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2019 Research Showcase
HR Abstracts
Abstract Title : Characterizing the Antibiotic Properties of a Natural Product Library
Abstract : It is imperative to find and characterize novel antibiotics in order to combat antibiotic resistance. However, in recent years, few new and clinically useful antibiotics have been discovered. In this project I characterized the antibacterial properties of a library of natural products. I evaluated the minimum inhibitory concentration of the compounds against a panel of Gram-positive and Gram-negative bacteria to determine the spectrum of activity and potency. To elucidate the mechanism of action, I used bacterial cytological profiling, a fluorescent microscopy technique that allows visualization of the physiological response of bacteria to a given treatment. Results indicate that many of the compounds have a potency comparable to currently used antibiotics. Each of the major biosynthetic pathways is inhibited by at least one compound in the library. Some of the compounds could be clinically relevant, but further work is needed to determine the precise target of many of these compounds.
Abstract Title : Nanoluciferase as a Genetic Reporter for Measuring Activation of the Intracellular Pathogen Response Pathway
Abstract : The Intracellular Pathogen Response (IPR) is a newly identified transcriptional response in the nematode C. elegans that helps in tolerating proteotoxic stress, including intracellular infection. The gene pals-5 is one of the most highly induced IPR genes, making it an effective IPR reporter when its promoter is coupled to the highly sensitive bioluminescent reporter protein Nanoluciferase. Here, we used transgenic animals that express Nanoluciferase under the control of the pals-5 promoter to test triggers of the IPR, including those that perturb proteostasis and other core cellular processes. These studies should provide new insights into how animals cope with proteotoxic stress.
Abstract Title : Investigating Small Molecule-Induced Blood-Flow Gene Expression Pattern in Endothelial Cells
Abstract : Endothelial cells (ECs) line the interior of the entire circulatory system, and EC dysfunction is commonly associated with the development of vascular diseases. In the Lopez-Ramirez Lab, previous work has demonstrated that KRIT1 (Krev interaction trapped protein 1) and transmembrane protein HEG1 (Heart of Glass) directly interact with each other to downregulate the gene expression of transcription factors KLF2 and KLF4 (Kruppel-like factors), which are implicated in blood-flow induced gene regulation. Based on this information, we hypothesized that HKi001G, a HEG1-KRIT1 protein interaction blocker, would upregulate the gene expression of KLF2 and KLF4 when applied to ECs, thereby mimicking blood-flow gene expression with vasoprotective activities. During this experiment, we treated brain endothelial cell-line hCMEC/D3 with HKi001G at different concentrations and durations to investigate its efficacy. Our results indicate that both increasing HKi001G concentrations and durations lead to upregulation of KLF2 and KLF4 expression.
Abstract Title : Using shRNA Macrophage Lines to Test GIV's Function in Inflammatory Diseases
Abstract : Chronic inflammation is a key component of many devastating diseases, including inflammatory bowel disease, diabetes, and cancer. Despite the importance of inflammation as a protective immune process required for tissue homeostasis, dysregulation in key cellular signaling cascades can lead to unconstrained long-term inflammation, which can damage tissue and cause disease. Understanding the regulatory elements involved in maintaining the balance between pro- and anti-inflammatory states and the molecular events that can trigger disease is integral for developing novel therapeutic strategies to target these illnesses. My mentor's lab has recently identified the non-receptor guanine nucleotide exchange factor GIV/GIRDIN as a novel pivot point between pro-/anti-inflammatory signals and a possible target for therapeutics. Specifically, GIV interacts with the trimeric G-protein Gi and Toll-like receptor 4 (TLR4) to potentially regulate macrophages, which have been implicated in inducing inflammatory cytokine production and driving inflammatory disease when dysregulated. TLR4 is a well-studied pattern recognition receptor on the surface of macrophages that initiates the immune response when activated by specific pathogen-associated molecular patterns. Previous work in the lab has identified a motif in GIV’s C-terminus that binds certain modules in TLR4 downstream of ligands like lipopolysaccharide (LPS) and integrates these signals into the G-protein signaling pathway. I seek to explore and characterize the role of GIV in balancing the pro-/anti-inflammatory signaling cascades that regulate macrophage inflammatory responses. To accomplish these goals, I used a macrophage cell model previously developed in the lab using short hairpin RNA (shRNA) to remove GIV and rescued the phenotype using two methods: transfection with a recombinant plasmid with the C-terminus of GIV and transfection of a cell-permeable exogenous recombinant protein. This experimental system will allow me to interrogate the precise mechanism by which GIV regulates the pro-/anti-inflammatory switch in macrophages and provide valuable insight into the development of a therapeutic target.
Abstract Title : STAT3 suppresses fatty acid re-esterification in adipocytes via interaction with GPAT3
Abstract : Stimulation of the sympathetic nervous system promotes the secretion of catecholamines from nerve terminals in adipose tissue. Catecholamine signaling in adipocytes promotes the mobilization of energy-rich triglycerides through a process of energy expenditure called lipolysis which results in the breakdown of triglycerides to free fatty acids (FFA) and glycerol. The resulting FFAs are secreted from the adipocytes to be taken up and oxidized for energy throughout the body. However, adipocytes reuptake some of the secreted FFA and either oxidizes them for energy or re-esterify them for storage as triglycerides. The Reilly lab has found that the oxidation of FFA produced by lipolysis in adipocytes depends on the suppression of FFA re-esterification by STAT3. Due to increased re-esterification and reduced oxidation of fatty acids in the white adipose tissue, adipocyte-specific Stat3 KO mice were found to be more sensitive to diet-induced obesity. During my research, I discovered that STAT3 interacts specifically with Glycerol-3-phosphate acetyltransferases-3 (GPAT3), which catalyzes the first-committed, and rate-limiting step of FFA re-esterification in adipocytes. Moreover, since STAT3 is activated by its phosphorylation, I investigated the role of the phosphorylatable residues on the interaction of STAT3 with GPAT3 and found that their mutation affects STAT3 interaction with GPAT3. Therefore, the interaction of STAT3 with GPAT3 may be regulated by STAT3 phosphorylation.
Abstract Title : First Steps to New Antibiotics
Abstract : Despite the initial effectiveness of extant antibiotic treatments, we have nearly exhausted their capabilities due to increasing numbers of resistant bacterial strains. If we wish to continue employing antibiotic treatments, it is essential that new antibiotics are identified and that their mechanism of action is determined. Bacterial Cytological Profiling is a fluorescence microscopy technique used to screen potential antibiotics by characterizing their effect on cellular pathways. Using this technique, two small molecules with low minimum inhibitory concentrations have been identified as DNA replication inhibitors and folate synthesis inhibitors. Resistant mutants against both compounds have been isolated, and Next Generation Sequencing revealed that these resistant mutations to the DNA synthesis inhibitor lie in gyrB—a gene encoding a subunit of DNA gyrase—and resistant mutations to the folate synthesis inhibitor lie in several genes associated with sulfonamide drug resistance. This project aims to investigate how these compounds are interacting with the proteins encoded by these affected genes, and how these interactions inhibit the growth of bacteria.
Abstract Title : Identifying Genomic Variants Associated with Stomatal Insensitivity in Response to Elevated CO2 Level in Brachypodium Distachyon & Arabidopsis Thaliana Guard Cell Transcriptome Expression Database
Abstract : In this study, I use Unix-based software programs to identify potential genomic variants that lead to stomatal insensitivity in the grass species Brachypodium distachyon in response to elevated CO2 level. I built a genomic variant identification pipeline. The pipeline uses tools in the Genome Analysis Toolkit (GATK) to identify variants in whole-genome sequencing data of Ethyl-methanesulfonate (EMS) mutant lines that show a mutant phenotype and uses SnpEff to annotate identified variants with gene-identifier names. After filtering-out common false positive variants, by running the pipeline on six EMS mutant lines with other phenotypes, I discovered 164 and 143 potential mutations causing variants in two mutant lines with promising phenotypes. I also collected a set of common false-positive variants and regions with rich false positive variants, using eight independent genome sequences of the parent line. Additionally, I built an Arabidopsis transcriptome expression database to facilitate further studies on stomatal biology and genetics.
Abstract Title : Intra-retinal Circuit of Intrinsically Photosensitive Retinal Ganglion Cells in the Mouse Retina
Abstract : Using a genetic mouse line (Opn4/Cre Knock-in mice), serial blockface electron microscopy, and a viral vector (MiniSOG), we have reconstructed the circuit structure of Intrinsically Photosensitive Retinal Ganglion Cells, or ipRGCs, within sections of the mouse retina. By studying the connections and cellular structure of ipRGCs, we increase our understanding of how ipRGCs may mediate non-image forming vision functions such as circadian photoentrainment and pupil constriction. Specifically, we studied the frequency of synaptic input along the dendrites of ipRGCS and the sources of these synaptic inputs. We report that several types of bipolar cells and amacrine cells synapse onto ipRGC dendrites with varying frequency depending on the location of the dendrite in the retina and the ipRGC subtype on the postsynaptic side. We also investigated the localization patterns of different neural processes within the retinal volume and discovered fasciculation for ipRGC axons in the ganglion cell layer of the retina.
Abstract Title : A Unique Epitope-Specific CD4T Cell Response is Crucial in Resolving Persistent MCMV infection 
Abstract : Prior research has shown that CD4T cells are critical for resolving persistent cytomegalovirus (CMV) infection. CD4T cells recognizing an epitope derived from the CMV m09 protein, which expand at later time points compared to canonical CD4T cells are particularly important for resolving CMV replication in the salivary gland and driving the virus into latency. These ‘late rising’ m09 cells display a unique phenotype. Hence, we sought to compare m09-specific CD4T cells to canonical CD4T cell specific for a m25- derived viral epitope, using data derived from bulk-cell RNA sequencing of these two T cell populations. Our results show that these two subsets of CD4T cells show unique gene expression profiles that vary by the time of infection.
Abstract Title : Assembly of a Nanobody-Based Fluorescent Sensor for the Neuropeptide Substance P
Abstract : The neuropeptide substance P (SP) is secreted by nerves and inflammatory cells and acts by binding neurokinin receptors. Because it has a high affinity for its receptors, only low concentrations accumulate in extracellular fluids upon secretion. Thus, determining SP release sites and the timescale of signaling is very challenging. We, therefore, set out to develop a genetically-encoded fluorescent sensor of SP. Our design is based on a SP-binding nanobody coupled through circularly-permuated GFP (cpGFP) to a low-affinity tethered SP variant (SPv), such that fluorescence is enhanced when endogenous SP displaces SPv. The sensor scaffold and a control construct lacking SPv were constructed using a combination of Gibson assembly and restriction enzyme-based cloning. The resulting constructs were then expressed in e. Coli, purified, and analyzed by fluorescence spectroscopy.
Abstract Title : Effect of Gamma-Secretase Modulator on the Level of Autophagy Activation in Cells with Mutations for Early-onset Familial Alzheimer’s Disease
Abstract : Amyloid beta (Abeta) plaque formation, which is a hallmark of Alzheimer’s disease (AD), can be caused by autophagy and endosome dysregulation due to inefficient cleavage of amyloid precursor protein (APP) by gamma-secretase. This APP processing maintains the normal level of the endosomal and autophagic activity in cells with familial AD mutations, such as APP and presenilin (PS-1 and PS-2) mutations. Gamma-secretase modulator (GSM) allosterically increases the efficiency of APP processing by reducing the production of amyloidogenic Abeta peptides. We hypothesize that GSM treatment results in decreased endosomal and autophagic activity in familial AD neurons. In this study, neurons with APP, PS-1, and PS-2 mutations were treated with GSM. The endosome markers, Rab5 and Lamp2 were analyzed by immunofluorescent imaging and biochemistry. The autophagy markers, LC3 and P62 were evaluated by western blot. We predict that autophagic and endosomal activity will restore to normal function with GSM treatment.
Advisor : JU CHEN
Abstract Title : The Role of NEDD4 Protein in Cardiac Function and Disease
Abstract : Neuronal precursor cell-expressed developmentally downregulated 4 (NEDD4) is a member of the NEDD4 family of HECT E3 ubiquitin ligases. NEDD4 is highly expressed in cardiomyocytes. However, the specific role of NEDD4 in cardiomyocytes is unknown. To study the role of NEDD4 adult cardiomyocytes, we have generated a Nedd4 flox mouse model and crossed it with tamoxifen-inducible MHC-MerCreMer mice to generate an inducible Nedd4 cardiomyocyte-specific knockout mouse model (Nedd4 icKO). In this study, we performed comprehensive molecular and cardiac physiology studies on Nedd4 icKO mice. Our results show that Nedd4 icKO mice displayed cardiomyopathy after tamoxifen-induced gene deletion. Utilizing the Nedd4 icKO mice model, we will be able to gain significant insight into mechanisms by which loss of NEDD4 in adult cardiomyocytes results in cardiomyopathy. Ultimately, this will bring us closer to our long-term goal of identifying new therapeutic targets for human cardiomyopathy.
Abstract Title : Cross leveraging maize and sorghum to understand biochemical layers of immunity
Abstract : In planning for increased production to match the increasing world population, large-scale and long-term maize (Zea mays) and sorghum (Sorghum bicolor) monocultures will predictably experience increased biotic and abiotic stresses. With a recent evolutionary divergence, these two important crops are among the worlds most closely related and are certain to share specialized metabolic pathways that play central roles in stress resilience. To elucidate conserved and unique pathways, we performed identical fungal-elicitation treatments on plant tissues of similar developmental stages and performed GC/MS and LC/MS-based metabolomics. Maize contains a diverse array of inducible oxygenated terpenoids while few have been characterized in sorghum. A candidate sesquiterpenoid present in both species was pursued using numerous analytical and informatic approaches yet yielded an alternative identification. Current efforts are focused on the regulation of sesquiterpenoids in both plants and the identification of terminal Cytochrome P450 Cyp81-derived products in the core maize zealexin pathway underlying pathogen resistance.
Abstract Title : Enhancing Specificity of Intrathecal Gene Therapy for Alzheimer’s Disease
Abstract : Numerous preclinical gene therapies for Alzheimer’s disease have been developed including proteolytic enzymes, anti-inflammatory genes, anti-tau or anti-amyloid antibodies, and growth factors such as brain-derived neurotrophic factor (BDNF). Alzheimer’s disease ultimately affects the entire cerebral cortex but widespread cortical gene delivery by direct brain injection is not possible. Intrathecal and intravenous infusion of Adeno-Associated Virus 9 (AAV9) are promising methods to drive widespread gene transfer to the cortex but off-target gene transfer may cause adverse side effects. We tested multiple strategies to target gene expression to cortical neurons including cell-specific promoters and microRNA binding sites. We found that the cortex-specific CTX-1 promoter successfully restricted gene expression to cortical neurons. Intrathecal or intravenous infusion of AAV9 carrying this promoter could achieve widespread and specific gene therapy of the cortex through a single and minimally invasive injection.
Abstract Title : Glyoxalase 1 Inhibition is a Potential Novel Therapeutic Target in Chronic Ethanol Withdrawal
Abstract : Approximately 15.1 million adults and 623,000 adolescents suffer from alcohol use disorder (AUD) in the United States alone. When chronic alcohol users cease consumption of alcohol, they often experience physical withdrawal symptoms ranging from headaches and nausea to hallucinations and delirium tremens. Withdrawal can also include behavioral symptoms, including anxiety and depression. These withdrawal symptoms are severe enough to compel chronic users to continue consuming alcohol or cause chronic users attempting to cease alcohol consumption to relapse. Thus, research on novel targets for alleviating the effects of alcohol withdrawal has the potential to aid millions of individuals currently suffering or recovering from AUD. The enzyme glyoxalase 1 (GLO1) has been linked to anxiety, depression, and seizure susceptibility, similar symptoms to those of alcohol withdrawal. GLO1 metabolizes methylglyoxal (MG), therefore blocking the accumulation of MG concentration in the brain. MG has been shown to be a competitive partial agonist at GABAA receptors. Manipulations that increase MG levels (e.g. genetic knockdown of Glo1; treatment with a GLO1 inhibitor) reduce anxiety- and depression-like behaviors in mice and also reduce alcohol drinking. Mice were given daily injections of 2 g/kg ethanol mixed with an alcohol dehydrogenase inhibitor for 10 days to induce ethanol dependence. A separate control group received no ethanol treatment. Following the last ethanol injection, mice were given a GLO1 inhibitor or vehicle injection and tested for handling-induced convulsions (HICs). 24 hours after the last ethanol injection, mice were given another inhibitor or vehicle injection and tested for locomotor activity and anxiety-like behavior in an open field. Lastly, mice were immediately tested for depression-like behavior in a forced swim test. Control group mice received vehicle injections on both tests. Vehicle-treated withdrawal mice showed greater seizure severity than control mice. Inhibitor-treated mice exhibited a trend towards lower seizure severity compared to vehicle mice, while still having greater seizure severity than the control group. Both withdrawal groups had decreased locomotor activity compared to control mice. However, only vehicle-treated withdrawal mice showed significantly higher anxiety-like behavior compared to control mice. In contrast, inhibitor-treated withdrawal mice did not differ from the control group in anxiety-like behavior. These results explore the potential of GLO1 as a therapeutic target for treatment of some of the physiological (seizure susceptibility and severity) and affective (anxiety) changes of chronic alcohol withdrawal.
Advisor : DONG WANG
Abstract Title : Preliminary Analysis of Biochemical Activities of Rhp26 Mutations Involved in Cockayne Syndrome
Abstract : Cockayne syndrome (CS) is a fatal autosomal recessive neurodegenerative disorder, and CS patients are characterized by hallmark symptoms such as sunlight photosensitivity, microencephaly, and impaired nervous system development. CS is commonly associated with mutations in the Cockayne Syndrome group B protein (CSB), an important protein involved in transcription-coupled DNA repair. Specific CSB mutations have been identified in CS patients, but how these mutations affect the biochemical activities of CSB remains unknown. To address this question, we compiled CSB mutations observed in CS patients from clinical reports and generated these mutations in Rhp26, the Schizosaccharomyces pombe homolog of CSB. Here it is demonstrated that two of the Rhp26 mutants, N475D and L774P, show decreased DNA binding and a significant loss in chromatin remodeling activity. Understanding how these Rhp26 mutants function biochemically can lead to a better understanding of transcription-coupled DNA repair and the pathology of Cockayne Syndrome.
Abstract Title : Highly Conserved Enhancer Grammar
Abstract : Enhancers are elements within the genome that act as switches to control the location and timing of gene expression. Thus, enhancers are fundamental for successful development. The functional features of an enhancer are “binding sites”, sequences that recruit specific transcription factors to allow activation of gene expression. The order, orientation, and spacing of these sites — collectively known as “enhancer grammar” — is thought to be important for the activity of an enhancer. We bioinformatically identified an enhancer for the gene Hes1 that has conserved its grammar for over 500 million years across all deuterostomes and some protostomes. We tested this enhancer from 4 protostomes, including scorpion, and 6 deuterostomes for activity in Ciona intestinalis embryos. The conserved enhancer grammar encodes a conserved expression pattern in the developing nervous system. Further studies of this rare conservation could uncover important principles governing enhancer grammar and help identify how grammar confers fitness during evolution.
Abstract Title : A Novel Platform to Uncover Pro-Arrhythmogenic Pathways Driving Arrhythmogenic Right Ventricular Cardiomyopathy
Abstract : Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC) is a genetic cardiac disease that causes arrhythmias and sudden death in young athletes, often triggered by exercise. Mutations within genes of the desmosome, a mechanical cell-cell anchoring junction, account for 40% of ARVC cases. Plakophillin-2 (PKP2) is the most mutated desmosomal gene; however, there are limited insights on mechanisms driving ARVC due to PKP2 mutations. We generated a novel PKP2 mutant mouse that recapitulates the adult disease spectrum of ARVC. We hypothesize that neonates represent a pre-disease stage to mine for early pro-arrhythmogenic pathways underlying ARVC at baseline and adrenergic stress (also triggered by exercise). We show that neonatal PKP2 mutant cardiomyocytes display loss of desmosomal proteins (desmoplakin, desmoglein-2) and the calcium regulator, SERCA2. These defects may underlie the adrenergic stress-induced arrhythmias that we also observed in neonatal PKP2 mutant cardiomyocytes. We provide a novel model to uncover mechanisms driving the early electrical dysfunction in ARVC.
Advisor : CHIH-YING SU
Abstract Title : Morphometric Analysis of Compartmentalized Olfactory Receptor Neurons in Drosophila
Abstract : In Drosophila, ephaptic interaction, or direct electrical interaction, mediates lateral inhibition between olfactory receptor neurons (ORNs) housed in the same sensillum. Interestingly, the strength of such lateral inhibition is asymmetric in that the larger spike A neuron can exert greater ephaptic influence onto the smaller spike B neuron. Mechanistically, this functional asymmetry arises from differences in the passive electrotonic properties of neurons, such as input resistance. This leads us to hypothesize that compartmentalized ORNs are of different sizes. The goal of my research is to systematically characterize the morphometric features of grouped ORNs in different sensillum types. I will perform computer-based segmentation of images acquired by serial block-face electron microscopy. Based on the 3D models, I will subsequently measure the size, length and surface area of grouped ORNs. My study is expected to yield critical information for us to understand how neuronal size influences the strength of ephaptic interaction.
Abstract Title : Targeting Aberrant RNA Splicing of MCL-1 to Prevent Malignant Regeneration in Multiple Myeloma
Abstract : Multiple myeloma is a form of cancer that is characterized by malignant plasma cells which produce abnormal antibodies that can cause symptoms such as renal damage, bone pain, and hypercalcemia. There is currently no known cure for multiple myeloma and knowledge is limited regarding the molecular basis of the disease as it relates to RNA splicing alterations. Splicing is a form of RNA processing in which removal, or lack thereof, of introns creates novel RNAs which can produce unique proteins. This project explored the role of aberrant splicing of the pro-survival gene MCL-1, which is correlated with disease progression and drug resistance in multiple myeloma. Cell viability and self-renewal were evaluated through both genetic modulation of the splicing factor SF3B1 and pharmacological modulation using a small molecule targeting the spliceosome complex in human myeloma cells. These findings could provide an avenue to explore novel therapies for multiple myeloma treatment.
Abstract Title : The Role of HPV in Shaping the Tumor Immune Landscape
Abstract : Immunotherapy has emerged as the most promising treatment for cancers. However, failure of a large percentage of patients remains the ultimate obstacle to successful treatment. Etiology-associated dysregulation of genes involved in the immune system may be central to the differential clinical response. To understand the role of human papillomavirus (HPV) in shaping the tumor immune landscape, we conducted a comprehensive investigation of immune-associated (IA) gene expression in HPV-associated cancers. Using next-generation RNA sequencing datasets, we identified a unique panel of HPV-dysregulated IA genes. To investigate the molecular mechanism for their dysregulation, we used in silico methods to explore their potential regulation by genomic and transcriptomic alterations, aberrant microRNA expression, and DNA methylation. Finally, we validated the dysregulation of several IA genes in vitro using HPV E6/E7-transfected cell lines. The panel of IA genes we identified may elucidate the HPV-mediated molecular mechanism of tumor immune evasion, and ultimately contribute to better treatment modalities for HPV-associated cancer patients.
Abstract Title : A Role for TAAR1 Genotype Associated with Methamphetamine Intake in Dopaminergic Changes
Abstract : Multiple factors contribute to individual susceptibility to drug addiction. Mouse lines MAHDR (High) and MALDR (Low) were bred for the amount of Methamphetamine (MA) they voluntarily consumed. MAHDR mice exhibit a homozygous TAAR1 gene SNP, making it non-functional. We tested the role of the TAAR1 mutation in dopaminergic and inflammatory pathway dysfunction, induced by the TAAR1 agonist MA, by measuring transcription of dopamine receptor DRD1, dopamine transporter DAT, and CCL5/RANTES chemokine in both groups at multiple time points after intraperitoneal MA. MAHDR mice showed an overall delayed response in major reward pathway regions such as pre-frontal cortex (PFC) and nucleus accumbens (NAc), characterized by increased DRD1 and DAT levels, detectable at 30min after MA. CCL5/RANTES was decreased in MALDR NAc at 30min, but increased in MAHDR ventral mid-brain (VMB) at 60min. These results suggest a role for TAAR1 in the susceptibility to MA intake and addiction, and its consequences.
Abstract Title : Complementary adaptations to bioinformatics approaches facilitate the untangling of maize defense biochemistry
Abstract : Small-molecule specialized metabolites constitute an important biochemical layer of plant immunity for pathogen defense. Maize (Zea mays) produces a complex mixture of antimicrobial isoprenoids in response to pathogen elicitation, including a genus specific family of acidic sesquiterpenes derived from β-macrocarpene, termed zealexins. Candidate zealexin biosynthetic genes are among the most dynamically regulated processes known in maize during pathogen attack. In this study we combined forward genetics, reverse genetics, bioinformatics and enzyme co-expression studies to define enzymes and catalytic promiscuity. We demonstrated the sufficiency of three cytochrome P450 enzymes located in a CYP71Z class gene cluster on chromosome 5, termed Zx5, Zx6 and Zx7, to independently catalyze oxygenation reactions of diverse olefin substrates, yielding different combinations and ratios of zealexin A1, D1, D2, β-costic acid and α-santalenoic acid. These enzymes represent an important biosynthetic node for maize defense biochemistry with many parallel defense pathways utilizing the same gene cluster.
Abstract Title : A Ubiquitination-Dependent Substrate-Induced Endoplasmic Reticulum Membrane Stress
Abstract : Endoplasmic reticulum-associated degradation (ERAD) is a form of protein quality control that degrades misfolded proteins in the cell. In ERAD, proteins are ubiquitinated, removed from the ER to the cytosol in a process called retrotranslocation, then degraded in the cytosol. Dfm1, a yeast ER membrane protein, is a key factor in retrotranslocation. We have discovered that in strains lacking Dfm1, expression of various ERAD substrates leads to a profound growth stress originating at the ER. Here we characterize this novel stress, including the molecular features of the proteins that cause it and the cellular systems that participate in both its generation and abatement. We have identified substrate ubiquitination as a key factor in inducing the growth stress.
Advisor : LI-FAN LU
Abstract Title : The Role of NLRP3 in Gut-Resident Regulatory T Cells
Abstract : Regulatory T (Treg) cells are a subset of adaptive immune cells known for their immunosuppressive functions. RNA-seq analysis performed on mice during systemic autoimmune inflammation revealed high expression levels of Nod-like receptor protein 3 (NLRP3) in gut-resident Treg cells. NLRP3 is part of a well-characterized inflammasome commonly associated with innate immune cells. To determine the role of NLRP3 in gut-resident Treg-mediated immunosuppression, we obtained a mouse model with a Treg-specific, constitutively active form of NLRP3. After immunologically challenging these mice via Citrobacter rodentium and Toxoplasma gondii, our data suggests that gut-resident Treg cells with a constitutively active form of NLRP3 have impaired control over Th17 responses but enhanced control over Th1 responses. Next, we plan to examine the efficacy of Treg cells in the presence of constitutively active NLPR3 or in the absence of NLRP3 to inhibit tumor-promoting inflammation by utilizing a well-established in vivo model of colorectal cancer.
Abstract Title : The Effects of Fear Learning on Sensory Representations in Secondary Auditory Cortex
Abstract : Sensory representations in our brain are known to change with experience. During auditory fear conditioning, pairing a neutral tone with an unpleasant stimulus (i.e. mild shock) changes tone responses in the auditory cortex. However, it remains unclear what changes in cortical input underlie this plasticity. The lateral amygdala (LA) plays an important role in fear and emotion processing, and sends dense projections to the auditory cortex. Thus, projection cells in LA are candidates for mediating changes in auditory cortex sound representations. To investigate this possibility,we used in vivo two photon calcium imaging in head-fixed mice to simultaneously image activity of LA projections and layer 2/3 pyramidal cells in auditory cortex before and after fear conditioning. Our results show that fear conditioning leads to long term changes in sound-evoked LA activity that may contribute to learning-related changes in tone responses of L2/3 principal cells in auditory cortex.
Abstract Title : Investigating a novel lysophosphatidic acid secretion pathway and its role in cancer progression and stress tolerance
Abstract : Cancer stem cells (CSC) represent a small, yet aggressive metastatic sub-population within the tumor characterized by their plasticity, drug resistance, stress tolerance, tumor initiation, and metastatic capacity. We found that CSC can secrete lysophosphatidic acid (LPA), a bioactive lipid that is known to induce tumor progression and metastasis. We identified integrin αvβ3 as a cancer stem cell marker that contributes to the synthesis of LPA by recruiting phospholipase A2 to the plasma membrane, a critical enzyme in the biosynthesis of LPA. Once secreted, LPA can reprogram surrounding cancer cells toward a more stem-like fate through its six LPA receptors. In fact, LPAR4 is selectively upregulated in pancreatic carcinoma cells after exposure to microenvironmental stress or drug treatment. LPAR4 expressing cells gain stem-like and metastatic properties. Taken together, these findings identify a mechanism whereby CSC through the secretion of LPA can enhance pancreatic cancer progression and reveal potential therapeutic targets to decrease the progression of this disease.
Abstract Title : Optimizing Methodologies for High-Quality Single-unit Neurophysiological Recording Coupled with Optogenetic Manipulation
Abstract : Measuring the activity of single neurons, or “units” allows researchers to relate spiking activity in a certain brain area with behaviors of interest. By coupling single-unit recording with optogenetic perturbations, a method of activating or inhibiting spiking activity, researchers can observe how neural activity changes and infer causal relationships between neural activity and behavior. There are relatively few cost-effective and standard methods for coupling these technologies. The goal of my current research is to develop a device that enables effective single unit recording coupled with optogenetic manipulation at customizable location. This new system is designed by fixing tetrode-wires capable of measuring single unit activity together with a fiberoptic cannula into a metal cannula. I have found that this method is effective for the collection of single unit activity. Future efforts will be made to minimize signal noise, improve the longevity of neural recording, and optimizing the system for combined optogenetic manipulations.
Abstract Title : Investigation of Protein Structural and Evolutionary Relationships Between ABC Superfamily Types
Abstract : The ATP-binding Cassette (ABC) superfamily consists of transporters that use ATP as energy source. Previous studies based on primary protein sequence suggested that ABC exporters evolved independently at least 3 times. Given the increasing availability of structural data within the superfamily, we are now able to investigate whether 3D structural similarities agree with the conclusions attained by sequence analyses. In our strategy, all available protein structures in the Protein Data Bank (PDB) for the three ABC types were cut into bundles of 4 transmembrane a-helices (4HB) and the alignments within and between ABC types were examined. Clustering and phylogenetic analyses will also be performed for the membrane constituents and ATPases to determine whether they have co-evolved. Our results will provide insights on the evolutionary relationships between ABC types, and serve as a guide for future studies of the ABC superfamily.
Abstract Title : Smooth Muscle Cell Heparan Sulfate Proteoglycans Mitigate Atherosclerosis Formation
Abstract : Atherosclerosis is a major cause of cardiovascular disease, which kills 17 million people worldwide annually. Atherosclerosis is an inflammatory response initiated by cholesterol buildup in the artery walls. Sub-endothelial proteoglycans produced by smooth muscle cells (SMCs) in the artery wall trap low-density lipoprotein (LDL) cholesterol. This project examines how vascular SMC heparan sulfate proteoglycans (HSPGs) affect the development of atherosclerosis. To test this, we use 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 SMC HSPGs. Weekly injections of LDL receptor anti-sense oligonucleotides and a high fat diet were used to generate hypercholesterolemia-induced atherosclerosis in both the wild type and Ndst1f/fSM22aCre+ mice. Analysis of the lesions so far show that loss of vascular SMC HSPGs promotes atherosclerosis lesion size and remodeling. Further studies will examine how HSPGs affect SMC proliferation, migration and inflammation.
Advisor : DR. REUBEN J. SHAW
Abstract Title : Bridging the Gap Between Energy Sensing and Mitophagy: Characterization of the Regulation of Mitophagy Receptors by ULK1 and TBK1
Abstract : Unc-51 like autophagy activating kinase-1 (ULK1) is a critical protein for activating autophagy and regulating mitochondrial autophagy (mitophagy). ULK1 is also known to be a substrate of AMP-activated Protein Kinase (AMPK), a highly-conserved master regulator of cell metabolism and energy homeostasis in eukaryotes. Previous studies have shown that AMPK affects mitophagy via ULK1, however, the mechanism remains to be elucidated. LC3-Interacting Region (LIR) is a conserved amino acid sequence that allows binding to Atg8 family proteins. The list of LIR-containing protein includes several mitophagy receptors that connect the mitochondria to Atg8 proteins in the autophagosome. Previous studies have shown that phosphorylation at the serine before the conserved LIR sequence increases LIR’s affinity to LC3B and alters the function of the LIR-containing protein. For example, ULK1 phosphorylates FUNDC1 at the serine before the LIR sequence, increasing FUNDC1 and LC3B binding affinity and promotes mitophagy. Interestingly, TBK1 was reported to have a very similar optimal substrate motif as ULK1 and the two kinases may phosphorylate the same serine in a given target protein in response to distinct stresses. With these discoveries, the project aims to understand the regulation of the serine-LIR-containing mitophagy receptors by ULK1 or TBK1. We will focus on a list of mitophagy receptors including Bnip3, NIX/Bnip3L, FUNDC1, Optineurin, and BCL2-L-13. We will use mass spectrometry-based methods, biochemistry experiments, and in vitro site directed mutagenesis to uncover potential interactions. The findings will enhance our understanding of the link between cellular energy status and mitochondrial homeostasis.
Abstract Title : Modelling Neuronal Circadian Rhythms in Bipolar Disorder using Human Induced Pluripotent Stem Cells
Abstract : Bipolar Disorder (BD) is a common psychiatric illness characterized by manic and depressive mood episodes and profound disturbances in circadian rhythms, manifesting in irregular sleep and activity cycles. The molecular mechanisms underlying BD and the associated rhythm disturbances remain mostly unknown. We differentiated pluripotent stem cells (iPSC) from BD patients and healthy controls into neural progenitor cells (NPCs) and neurons. We then recorded cellular circadian rhythms using Per2-luc, a bioluminescent reporter of circadian clock activity. Our preliminary data indicate that circadian rhythms in NPCs and neurons are weaker and damp faster in cells from BD patients vs. controls. Single cell Per2-luc imaging validated this result in NPCs. Ongoing work is investigating the ways in which neuronal circadian rhythms in BD vary, and whether pharmacological intervention can alter the BD-associated reduction in rhythm. The results of this work may inform future efforts to develop new drug treatments for BD.
Abstract Title : Differences in Apolipoprotein E ε-4 Genotype in Early vs. Late-Onset Alzheimer’s Disease
Abstract : Alzheimer’s Disease (AD) is a neurodegenerative disorder thought to be a primary cause of dementia. Apolipoprotein Allele 4 (ApoE4) is thought to be a major risk factor for the disease. We suspect that the distribution of ApoE genotype might vary based on age of onset. To address this question we grouped our data into Early Onset (Age ≤ 60, N=163) and Late Onset (Age ≥ 70, N=629) for analysis, omitting cases where patients were known to have an Autosomal Mutation that is causative of AD such as in Amyloid Precursor Protein or in Presenilin 1 and 2 and controlling for education and sex which had statistically insignificant differences between the two groups. Based on a Chi-Square analysis comparing patients without ApoE4 (E3/E3) versus patients with ApoE4 (E3/E4 and E4/E4), we concluded there was not a statistically significant difference in ApoE allele distribution (p=0.598). Because the ApoE2 allele is a known protective factor against AD whose interaction with the ApoE4 allele is unknown and because the number of patients with ApoE2 was small, these were omitted from Chi-Square analysis. We concluded that the ApoE allele distribution between Early and Late onset AD patients are similar.
Abstract Title : The role of PTP4A1 in systemic sclerosis
Abstract : Previous study in the Bottini Lab shows that the expression of Protein Tyrosine Phosphatase IVA I (PTP4A1) is elevated in dermal fibroblast of patients with systemic sclerosis, a fibrosing autoimmune disease, correlating with stimulation of pro-fibrotic genes. PTP4A1 promotes fibrosis via enhanced activation of the kinase SRC, its downstream RAS-ERK pathway and the transcription factor SMAD3 in fibroblasts. We are interested in understanding a key physical interaction between PTP4A1 and the SRC kinase at the molecular level. We have purified sufficient amounts of SRC and PTP4A1 proteins in active form from E.coli. Co-precipitation assays was successfully established and demonstrated direct binding between PTP4A1 and SRC. We are in the process of applying the assay to assess how different variables, including the phosphorylation status of SRC, the oxidation state of PTP4A1, and the contribution of different domains of SRC, affect the interaction. This knowledge can potentially help in the search of novel treatments for systemic sclerosis.