certain genes was identified to be unique or absent in a single PTC subtype when compared to the other subtypes. Analysis of mutations identified a positive correlation between mutation at the 1q42 locus and dysregulation of certain immune associated genes in the classical PTC subtype. Functional analysis of the common and unique genes and mutations may reveal underlying pathways that differentiate PTC subtypes from one another or serve as potential targets for immunotherapy.
Abstract Title : IL-6 activated STAT3 promotes oxidative metabolism in obesity
Abstract : Rapidly increasing rates of obesity pose a significant threat to public health, yet pathways in weight loss are still poorly understood. In this study, we explore the novel role of IL-6 and its target, STAT3, in the metabolic regulation of adipose tissue in response to sympathetic nervous system activation. First, we treated diet-induced obese mice with CL-316,243 (CL) to mimic sympathetic activation. Consequently, we observed a robust increase in serum IL-6 and STAT3 phosphorylation in liver and adipose tissue. To isolate IL-6 specific effects, mice were treated with an IL-6 neutralizing antibody (NA) prior to CL injection, resulting in decreased levels of phosphorylated STAT3. Surprisingly, metabolic rate was significantly attenuated in the presence of NA, suggesting a relationship between STAT3 activation and oxidative metabolism. Our findings indicate that catecholamine-stimulated IL-6 secretion activates STAT3 in an autocrine manner. Characterizing the IL-6/STAT3 signaling axis could provide the foundation for researchers to develop new therapeutic treatments to combat the obesity epidemic this country is facing.
Abstract Title : Combining Stiffness and Stretch to Study Cardiac Fibroblast Pro-fibrotic Activity
Abstract : Cardiac fibrosis is prevalent in many forms of heart disease and results from the excessive accumulation of extracellular matrix (ECM). When the heart is subjected to mechanical stress, it induces a signal in cardiac fibroblasts that enhances collagen production and induces differentiation into myofibroblasts. Myofibroblasts are characterized by expression of smooth muscle α-actin (SMA) enabling them to “pull” on the newly synthesized collagen and thereby remodel the heart. Although these properties initially provide structural support to the mechanically-stressed heart, persistant collagen production and myofibroblast activity will lead to myocardial stiffening and thereby compromise cardiac diastolic function. To date, there is no effective treatment for cardiac fibrosis reflecting incomplete understanding of the underlying mechanisms that convert mechanical stress into pro-fibrotic activity of cardiac fibroblasts, as well as differential effects of stretch and stiffness. Previous data showed stiffness-dependent responses to stretch between cardiac fibroblasts plated on hydrogels with stiffness in the lower (3 kPa) and upper (8 kPa) physiological range. We here study the pro-fibrotic responses to stretch in cardiac fibroblasts plated on substrates with stiffness corresponding to the healthy (4.5 kPa) and fibrotic (40 kPa) heart. Cardiac fibroblasts were isolated from mouse ventricles and plated on collagen-coated polyacrylamide (PA) gels with stiffness of 4.5 kPa or 40 kPa. Immunocytochemistry for SMA fibers demonstrated lack of SMA fibers at 4.5 kPa while prominent SMA fiber formation at 40 kPa. Staining for the focal adhesion protein vinculin revealed smaller focal adhesions in cardiac fibroblasts on 4.5 kPa compared to 40 kPa indicating differences in these important mechanotransduction sites. To examine the effect of stretch on cardiac fibroblasts plated on 4.5 and 40 kPa stiffness, PA gels were attached to polydimethylsiloxane (PDMS) stretch membranes and cardiac fibroblasts stretched (10 % area increase) for 24 h. mRNA was extracted to measure collagen 1a1, collagen 1a2, collagen 3a1, fibronectin, SMA, lysyl oxidase and osteopontin expression. In response to stretch, cardiac fibroblasts plated on gels with fibrotic stiffness exhibited less increase in fibrotic gene expression compared to cardiac fibroblasts on gels with physiological stiffness. Preliminary results indicate rapid upregulation of collagen 1a1 30 min after stretching when cardiac fibroblasts are plated on 4.5 kPa. In contrast, collagen 1a1 was downregulated at this time point when cardiac fibroblasts were plated on 40 kPa, indicating that the response to stretch is highly dependent on substrate stiffness and the initial phenotype of the cardiac fibroblast. It will be interesting to more closely examine the underlying mechanotransduction pathways that are activated at different stiffness and thereby possibly enable selective targeting of myofibroblasts.
Abstract Title : Cracking open the Black Box of Neural networks
Abstract : Artificial neural networks (ANNs) have been central to machine learning research over the past few decades. Although they have performed extraordinarily well to solve multiple problems, they have been termed "black-boxes" due to a lack of understanding of the underlying principle causing ANNs to work well, other than a vague inspiration drawn from the architecture of the brain. We create a neuro-mimetic, dynamic artificial neural network, adding delays, decays and geometric information to each neuron, modeling the biological brain much more closely compared to traditional ANNs. We then analyze this complex system's steady-state patterns to understand how these networks learn. More specifically, we analyze how this dynamic network evolves and behaves when learning new input. Instead of tuning thousands of parameters to optimize a cost function, we make classification decisions by directly comparing steady-state patterns generated by the network for different inputs. Furthermore, we provide a framework and a metric to introduce a new classification method requiring minimal training compared to traditional techniques.
Abstract Title : Fibrotic Scarring in Neurological Disorders
Abstract : Neurological disorders are an important area of study because they make up a large percentage of the world’s disease burden and their pathologies are not fully understood. Fibrosis is a process that forms scar tissue in an organ or tissue and in the central nervous system (CNS) is mainly studied in the context of spinal cord injury. The scar is formed in order to protect the tissue from further injury but may prevent repair. We were interested in studying what neurological disorders caused fibrotic scarring to occur, so we collected tissue from mice modeling several diseases affecting the CNS and analyzed the presence of scar tissue and scar-forming cells. Specifically, we studied mice induced with Experimental Autoimmune Encephalomyelitis (EAE), a model of Multiple Sclerosis (MS), SOD 1 mutant mice to model Amyotrophic Encephalomyelitis (ALS), stroke, and LPS injections to model sepsis. Col1-GFP mice were used to label scar forming cells and the tissue was stained for collagen 1. We found that scarring was present in the spinal cords and brains of mice affected with EAE and stroke, respectively, but not in the brains and spinal cords of ALS and LPS mice. The EAE mice demonstrated massive amounts of scarring so this disease was selected for further study. Multiple Sclerosis (MS) is a demyelinating autoimmune disease that involves progressive decrease of motor function. When viewing tissue from the EAE model, most of the demyelination was observed in the spinal cord, where most of the white matter in the mouse resides. Since the spinal cord is a central part in motor function, EAE induction leads to motor symptoms that can be scored on a defined scale. In EAE mice we looked at scar progression and compared it to how the disease itself progressed. Mice were sacrificed at different time points in EAE progression and their spinal cords were collected to analyze the number of GFP positive cells present in demyelinating regions. It was observed that GFP+ cells peaked around 10 days after symptoms began, just following the peak motor symptoms in EAE. Results showed the presence of GFP+ cells continued months after symptoms first appeared. Further studies on the role fibrotic scarring has in EAE might lead to better understanding of disease progression.
Abstract Title : Cardiac fibroblast (CF) activation under high glucose treatment and its implications for fibrosis
Abstract : Cardiac fibroblasts are the main cell type that produces extracellular matrix proteins which provide a structural support for myocytes, distribute mechanical forces through the heart, and thus mediate tissue contraction. When myocardium becomes injured, CFs are activated into myofibroblasts and produce excessive collagens in order to repair or compensate for the loss of myocytes. This process can become pathologic when there is an uncontrolled and excessive collagen production, causing an imbalance in homeostasis leading to a stiffened heart. In this study, fibroblasts from hearts of three-month old rats are isolated, cultured and then treated under normal and high glucose conditions (to simulate diabetes). Preliminary results demonstrate that CFs treated with high glucose show an increased production of collagens I and III as seen in diabetic patients. This project will serve as a model system to test the response of CFs to various agents that can modulate fibrosis.
Abstract Title : Ligand-dependent regulation of 3D chromosomal architecture and subnuclear structures
Abstract : The functional specialization of cells in metazoans requires precise genomic transcriptional responses to developmental and environmental signals. These responses are largely mediated by enhancers, which are cis regulatory DNA elements. The primary model for their mechanism of action on transcriptional activation involves “looping” of enhancers and proximal promoter sites through cooperative clustering of transcriptional factors and coactivators. Nuclear estrogen receptor α (ERα) translocates to the nucleus upon binding of its natural ligand, 17β-estradiol (E2), and activates its bound enhancers and their associated target genes that ultimately impacts chromosomal conformation in three-dimensional nuclear space. As an established regulator of gene transcriptional programs, ERα is an ideal model candidate for investigating the consequence of a ligand-dependent enhancer activation on the dynamic chromosomal architectural rearrangements. ERα binds to ~8500 EREs located in enhancers, of which the ~1300 most robust enhancers adjacent to E2 upregulated coding genes cause increased enhancer-promoter looping in the regulated transcriptional programs. Current data has revealed that E2 induced proximity of a cohort of the most high-induced, “first tier”, ERα-bound enhancers, some >27-30mB apart by linear distance, in Chromosome 21 of our model cell line MCF7 unexpectedly confer robustness to the interacting enhancers. We predict that there are transient shifts in 3D chromatin architecture upon ERα recruitment, forming a dynamically E2-regulated “connectome” required for the transcriptional robustness of interacting enhancers. Furthermore, previous findings indicate that at least a subset of enhancers require association with specific subnuclear architectural structures to be functionally active. We hypothesize that these most robustly active enhancers co-localize with a specific subnuclear structure to permit concentration of coactivators and transcriptional machinery required for optimal enhancer activation. To reveal the relative kinetics of dynamic chromatin rearrangements and assess the predicted E2 stimulated “connectome” of enhancers in 3D space in respect to subnuclear organelles, we will develop a real time imaging approach.
Abstract Title : YAP and MRTF-A, transcriptional co-activators of RhoA-mediated gene expression, are critical for glioblastoma tumorigenicity.
Abstract : The role of YAP (Yes associated protein 1 gene) and MRTF-A (Megakaryoblastic Leukemia gene, MLK1), two transcriptional co-activators regulated downstream of GPCRs (G-protein coupled receptors) and RhoA, in growth of glioblastoma cells and in vivo GBM tumor development was explored using human glioblastoma cell lines and tumor initiating cells derived from patient derived xenografts (PDX). Knockdown of these co-activators in PDX cells using shRNA significantly attenuated in vitro proliferation and stemness assessed by limiting dilution and neurosphere formation. Orthotopic xenografts of the MRTF-A and YAP knockdown PDX cells formed significantly smaller tumors with lower morbidity than wild-type cells. In vitro studies of cellular responses to the GPCR agonist sphingosine 1-phosphate (S1P) demonstrated that YAP was required for glioblastoma cell invasion and migration, whereas MRTF-A was required for cell adhesion. S1P-stimulated proliferation was abolished by knockout of either YAP or MRTF-A. Gene expression analysis by RNA-sequencing of S1P-treated MRTF-A or YAP knockout cells identified 44 genes that were induced through RhoA and highly dependent on YAP, MRTF-A, or both. Knockdown of F3 (tissue factor gene; TF), a target gene regulated selectively through YAP, blocked cell invasion and migration, whereas knockdown of HBEGF (Heparin binding EGF-like growth factor), a gene selectively induced through MRTF-A, prevented cell adhesion in response to S1P. Proliferation was sensitive to knockdown of target genes regulated through either or both YAP and MRTF-A. Expression of these genes was also decreased in tumors from PDX cells lacking YAP or MRTF-A, indicating that these transcriptional pathways are regulated in the in vivo GBM tumor environment and suggesting that their activation through GPCRs and RhoA contributes to growth and maintenance of human GBM.
Abstract Title : Platelet-mediated Innate Immunity against B. anthracis
Abstract : Bacillus anthracis is a Gram-positive, spore-forming bacterium that is the causative agent of anthrax. It is an obligate pathogen that has been unfortunately used as a biological warfare agent due to its high virulence and its ability to cause widespread infection in both humans and livestock. Previous research on the pathogenesis of anthrax infection has largely focused on the bacteria’s antiphagocytic poly-D-glutamic acid capsule, as well as its plasmid-encoded exotoxins: protective antigen (PA), lethal factor (LF) and edema factor (EF). Here we explore novel virulence functions of the chromosomally-encoded cholesterol-dependent pore-forming cytolysin anthrolysin O (ALO), and the mechanism(s) by which ALO contributes to B. anthracis disease pathogenesis. Using the B. anthracis Sterne strain (lacking capsule), we observe that circulating platelets, increasingly recognized as a contributor to inflammatory and immune processes in the host, directly kill B. anthracis in vitro and are critical for host defense against B. anthracis infection in vivo. We further show that ALO aids in the pathogen bloodstream persistence by subverting platelet-mediated microbial activity. A significant delay in time between exposure to anthrax and clinical manifestations suggests a prominent role of innate defenses in attempting to contain the infection before medical therapy, and research into improving platelet antimicrobial function could provide new therapeutic avenues against anthrax infection. CO-PRESENTER: IDA PRICE
Advisor : STANLEY LO
Abstract Title : Using Social Network Analysis to Measure Student Interactions and Dynamics in Group Work
Abstract : Group work underpins many evidence-based instructional practices. In existing literature, qualitative discourse analysis has largely been used to examine student discussions and interactions. In this study, we developed a quantitative methodology to examine student interaction dynamics in group work through observations of 15 groups of 4-8 people in peer-led learning environments at a private research university. Data were collected in a question-and-response format which sequentially tracks student talk order. Treating the discussion as a network, we analyzed this data and adapted traditional social network parameters (including centrality, density, & average degree) to quantify student interactions in group work. Our results show three paradigms. In a group of five, the peer facilitator was highly central; the density of the discussion network is 0.4, and the average degree is 1.6, indicating that few students are talking with other students. In a group of six, the peer facilitator was not highly central; the density of the discussion network is 1.0, and the average degree is 5.0, indicating that students are interacting with one another. Third, in a group of eight, there were two subgroups divided on gender. Our research provides quantitative insight into the creation of more inclusive learning environments.
Abstract Title : Rat Decision Making in Weighing Quality against Quantity when Evaluating Rewards
Abstract : In nature, rats must assign values to different energy-consuming activities, such as eating, drinking, foraging, and more. However, how rats compare the value of non-commensurate actions is still unknown. To better understand this decision-making phenomenon, the experiments in this research project focus on observing the rat’s decision making in regards to different qualities of water. Rats prefer plain water to sour water. Thirsty rats will be offered a choice to work for either a larger quantity of sour water or a smaller amount of plain water. The goal is to see if a larger reward size will overcome the rat’s instinctive aversion to sour water. Current obtained data indicate the rat’s strong aversion to choosing sour water. More experiments are needed to determine if a larger reward size will overcome this aversion. Information gained from this research will help develop a reliable assay for comparing liquid reward values. This assay can be used to better elucidate how the brain makes decisions, an important question with many social and economic implications.
Abstract Title : Determining the Role of Candidate Wound Signature Molecules in Activation of 𝛾𝛿T Cells Using CRISPR/Cas9
Abstract : Background: Chronic non healing wounds are becoming increasingly common, but unfortunately it is not fully understood what cellular interactions are causing this trend. It is known that 𝛾𝛿T cells are crucial for homeostasis and repair of the skin epithelial tissue, and dysregulation of these cells results in failure to regenerate or delay of repair processes. 𝛾𝛿T cells require both stimulation of T cell receptors and additional accessory molecules to be activated. Many of these crosstalk pathways are known, however we believe there still remain some molecules that are unknown. We have identified a set of molecules that may be crucial in activation of the gamma delta T cell wound healing functions through an RNA-seq analysis of genes upregulated following wounding. Our set of candidate molecules include HSPA8 and CRISPLD2, two keratinocyte cell stress ligands, and CD316 and ICAM-1, the binding receptors in T cells we believe may be important for their activation. The goal of this study is to determine if these candidate molecules are important in 𝛾𝛿T cell activation by using CRISPR/Cas9 gene editing to knock out these molecules. Methods: 8 single guide RNA (sgRNA) for CRISPR/Cas9 were designed for each gene, inserted into Cas9 expressing plasmids, and sequenced. The efficacy of the sgRNA-CAS9 complex cutting was tested using EGxxFP plasmids and HEK293T cells. The most efficient sgRNA-CAS9 complexes were transfected into PAM2.12 cells (a keratinocyte cell line). PCR was performed on target genes and mutations identified via shifts in band size. Using FACS we quantified co-stimulatory molecule expression in PAM2.12 cells. Cells with mutations in the gene of interest are currently being used in functional assays to determine the effect of these mutations on the activation of T cells. Results: EGxxFP experiments showed that the sgRNA-Cas9 guides for CD316, HSPA8, CRISPLD2, and ICAM-1 were successful in cutting the target gene sequence. PCR results from PAM2.12 cells transfected with sgRNA-CAS9 complexes for HSPA8 and CRISPLD2 both indicated successful mutation of endogenous genes. FACS results for both HSPA8 and CRISPLD2 sgRNA-CAS9 complex transfected PAM2.12 cells indicated presence of each protein respectively. Conclusion: Sequence results show that we successfully mutated the HSPA8 and CRISPLD2 genes in PAM2.12 cells, however there was no visible effect on their protein expression or function. For future experiments, we will co-transfect two sgRNA-CAS-9 complexes targeting different regions of each protein to see if we can successfully knock out the protein expression in PAM2.12 cells for use in our functional assay.
Abstract Title : Improved RNA-hairpin recognition in the RNA-TAG system
Abstract : Biochemical modification of RNA substrates could aid in elucidating biological function of RNA and protein, for example by pulling down RNA associated proteins or RNA-imaging in vivo. Previous work has been done on the RNA-TAG system which uses an enzyme called tRNA Guanine Transgycosylase (TGT) to label a 19-nucleotide hairpin motif. A specific guanine in the hairpin is replaced for a functionalized PreQ1 probe. Initial work with this system used E. coli derived TGT. While this enzyme achieves good labeling efficiency in vitro, it also labels endogenous ‘off target’ mRNA abundant in all cells. We found, using qPCR, increased labeling specificity from a variant of the TGT enzyme from the bacteria Z. mobilis. Zm TGT has nearly 1 order of magnitude greater specificity toward the ‘on target’ over the ‘off target’ labeling when compared to E. coli. We confirmed in vitro. We also explored the structure function relationship in this system we also generated enzyme and RNA-hairpin mutants and characterized their activity in vitro through labeling and gel shift assay.
Abstract Title : Characterizing Wnt16 signaling with noncanonical Wnt reporter assays
Abstract : Wnt genes encode secreted signaling proteins that regulate several developmental processes including the development of blood stem cells. Wnt signaling has been grouped into two families, canonical (β catenin dependent) and noncanonical (β catenin independent). Previous work in the laboratory has shown that Wnt16 is required for blood stem cell specification in zebrafish, but does not signal through β catenin. Similar experiments have shown that Fzd5 is also required for blood stem cell specification independent of β catenin and has been identified as a candidate receptor for noncanonical Wnt16 signaling. My project focuses on developing noncanonical Wnt reporter assays in order to demonstrate that Wnt16 signals through Fzd5 through one of the noncanonical pathways (PCP or Ca2+). The activation of these noncanonical pathways was quantified in vitro using luciferase reporter assays in order to characterize the Wnt16-Fzd5 signaling interaction. Our results demonstrate that in vitro Wnt16 interacts with Fzd5 to activate the PCP pathway, but not the Ca2+ pathway. By better understanding the roles that Wnt signaling plays in regulating blood stem cell development, we hope to develop protocols to develop blood stem cells in vitro in order to replace current leukemia treatments.
Abstract Title : What Strategies Do Students Use on Free Response Test Questions?
Abstract : When students have to generate their own responses, subsequent retention of information is greater than when students have to recognize a correct response, as is the case with multiple-choice questions (Kang, et al., 2007; McDaniel et al., 2007). While previous studies have looked into the strategies students use when preparing for a test (Hartwig, et al., 2012; Ross, et al., 2006) no studies have assessed the strategies that students use when taking the test itself. This study was designed to address the following questions: What strategies do students use during open-ended exam questions? Why do students use those strategies? Do the strategies used change in response to metacognitive reflection assignments? To answer question one, survey data was collected after two midterms in BIPN 134 (Human Reproduction) in which we asked students what strategies they use during open-ended questions. To answer question two, we asked them why they may have included extraneous information on a reflection assignment. Question three was analyzed by comparing the different binned responses that students gave to the study strategy question after midterm one and two. Current data collected suggests that students alter their test taking strategies from midterm one to midterm two based on difficulty perception and time constraints felt during midterm two; we find that students tend to shift towards methods of improving time and conciseness on midterm two.
Abstract Title : Identification of New Antibiotics with Cell Wall-targeting and Anti-cancer Mechanisms
Abstract : Developing trends of antibiotic resistance pose one of the greatest threats to modern medicine, and for this reason, there is a pressing demand for new antimicrobials with unique molecular targets. In particular, the cell wall has proven to be an optimal target for clinically used antibacterials, as its absence in mammalian cells fosters an acute, targeted elimination of infectious bacteria in patients treated with cell wall-targeting antibiotics. In this study, I used bacterial cytological profiling (BCP) to screen a number of compounds from the National Cancer Institute's compound library in an attempt to identify antibiotics that target the cell wall of Escherichia coli. Of the compounds screened, I identified six very promising cell wall inhibitors and one DNA intercalator with certain anti-tumor activity via the inhibition of topoisomerase II. Many of the cell wall-targeting antibiotics that I have identified produced highly unique cell morphologies, differing substantially from those produced by the clinically used penicillins, carbapenems, and cephems. This uniqueness suggests novel and potentially undiscovered targets of these new antibiotics.
Advisor : YANG
Abstract Title : Development of Protein Loaded Hydrogels for Delivery to Peripheral Nerve
Abstract : Neuropathic pain is a type of chronic pain resulting from sensory nerve damage in the peripheral nervous system. This disorder is untreatable and is expected to rise due to aging of the global population, increased rates of Diabetes Mellitus, and improved survival of cancer patients after chemotherapy. Proposed treatment for this condition includes the use of biologics like the hemopexin domain of matrix metallopeptidase 9 (MMP 9 PEX). MMP 9 PEX is a protein which has been found to promote Schwann cell migration, believed to regenerate damaged nerve fiber, and therefore could potentially be used for treatment of neuropathic pain. To deliver a protein like MMP 9 PEX to a peripheral nerve, alginate hydrogel has been used in the past; however, calcium ions that are used to crosslink the hydrogel have been shown to induce an inflammatory response. One way to circumvent high calcium concentration is to dope alginate gels with agarose to produce composite gels. Here we study the effects of agarose and calcium concentration on the preparation of protein loaded alginate-based hydrogels for MMP 9 PEX delivery to a peripheral nerve. Recombinant glutathione S-transferase (GST), a model protein for MMP 9 PEX, was first produced and chemically modified with a fluorescent tag, 5(6)-carboxyfluorescein. The fluorescent GST was then loaded in alginate and agarose-alginate hydrogels and its release was measured in vitro by monitoring fluorescence. Analysis of GST kinetic release profiles was finally used to identify the optimal hydrogel formulation for in vivo application.
Abstract Title : Evaluating clinical texts using Natural Language Processing to predict patient neurological disorders
Abstract : Neurological disorders affect up to 1 billion people worldwide, and include disorders of the nervous system, such as vertigo, neurotology, and migraines - of which affects at least 1 in 4 United States households. However, compared to the influx of patients who require urgent consultation, trained medical personnel are in short supply. The untreated condition subsequently worsens, sometimes to disability, with the increasing wait time. In order to prevent avoidable disabilities and critical health declines, we used Natural Language Processing to analyze clinical records from the UCSD Medical Center. In identifying correlations between subjective patient symptoms and doctor assessments within the structure of the text, we will later translate our findings into the foundation of a conversational agent intended for patient neurological evaluation and follow-up. Keywords: machine learning, natural language processing, clinical data, symptoms. CO-PRESENTERS: JENNY LEE, RAGHAV METHA
Abstract Title : The Expression of DRA Following Enteric Infection
Abstract : Salmonella is the most burdensome foodborne illness worldwide, yet despite its prevalence, the mechanism to which Salmonella causes diarrhea is not entirely known. Intestinal ion transporters play important roles in the water absorption in the intestine and can be a therapeutic target in Salmonella-induced diarrhea. Previously, it has been found that Salmonella infection causes a decreased expression of the chloride/bicarbonate exchanger SLC26A3 (DRA; Down-Regulated in Adenoma) in the Caco-2 cell line. To better understand the mechanism of Salmonella infection in intestinal epithelial cells, a stem cell based enteroid model has been developed that mimics the crypt-villus axis of the intestines. With this model, it has been found that DRA is downregulated after Salmonella infection, similar to that seen in vivo.
Advisor : ENFU HUI
Abstract Title : Understanding the T Cell Receptor (TCR) Architecture Using a Membrane Reconstitution System
Abstract : T cells are a type of lymphocyte that are able to recognize and destroy pathogens and tumor cells. The initial step in T cell activation is the binding of antigen peptide to the TCR, which causes Lck mediated phosphorylation of its associated CD3 chains, including a ζζ homodimer, a γε heterodimer, and a δε heterodimer. It is well established that ζζ homodimer recruits ZAP70, a key cytosolic kinase, upon tyrosine phosphorylation. While much research has focused on CD3ζ, the roles of other CD3 chains are less well understood. It is not clear why they are needed, what cytosolic proteins they recruit, and why they assemble into heterodimers. To begin to address this question, we sought to reconstitute the CD3 chains, together with the kinase Lck, onto liposomes, which mimic a cell membrane. This reconstitution system has allowed us to measure direct protein-protein interactions in a well-defined membrane environment. Using our recently developed fluorescence resonance energy transfer (FRET) assay, I compared the abilities of individual CD3 chains to recruit ZAP70 and several other T cell signaling proteins. Furthermore, using a chemically induced heterodimerization system, we assembled the γε heterodimer or δε heterodimer on liposomes and compared their abilities to recruit signaling proteins. Our data suggest that ZAP70 is the major effector for all CD3 chains and the dimerization of CD3 chains might lead to recruitment of additional cytosolic factors.
Abstract Title : Genomic and Proteomic Analysis of the Novel Streptomyces Phage Darolandstone
Abstract : The annotation of phage genomes allows us to better comprehend the mechanisms of phage infection and evolution. The Streptomyces phage Darolandstone is one of the bacteriophages our class isolated in order to have its genome sequenced and annotated. We were able to annotate this genome using an annotation software and various bioinformatics tools with the objective of identifying the most likely start sites of the genes and determining the functions of the proteins they encode for. By considering factors such as the coding potential, gaps and overlaps found between genes, and comparisons to the NCBI BLAST database, we were able to identify 54 putative open reading frames (ORFs). Of the 54 ORF’s, we were able to assign functions to 20 genes while the remaining 34 ORFs code for hypothetical proteins with no known function. In addition, we utilized mass spectrometry data from the phage lysate to substantiate certain function assignments, mainly those of structural proteins. Our findings indicate that Darolandstone is closely related to the Streptomyces phage Raleigh, although similarities to other phages suggest a broader relationship between Streptomyces bacteriophages. CO-PRESENTERS: Cianna Calia, Aaron Oliver, Keeley Martinez, and Noorhan Amani.
Abstract Title : A Global Transcriptional Network Connecting Noncoding Mutations to Changes in Tumor Gene Expression
Abstract : Although cancer genomes are replete with noncoding mutations, the effects of these mutations remain poorly characterized. Here we perform an integrative analysis of 930 tumor whole genomes and matched transcriptomes, identifying a network of 193 noncoding loci in which mutations disrupt target gene expression. These ‘somatic eQTLs’ (expression quantitative trait loci) are frequently mutated in specific cancer tissues, and the majority can be validated in an independent cohort of 3,382 tumors. Among these, we find that the effects of noncoding mutations on DAAM1, MTG2 and HYI transcription recapitulate in multiple cancer cell lines, and that increasing DAAM1 expression leads to invasive cell migration, in vitro. The somatic eQTL network is disrupted in 88% of tumors, suggesting widespread impact of noncoding mutations in cancer.
Abstract Title : Wnt-Fzd Specificity in Hematopoietic Stem Cell Development
Abstract : Hematopoietic Stem Cells (HSCs) are blood stem cells, and one candidate pathway for HSC development is the Wnt-β-Catenin (Wnt hereafter) pathway. Our laboratory previously demonstrated Wnt signaling is necessary for proper HSC development through the Wnt ligand Wnt9a and the Frizzled (Fzd) receptor Fzd9b. My research characterizes how features of the Fzd receptor affect Wnt ligand signaling through the Fzd receptor. Our laboratory utilized luciferase reporter assay for Wnt signaling to demonstrate that Wnt9a signals through Fzd9b but not Fzd8a. We made Fzd9b (signaler) constructs that have one or more domains replaced by the corresponding domain in Fzd8a (non-signaler). The function of these chimeric Fzd proteins was characterized in vitro by quantifying Wnt signaling in cells using the luciferase reporter assay. It was determined that the ICL-3 and CTT confer all of the signaling specificity between Fzd9b and Wnt9a. Future experiments will confirm these specificity effects in vivo using a zebrafish model.
Abstract Title : Generating Substrates of The Ubiquitin Proteasome System With Optics
Abstract : Eukaryotic cells have evolved exquisitely specific systems that recognize and degrade misfolded proteins. Collectively, these systems are called protein quality control (PQC). A major arm of PQC is managed by the ubiquitin proteasome system (UPS). Decades of research show that the UPS rapidly recognizes and degrades a wide variety of model substrates, such as truncated and mislocalized proteins. However, model substrates resulting from individual point mutations are rare and difficult to find. Systematically screening single point mutants for degradation is impractical because it requires tremendous throughput. Fortunately, GFP-tagged model substrates show diminished fluorescence because they misfold and degrade in the UPS. Using degradation to indicate which mutants cause misfolding, we developed a screen to study the structural consequences of point mutations on proteins. The coding region of a well-characterized protein with a known crystal structure was randomly mutagenized via PCR-mediated random mutagenesis and fused to GFP to create a library in yeast. The random mutant library was amenable to optical screening. To improve throughput, we built an ergonomic workstation that illuminates GFP and allows us to conduct the screen quickly. We have screened upwards of 12,000 mutant colonies at once, which have yielded ~30 PQC substrates to date.
Abstract Title : Understanding Students' Knowledge Frameworks about Biology Research
Abstract : A major objective in exposing students to research and inquiry experiences during their undergraduate courses is for them to adopt scientific thinking behaviors and more developed and expert-like knowledge structures about biology and research. Knowledge structures are ways of organizing information and experiences. For instance, novice knowledge structures about research may simply be that research is the scientific method, whereas more sophisticated knowledge structures may include further connections between data, questioning, and iteration with research. These behaviors and mindsets may be developed by engaging in scientific inquiry and during research in laboratory courses. Word Association Tests are useful tools to map knowledge structures. This project aims to use word association tests to determine: 1) What knowledge structures do students have relating to scientific inquiry (research) when they enter into the biology program, and in upper division biology? 2) How do the course-based research experiences and inquiry lab experiences we offer change student knowledge structures? 3) How do the knowledge structures of experienced scientists compare to that of students? Data collected from more than 500 undergraduates engaging in either a course-based research experience (CURE) or an inquiry-style laboratory course (non-CURE) reveal varied knowledge structures that are particularly influenced by course-based research experiences. We also find very distinct differences between students and expert knowledge frameworks, particularly related to experimental design. Implications of these findings for teaching, design of CUREs, and understanding student thinking will be discussed.
Abstract Title : Improving Cyanobacteria Strain Fitness in Outdoor Ponds
Abstract : Cyanobacteria are a type of bacteria that generate their energy via photosynthesis. They are the only photosynthetic prokaryote that are able to produce oxygen. Because of this environmental friendly characteristic, cyanobacteria are potential candidate for biofuel source. Despite their useful application, efficient growing of cyanobacteria may be difficult. Today, cyanobacteria are often grown in outdoor ponds, which means they are constantly exposed to the natural light and dark condition. Since cyanobacteria is a photosynthetic organism, they need light as food source to grow. Therefore, the constant light and dark natural cycle threatens the cyanobacterial growth. To address this issue, we hope to find the cyanobacterial mutation that allow cyanobacteria to grow faster under light-dark cycle by knocking out individual genes and study their survival performance after exposure to light/dark condition.
Abstract Title : "What’s in an argument?" Using a simplified Toulmin framework to quantify student arguments
Abstract : Scientific argumentation is a crucial skill to master for developing students and researchers. However, argumentation practice has not traditionally been taught in the classroom setting, leading to deficits in this skill among students. Our project involves developing a framework to quantify and examine the content of student arguments. Using this framework, samples of student arguments taken from undergraduate genetics courses in two years were quantified. We assessed these arguments by breaking them down into sentences and ideas, so that we were able to determine the proportion of claims made, evidence used, and mechanisms proposed, using the Toulmin model of argumentation. Current findings revealed that students have proportionally higher usage of evidence compared to the other categories, and this trend was consistent among student populations from two years. These results act as a baseline to compare student populations and could allow insight about how to better teach argumentation in our classes. Our framework works as an assessment tool for analyzing arguments, and could be used to measure the effect of educational interventions on argumentation, and to compare the compositions of arguments between novices and experts. CO-PRESENTER: JOSEPH LEE
Abstract Title : Engineering Entry Inhibitors to More Potently Bind HIV Envelope Proteins
Abstract : Human immunodeficiency virus 1 (HIV-1) attacks the immune system, currently affecting over 36.7 million people worldwide. Despite intensive research, no prophylactic vaccine has been developed to date. An alternative strategy to prevent HIV infection involves passive administration of neutralizing agents capable of providing sterilizing immunity. HIV-1 gains entry into a target cell via interaction between its viral envelope glycoprotein (Env) and the cellular receptor, CD4. The purpose of this project was to optimize an artificial entry inhibitor that competitively inhibits interaction of Env and CD4, for increased binding breadth and potency. Utilizing directed evolution via yeast surface display, variants of a candidate inhibitor were screened through fluorescent-activated cell sorting (FACS) for constructs with increased affinity against diverse HIV envelope proteins. These selected variants with optimized binding profiles might provide improved protection against HIV infection and might help suppress viral load in infected individuals, and are therefore promising candidates for future HIV prevention or therapy strategies.
Abstract Title : Smooth Muscle Cell Heparan Sulfate Proteoglycans Mitigate Atherosclerosis Formation
Abstract : Atherosclerosis is the buildup of fatty deposits, mostly cholesterol, in the artery walls. It is a major cause of cardiovascular disease that is responsible for the death of 17 million people worldwide every year. Atherosclerotic lesions initiate by the trapping of remnant lipoproteins, mainly low-density lipoprotein (LDL) cholesterol, by proteoglycans in the underlying matrix of damaged or activated arterial endothelium. The body tries to defend against the accumulation of LDL by recruiting macrophages to remove the cholesterol particles. Cholesterol-rich lipoprotein remnants get internalized via phagocytosis or scavenger receptors without constraints and induce massive cholesterol accumulation turning macrophages into lipid droplet-laden cells, called foam cells. The accumulation of foam cells leads to fatty streaks and plaques. The expansion of plaques leads to reduced blood flow and blood clots, which can cause life-threatening strokes or heart attacks. The goal of this project is to see how the vascular smooth muscle cell (SMC) heparan sulfate proteoglycans (HSPGs) affect the development of atherosclerosis in mice. To test this we used mice lacking the HSPG biosynthetic gene GlcNAc N-deacetylase/N-sulfotransferase 1 (Ndst1) selectively in SMCs by crossing Ndst1f/f mice with SM22aCre+ mice. Ndst1 inactivation results in significantly undersulfated HSPGs. Weekly injections of LDL receptor (LDLR) anti-sense oligonucleotides (ASO) for 10 weeks and a high fat diet for the final 8 weeks were used to induce hypercholesterolemia and the associated atherosclerosis in both the wild type mice and Ndst1f/fSM22aCre+ mice. Lack of SMC Ndst1 did not affect body weight gain, livers steatosis, circulating plasma cholesterol and triglycerides nor did it alter the plasma lipoprotein profile. Atherosclerotic lesions quantification in the aortic root showed a 63% increase in lesion formation in Ndst1f/fSM22aCre + mice compared to wild type mice treated with the LDLR ASO. Analysis of the lesions suggest that the atherosclerosis lesions in Ndst1f/fSM22aCre + mice have an increase in SMC content. The results suggest that HSPG produced by SMC in the artery wall are atheroprotective and do not promote the trapping of LDL particles. Further studies will examine the intricate workings of how HSPG can affect SMC biology relevant for atherosclerosis development, including proliferation, migration and inflammation.
Abstract Title : The ELMO1, A Microbial Sensor Regulates Bacterial Clearance and Endo-Lysomal Signaling
Abstract : Intestinal antigen presenting cells (APCs) are a major component of the mucosal immune system and maintain the gut homeostasis and the inflammation level. Microbial sensors present on APCs recognize microbes irrespective of their pathogenicity but little is known about their mechanism that leads to host cellular responses unique to bacterial pathogenesis. Previously, we found that BAI1 (Brain Angiogenesis Inhibitor 1) recognizes bacterial LPS from pathogenic and non-pathogenic bacteria. BAI1 binds cytosolic protein ELMO1 (Engulfment and cell motility protein 1), which facilitates the engulfment of bacteria via Rac1 activation and induces the pro-inflammatory cytokines and regulates bacterial clearance. Interestingly, ELMO1 interacts with Salmonella effector SifA involved in bacterial survival. Therefore, we hypothesized that ELMO1 is a cytosolic sensor that interacts with bacterial effectors involved in the pathogenesis of enteric infection, controls bacterial clearance and provides differential immune responses between pathogens and commensals. In silico analysis of bacterial database revealed that Salmonella effector SifA shares a WxxxE signature motif present in several effectors from other enteric pathogens, including IpgB1 in Shigella that binds ELMO1. This signature motif is not present in commensal gut microbes. Interestingly, mutation in the WxxxE motif of SifA abolished interaction with ELMO1.
Abstract Title : Isolation and Characterization of Bacteriophage from Soil
Abstract : Bacteriophage, viruses that infect bacteria, are one of the most abundant microbes in the universe. Because of their vast genetic diversity, there is a lot of potential in discovering new types of bacteriophage, which lends to our knowledge of the complexity of the microbial world. Due to the emergence of antibiotic resistant bacteria, alternative practices, such as phage therapy, can be utilized to treat bacterial infections. Our study focuses on isolating novel strains of bacteriophage that infect Streptomyces platensis bacteria due to its wide host range and abundance in soil. Our class collected soil samples from various locations around UC San Diego and the local San Diego area and enriched the soil with bacteria to amplify the amount of phage for future isolation. Through continuous purification of the phage, we were able to isolate the phage and then extract its DNA. Utilizing electron microscopy, we characterized the phage as either siphoviridae or myoviridae by analyzing their capsid and tail dimensions and overall appearance. Altogether, our class isolated and named approximately 25 different bacteriophage. Out of the 25 bacteriophages, 2 bacteriophage genomes, Hank144’s and Darolandstone’s, were sequenced and annotated. CO-PRESENTERS: Ella Chen, Lindsay Hill, Sarna Karpinska, and Jessica Pham.
Abstract Title : Discovery and application of the RNA-Targeting CRISPR Effector Cas13d in Transcriptome Engineering
Abstract : Discovery of the microbial adaptive immune system CRISPR (clustered regularly interspaced short palindromic repeats) has led to the development of DNA engineering technologies, with CRISPR-Cas9 being the most widely applied system. While Cas9 has a wide variety of applications in DNA-targeting and editing technologies, RNA-targeting technologies have remained relatively limited. Recently, our group analyzed prokaryotic genome and metagenome sequences and identified Cas13d, a family of RNA-guided, RNA-targeting CRISPR proteins. The initial computational pipeline yielded seven distinct orthologs, which we screened for RNA knockdown in human cells. This revealed variations in targeting efficiency, but one effector from Ruminococcus flavefaciens XPD3002 mediated 90% knockdown. Due to increased access to microbial genomic sequences through online databases, we sought to expand our computational search to identify more orthologous Cas13d proteins with greater efficacy in human cells. This search uncovered 44 new orthologs of Cas13d, which were cloned into mammalian expression constructs, and assessed for mRNA knockdown efficiency by flow cytometry. Moving forward, we will adapt these orthologs for various programmable RNA-sensing, binding, editing, and knockdown applications.
Abstract Title : Probing the Descending Pain Pathway
Abstract : Opioids are the primary treatment for severe pain, however these compounds are incredibly addicting and alternatives are needed to combat the growing epidemic of abuse. Elements of the descending analgesia pathway are ideal targets for developing alternative therapies as they are distinct from addiction circuitry. In order to identify constituent cell types in the descending analgesic pathway, we examined the expression of a myriad of neuromodulators known to affect endogenous analgesia using fluorescent in-situ hybridization in the Periaqueductal Grey (PAG). Co-localization of neuromodulatory molecules with markers for glutamate or GABA identified several putative cell classes that may underlie the analgesic actions of neuromodulators in the PAG. Current experiments aim to refine cell class definition based on the expression of multiple markers as well as their projections to downstream nuclei. Defining these cell classes may identify novel therapeutic targets for non-addictive pain-killers.
Abstract Title : Brain state-dependent modulation of sensory representations in layer 2/3 of primary auditory cortex
Abstract : Sensory processing in the neocortex is continuously modulated by changes in behavioral and cognitive state. Thus, deconstructing the complex and multi-faceted relationship between brain state and sensory responses is key to understanding how our brains represent the world around us. In deep layers of the primary auditory cortex (A1) of mice, the magnitude and reliability of sound-evoked responses have been shown to be maximal at intermediate states of arousal (McGinley et al., 2015). However, the manner in which brain state modulates sensory responses in superficial layers of A1—where the bulk of intracortical auditory processing is thought to occur—is less well understood. To address this question, we monitored pure tone-evoked responses using whole cell current clamp or two-photon GCaMP6s calcium imaging of layer 2/3 (L2/3) pyramidal cells in awake, head-fixed mice situated on a linear treadmill. We simultaneously measured fluctuations in arousal and locomotion via pupillometry and treadmill activity tracking, respectively. In contrast to previous observations in deep layers of A1, we found that the magnitude and reliability of L2/3 tone-evoked suprathreshold responses increased linearly with arousal, peaking at high states of arousal in the absence of locomotion. Increases in arousal also enhanced the amplitude and duration of tone-evoked excitatory postsynaptic potentials (EPSPs). Thus, the relative sparseness of sensory representations rapidly changed depending on the level of arousal. Furthermore, although changes in arousal did not alter the best frequency of individual cells, increases in arousal broadened frequency tuning. ­­We then used voltage-clamp recordings to study the excitatory and inhibitory postsynaptic currents (EPSCs and IPSCs) underlying tone-evoked responses. Surprisingly, increases in arousal were associated with modest decreases in short-latency, evoked EPSCs and IPSCs locked to tone onset. However, we observed that lateral inhibition generated by a slow, tone-evoked withdrawal of ongoing, recurrent excitation (“network suppression”, Kato et al., 2017) was strongly suppressed as arousal increased. These results reinforce the notion that A1 operates as an inhibition stabilized network and show that the modulation of recurrent activity underlies stronger and more broadly tuned tone-evoked responses in aroused brain states. McGinley, M.J., David, S.V., and McCormick, D.A. Cortical membrane potential signature of optimal states for sensory signal detection. Neuron. 2015; 87: 179-192. Kato, H.K., Asinof, S.K., and Isaacson, J.S. Network-level control of frequency tuning in auditory cortex. Neuron 2017: 95(2): 412-423. CO-PRESENTER: KIMI TAIRA. S.K. ASINOF,* P.A. LIN,* K.G. TAIRA, E.A. WESTEINDE, J.S. ISAACSON *These authors contributed equally.
Abstract Title : Genome Analysis of Streptomyces Phage Hank144
Abstract : Our research investigates the Streptomyces phage, Hank144, and its relation to other sequenced phages as well as the possibility of novel proteins coded for by its genome. To determine this, we annotated the phage genome using the program, DNAMaster, and performed mass spectrometry analysis. The annotation process consisted of our class of BIMM171B students dividing into pairs where half of the groups annotated open reading frames or ORFs 2-38 and the other half annotated ORFs 39-76. After annotation was complete, these pairs consolidated into groups of six to discuss the annotations made and confirm that the same conclusions had been reached, ensuring overall accuracy. The tools used during the annotation process were the auto-annotation using the annotation software DNAMaster, a coding potential graph generated by Genemark for Hank144, Protein BLAST, HHPred, and Phamerator. In order to decide the length of each ORF during annotation, we used of a hierarchy of start codon indicators consisting of 1) Genemark coding potential for Hank144, 2) the size of the gap or overlap with other ORFs, 3) whether or it was the longest ORF, 4) BLAST protein matches, and 5) the RBS score for any particular start codon. We determined from this analysis that were 75 ORFs in Hank144’s genome that codes for at least 27 unique protein functions.Using Phamerator, a genome comparison tool, we determined that Hank144 belonged to the cluster BD, consisting of 59 phage, and the subcluster BD2, consisting of 20 phage. Hank144 was most related to the Streptomyces phage, SqueakyClean. Our mass spectrometry analysis confirmed that some of the hypothetical proteins identified indeed coded for peptides. We also were able to determine some of the post-translational modifications of Hank144’s peptides. In the future, we hope to conduct further analysis of hypothetical proteins within the genome and determine how they function for the phage. CO-PRESENTERS: Marina Ramsey, Caroline Shi, William Bushnell, Neil Shende.
Advisor : JING W. WANG
Abstract Title : Exploring the Role of Sensory Information in Patterning Complex Social Behaviors in Male Drosophila Melanogaster using a High-Resolution Assay
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. In this research, we try to address 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. In this research, we made video recordings to obtain the tracks of movements of flies and applied machine-learning algorithms to classify each of stereotyped behaviors. We analyzed how male flies choose specific courtship or non-courtship behaviors to perform depending on relative position and movement of female flies. Furthermore, we used visual deprivation and genetic mutations to specifically eliminate single sensory cues and tested how these manipulations altered the behavioral strategy of the male fly. With this approach, we developed an understanding of how sensory information is constantly monitored under different contexts to pattern behavior for optimal outcomes. CO-PRESENTER: VICKY MAK
Abstract Title : Plagiarism Awareness Writing Assignments Reduce Plagiarism and Writing Problems in an Upper Division Laboratory Course
Abstract : In many of our courses, particularly laboratory courses, students are expected to engage in scientific writing. As instructors, we are often faced with the frustration of student plagiarism and common writing problems. Here, we describe a very simple intervention designed to help students become more aware of different types of plagiarism and writing problems, and how to avoid these problems. We will introduce types of plagiarism and writing problems commonly encountered, the intervention, and the results of our study. From the evaluation of 371 student reports from an upper division laboratory source, we found that implementation of the plagiarism intervention resulted in nearly 50% fewer instances of common type plagiarism and writing problems. We also observed nearly 40% fewer instances of severe plagiarism (e.g. several sentences copied from an external source). In addition, we found that the effects of this intervention last for several weeks after the students complete the plagiarism writing assignment. This assignment is particularly easy to implement, and can be a very useful tool for many instructors looking to improve student awareness of how to write in their own voice.