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2016 Research Showcase
Master's Abstracts
Abstract Title : Changes In Microbial Pathogenicity Caused by Cigarette Smoke Exposure
Abstract : Cigarette smoking is responsible for over 450,000 deaths per year, making it one of the leading preventable causes of death in the United States. Cigarette smoke has been associated with increased susceptibility to pneumonia and chronic obstructive pulmonary disease (COPD). Colonization and invasive infection by bacteria play key roles in both of these diseases. Here, we examine the effects of cigarette smoke on the virulence properties of Pseudomonas aeruginosa (PSA), which is one of the top three bacterial respiratory pathogens in adult smokers. PSA was grown in media with increasing concentrations of cigarette smoke extract (CSE) in order to determine effects on growth, hydrophobicity, resistance to reactive oxygen species (ROS), surface charge, resistance to killing by cells of host defense, and virulence in vivo. While exposure to CSE slowed PSA growth in a dose-dependent manner, it led to an increase in pathogenicity factors, as shown by alterations in hydrophobicity and biofilm formation, as well as increased resistance to killing by reactive oxygen species, such as hydrogen peroxide. These findings suggest that cigarette smoke exposure aids in the survival of PSA in phagolysosomes, a principal innate immune antimicrobial mechanism. When human neutrophils were infected with PSA, CSE exposure resulted in resistance to neutrophil killing. Additionally, the inhibition of PSA growth by CSE is consistent with smoke as an environmental stressor, facilitating differential gene expression to enhance bacterial survival. We conclude that cigarette smoke-induced resistance phenotypes in pathogens may be an important contributor to the vulnerability of cigarette smokers to infectious airway diseases. With more than 1 billion smokers worldwide, it is becoming increasingly critical to understand the consequences of cigarette smoking. We believe our study will be instrumental in the understanding of the effects of cigarette smoke on human health.
Abstract Title : Time Course of Brainstem Glial Activation in Rat Respiratory Centers Following Exposure to Chronic Sustained Hypoxia
Abstract : Neuron-glia communication in the central nervous system is emerging as an important component of neuronal signaling in respiratory control centers of the brainstem. To investigate glial cell contributions to respiratory signaling during hypoxia, I measured the time course of activation of microglia and astrocytes following different lengths of exposure to sustained hypoxia (10% O2). Rats were exposed to sustained hypoxia at the following time points: 30 minutes, 1-, 4-, 12- and 24-hours, 4- and 7-days. Activation of glial cells was measured in two respiratory control centers: the nucleus tractus solitarius (NTS) and the hypoglossal (XII) motor nucleus. Microglia (Iba-1 positive cells) show an active morphology after 1 hour of hypoxia in the NTS and the hypoglossal motor nucleus, displaying shorter and fewer branches, and return to the ramified state (longer filaments and extensive branching) after 4 hours of sustained hypoxia. The number of microglia stayed constant throughout activation. Astrocytes increase glial fibrillary acidic protein (GFAP) expression upon activation as measured by an increase in GFAP antibody immunofluorescent intensity after 4 hours of sustained hypoxia in the NTS and after 7 days in the hypoglossal motor nucleus. Astrocyte proliferation was observed after 7 days of sustained hypoxia in the hypoglossal motor nucleus. These results show that glial activation occurs sequentially with microglia activation preceding astrocyte activation. This finding implicates glia as a possible target for treatments in respiratory ailments. ADDITIONAL PRESENTER: Jennifer Stokes
Abstract Title : Projection-, cell-type, and stage-specific effects of cocaine on dendritic spine densities of excitatory inputs to the nucleus accumbens
Abstract : Drug addiction is a major global health problem due to a lack of effective treatments. Furthermore, the pathophysiology underlying the progression to drug addiction remains unknown. Studies using mouse models of cocaine addiction implicate medium spiny neurons (MSNs) of the nucleus accumbens (NAc) in this process. In particular, cocaine selectively modifies MSN dendritic spine densities depending on MSN expression of either dopamine receptor D1 or D2, suggesting a cocaine-induced reorganization of connectivity to the NAc. To determine whether cocaine alters spine densities in neurons that modulate MSNs, we utilized a method of monosynaptic retrograde tracing using an EnvA pseudotyped rabies virus (PRV); we injected the PRV into the NAc of either D1-cre or A2a-cre mice to infect D1 or D2-MSNs, respectively, such that PRV would fluorescently label neurons that directly synapse onto primary infected cells. The effect of cocaine (20 mg/kg) on spine density was measured in multiple stages: 5 day cocaine, 5 day cocaine followed by 2 week withdrawal, and 5 day cocaine followed by cocaine reinstatement at the end of 2 week withdrawal. Here, we characterize the different effects of cocaine exposure on dendritic modifications in a projection-, cell type-, and stage-specific manner. Our findings suggest cocaine differentially alters spine densities in brain areas that send excitatory projections to modulate NAc MSNs: the prefrontal cortex, the basolateral amygdala and the ventral hippocampus. These neuroanatomical results establish a framework to understand the circuit-basis of drug addiction. ADDITIONAL PRESENTERS: Daniel Knowland
Abstract Title : Functional Diversity of Human ATF6 Mutations Associated with Achromatopsia
Abstract : Achromatopsia is an autosomal recessive disorder characterized by cone photoreceptor dysfunction. We recently identified Activating Transcription Factor 6 (ATF6) as a novel genetic cause of achromatopsia. ATF6 is a key regulator of the Unfolded Protein Response. In response to endoplasmic reticulum (ER) stress, ATF6 migrates from the ER to Golgi where it undergoes regulated intramembrane proteolysis to release a cytosolic domain containing a bZIP transcriptional activator. The cleaved ATF6 fragment migrates to the nucleus to transcriptionally upregulate protein folding enzymes and chaperones. ATF6 mutations in achromatopsia patients include missense, nonsense, splice site, and single-nucleotide deletion or duplication changes found across the entire gene. Here, we comprehensively tested the functional consequences of achromatopsia-associated ATF6 mutations and found that they group into three distinct molecular pathomechanisms. Class 1 ATF6 mutants show impaired ER to Golgi trafficking and diminished regulated intramembrane proteolysis and transcriptional activity in response to ER stress. Class 2 ATF6 mutants bear the entire ATF6 cytosolic domain with fully intact transcriptional activity and constitutive induction of downstream target genes even in the absence of ER stress. Class 3 ATF6 mutants have complete loss of transcriptional activity due to absent or defective bZIP domains. Primary fibroblasts from patients with Class 1 or Class 3 ATF6 mutations show increased cell death in response to ER stress. Our findings reveal that human ATF6 mutations interrupt distinct serial steps of the ATF6 activation mechanism. Our findings suggest that increased susceptibility to ER stress-induced damage underlies the pathology of achromatopsia in patients with ATF6 mutations.
Abstract Title : Functional characterization of proteins essential for peroxisome biogenesis
Abstract : Recent discoveries suggest a role of the endoplasmic reticulum (ER) in peroxisome formation. Following the intra-ER sorting of peroxisomal membrane proteins (PMPs) to pre-peroxisomal vesicles (ppVs), the budding and fusion of these ppVs generate import-competent peroxisomes. This defines the paradigm of the de novo model of peroxisome biogenesis. Although many of the important PMPs in the de novo pathway have been identified, their role and the mechanistic details of their actions remain unclear. In this study we investigate the trafficking and subcellular localization of docking and RING subcomplex PMPs, Pex17 and Pex12, respectively. By performing subcellular fractionation procedures and fluorescence microscopy imaging of endogenously expressed Pex17-GFP, we demonstrate an exclusive ER origin of Pex17-GFP during the de novo pathway. Additionally, we identify a co-packaging of Pex12 with the docking subcomplex vesicles, in a Pex3-and Pex19-dependent manner. To further investigate the role of Pex3 in de novo peroxisome biogenesis, we have created and characterized several Pex3 mutations. Site-directed mutagenesis is revealing the essential role of Pex3 domains in peroxisome formation, although further analysis is required to uncover the mechanistic details of this complex process.
Abstract Title : Determining the Role of UPF3B in Proper Neurodevelopment
Abstract : Nonsense mediated decay (NMD) is a post-transcriptional RNA surveillance mechanism that targets mRNA transcripts bearing premature termination codons to signal them for decay. In addition, NMD also serves a dual role by targeting normal transcripts for degradation in a regulatory fashion. Loss of NMD factors in various species has been found to result in dysregulation of approximately 3%-15% mRNAs. Loss of the NMD factor, UPF3B, has been shown to dysregulate ~5% of the transcriptome. It was found that NMD factor UPF1 was down regulated to promote neural development and in support of NMD playing a critical role in neural development, mutations in the NMD factor UPF3B cause intellectual disability including mental retardation, autism, and schizophrenia. This works aims to identify the role of UPF3B in regulation of the transcriptome in various stages throughout neurodevelopment and potential implications on cellular processes through both in vitro and in silico methods. Presented is evidence to suggest that UPF3B regulates several transcripts governing pluripotency, cell fate, and cell cycle, to help maintain the pluripotent cell state. Demonstrating functional UPF3B is needed to carry out early developmental decisions required for proper neural development as well as outlining the importance of post-transcriptional gene regulation role in development.
Abstract Title : How important are honeybees as pollinators to natural plants?
Abstract : Pollinators provide vital services for natural systems, however pollinator diversity and abundance is threatened by habitat fragmentation and climate change. The honey bee (Apis mellifera), is the most prevalent pollinator in agricultural systems, but the importance of honey bees as pollinators in natural systems is largely unknown. As a generalist pollinator, the honeybee is expected to be less affected by habitat fragmentation and climate change than specialist pollinators. To fill this knowledge gap, we performed a meta-analysis of 73 studies which report frequency of floral visits by different pollinators to plants in natural communities worldwide (pollinator network studies) and evaluated 35 studies that report the efficiency of honey bees as pollinators relative to other floral visitors for both natural and agricultural plants. We found that where present the honeybee accounts on average for 18% of total floral visits and is the most important floral visitor worldwide due to its global distribution. Additionally, on average, the honeybee is no different in efficiency from the top non-Apis pollinator for plants studied. Therefore, the honeybee can generally be expected to provide adequate pollination services for most plants in natural communities, such as San Diego, where honey bees have become the predominant pollinator.
Abstract Title : Circadian Rhythm Amplitude and Neuronal Survival
Abstract : Bipolar Disorder is a serious neuropsychiatric disorder that causes impairment in mood regulation. Patients with Bipolar Disorder have structural brain atrophy, reduced hippocampal volume, and suffer from disturbances in circadian rhythms. The mood-stabilizer lithium is a mainstream treatment for the disorder, and has been shown to increase hippocampal gray matter volume in MRI studies. However, the mechanism of this neuroprotective effect is unclear. Lithium is also known to amplify circadian rhythms. Therefore, we sought to investigate whether modulation of circadian rhythm amplitude by molecular or pharmacological means impacts neuronal survival. An immortalized mouse hippocampal cell line was transfected with a Per2::luciferase reporter, and circadian rhythms were characterized in response to drugs suspected to increase or decrease rhythm amplitude, or after knockdown of BMAL1, an essential clock gene. The same interventions were then used on neurons to determine their effects on viability following a toxic glutamate exposure. We report, with a small sample of drugs, that those that increase rhythm amplitude tend to promote neuronal survival. Conversely, we have seen that drugs that decrease rhythm amplitude tend to decrease survival. However, BMAL1 knockdown reduced amplitude but increased neuronal survival. The data suggest that there is overlap between circadian rhythm and cell survival systems, but that the relationship is not simple. Non-specific drug effects may complicate the interpretation of some results. Further studies are needed to clarify the connection.
Abstract Title : Multiple loci contribute to synthetic lethality in the Zfp423 model of Joubert syndrome
Abstract : Zfp423 is a transcription factor with potential to integrate several canonical signaling pathways. Inactivating mutation in ZNF423 (human ortholog of Zfp423) results in Joubert Syndrom 19, a rare but severe brain malformation that often includes abnormalities in other organ systems. Complete loss of Zfp423 in mice presents a variety of phenotypes that depend on genetic background and other factors. We already identified Zfp423 as the gene mutated in nur12 mice. Interestingly, the survival of mutants on BALB/C is around 70%, while the survival of mutants on C57BL/6 is 0%. I am mapping the genetic factors by linkage in a cross between those two inbred strains, which limit the complexity and allow deep sampling to detect statistically significant effects on a major phenotypic outcome: neonatal death. To identify genes that contribute to lethality, we are using both chi-square tests and survival analysis to highlight genomic regions where C57BL/6 alleles are underrepresented among surviving mutant offspring from a cross between C57BL/6 and BALB/C.
Abstract Title : Driving forces of cell sorting during Hydra regeneration from aggregates
Abstract : How patterns form from an initially homogenous cluster of cells is a fundamental questions in developmental biology. Hydra is a powerful model for studying mechanisms driving pattern formation because of its ability to regenerate after dissociation into individual cells. The first step of regeneration involves segregation of the heterogeneous mixture of two cell types into their respective tissue layers. Two competing theories have been proposed to explain this sorting: the differential cell migration and the differential adhesion hypothesis (DAH). The first attributes cell sorting to differences in cell motility of the two tissue types, the second to differences in surface tension, similar to the breaking up of an emulsion of immiscible liquids. Using 2-photon time-lapse microscopy of 3D cellular aggregates, we show that no difference exists in the motility of the two types of epithelial cells, rejecting the differential cell migration model. In contrast, our preliminary data support the DAH since we find that (a) cell aggregates exhibit fluid-like behaviors, justifying the concept of surface tension, and (b) cell sorting dynamics seem to mimic the breaking up of an emulsion. Future measurements of tissue surface tension will allow us to test the DAH directly. In summary, utilizing a novel model organism, Hydra, we are able to gain insight into fundamental mechanisms of tissue organization that are indispensable for development.
Abstract Title : Investigating Transgressive Segregation by Hybridization in the Intertidal Copepod Species Tigriopus californicus
Abstract : Biologists have long thought hybridization to be a barrier to speciation. However, hybridization can result in a higher fitness; some hybrids have an expanded range of environmental tolerance compared to their parents, known as transgressive segregation. Hybridization experiments using the intertidal copepod species Tigriopus californicus have shown an expanded range of heat tolerance. This is not the case with all hybrids, however. In this study, we seek to predict which populations will hybridize to produce transgressive segregation. We predict that ecological similarity between populations will always result in transgression, despite genetic variability. We test this by hybridizing genetically divergent southern populations of T. californicus and comparing their range of heat tolerance with that of their parents. Because 9 of the 19 hybrid lines that were tested exhibited transgressive segregation despite genetic dissimilarity, our hypothesis was not supported; the environment may play a larger role than genetics. Further investigations using more varied populations will expand our knowledge of this topic.
Abstract Title : Rove Beetle (Staphylinidae) Diversity, Distribution, and Ecology in the Sonoran Desert Necrotic Cactus Niche
Abstract : The rotting cactus niche contains a high diversity of desert arthropods, yet only four studies of this diversity have ever been conducted. Rove beetles (family Staphylinidae) have the highest species richness in this system, yet little is known about their distribution, ecology, specificity, or life history. For my study, I collected over 5,000 rove beetles from four species of rotting cactus: cardón (Pachycereus pringlei, senita (Lophocereus schottii), and organ pipe (Stenocereus thurberi) from Baja California Sur, Mexico and California barrel cactus (Ferocactus cylindraceus) from Anza Borrego Desert State Park in Southern California. Altogether, I identified 21 morphospecies of rove beetles from six subfamilies. Species from the subfamilies Scaphidiinae and Osoriinae were found exclusively in cardón cactus. Comparison with previous studies showed that cactus size and chemistry were important indicators of diversity. I also found evidence that rove beetles in Anza Borrego Desert State Park were specific to rotting cactus and they were primarily diurnal. Together, these studies add to our understanding of rove beetles in the necrotic cactus niche and provide a baseline for future studies of necrotic cactus arthropods.
Abstract Title : Endosomal Abnormalities in Rodent Primary Neurons after Treatment by Soluble Aβ Oligomers
Abstract : Alzheimer's disease (AD) is a chronic neurodegenerative disease, and the most prevalent cause of dementia in the elderly. Previous studies by Cataldo and colleagues have shown that endosomal abnormalities are one of the earliest detectable pathologies observed in Ts65Dn mice, a model for studying Down Syndrome and AD. In addition, the changes in endosomal size and transport were reversed after the deletion of the extra copy of APP (Cataldo et al. 2003). Previous experiments in our lab by Dr. Sawa provide evidence that treatment of PC12 cells with soluble Aβ oligomers cause endosomal abnormalities. In these studies, we provide evidence that treatment of rat and mice primary hippocampal neurons with soluble Aβ oligomers causes an increase in early endosomal size and count.
Advisor : DR. DONG WANG
Abstract Title : Investigation and comparison of unnatural nucleotide incorporation during in vitro transcription
Abstract : The building blocks of DNA are widely known as the four deoxyribonucleotides, dA, dT, dG and dC, which form two specific base pairs. The phenomenon of sequence specific base pairing between the four nucleotide bases is consistent throughout the central dogma of biology, from DNA, to RNA, and subsequently to protein. However, recent efforts in synthetic biology have given rise to the development of synthetic nucleotides that form a third, unnatural base pair (UBP). The UBP formed by dNaM and dTPT3 was previously shown to be efficiently replicated and transcribed in vitro. We aim to investigate and compare the incorporation and elongation efficiencies of this UBP by RNA polymerase II (pol II), as well as E. coli RNAP and T7 RNAP, during in vitro transcription. Our studies show that, surprisingly, T7 RNAP shows the most discrimination in selectively incorporating rTPT3 opposite the dNaM template base, as well as the most efficient bypass of the pausing caused by dNaM and subsequent elongation. These results demonstrate exciting new prospects in the field of synthetic biology and continued efforts towards the development of a UBP that may be functionally stable within an organism.
Advisor : DR. WILBUR LEW
Abstract Title : CREB Regulated Transcriptional Co-activator 2 (CRTC2) in Improving Heart Function in Myocardial Infarction (MI)
Abstract : As the number one cause of death in the world, and the leading cause of death in the United States, heart disease claims more lives than all forms of cancer combined. Though several heart failure therapies are currently available, there remains other mechanisms left undiscovered, potential targets for novel treatments. Among other targets, β-adrenergic receptor (β-AR) signaling and calcium, are important factors in cardiac functioning and contractility that available therapies modulate. Here, we examined CREB Regulated Transcriptional Co-activator 2 (CRTC2), a known converging point between β-adrenergic receptor and calcium in pancreatic tumor cells. Upon comparison of survival and cardiac functioning of wildtype and transgenic CRTC2 mice in the setting of myocardial infarction, we provide evidence of trends favoring mice positive for the transgene, though significance is not yet obtained. Understanding of a β-AR and calcium point of convergence will enable a novel therapy that targets both systems, both of which are fundamental to cardiac function.
Abstract Title : Microbial Sensing by Host ELMO1 Linked to Inflammatory Diseases
Abstract : Gastrointestinal systems maintain homeostatic and dynamic interfaces between commensal flora, pathogenic microbes and host immune responses. The intestinal immune system maintains optimum levels of antimicrobial activity without inflammation from normal flora and induces the inflammatory responses. Microbes are recognized by specific receptors such as Pattern Recognition Receptors (PRRs). PRRs bind pathogens and commensals, yet in most cases inflammation is generated from pathogenic encounters. Previously, we found BAI1 (Brain Angiogenesis Inhibitor 1) is a PRR that recognizes bacterial Lipopolysaccharide. BAI1 binds ELMO1 (EnguLfment and cell Motility) which facilitates the engulfment of bacteria via Rac1 activation. ELMO1 is involved in several inflammatory diseases including Rheumatoid arthritis, Ulcerative colitis, Atherosclerosis and Type II diabetes. We hypothesized that the engulfment and sensing of enteric pathogens by ELMO1 regulates inflammatory responses. To delineate the role of ELMO1 in host inflammatory responses, ELMO1-depleted murine macrophage cell lines, intestinal macrophages, and ELMO1 deficient mice (total or myeloid-cell specific) were infected with Salmonella enterica serovar Typhimurium. The cytokine array and ELISA showed reduced levels of pro-inflammatory cytokines TNF-α, MCP-1 and IL-1β by ELMO1-depleted macrophages. ELMO1-dependent internalization was indispensable for TNF-α and MCP-1. Simultaneous inhibition of ELMO1 and Rac function abrogated TNF-α response to infection. The Real Time RTPCR of RNA isolated from ileum and spleen of infected ELMO1 KO mice showed reduced expression of TNF-α, MCP-1 and IL-1β. TNF-α is the major cytokine involved in Rheumatoid arthritis and inflammatory bowel disease. MCP-1 is a pro-inflammatory cytokine involved in monocyte recruitment. Its overexpression by macrophages lining blood vessel walls has been shown to increase the progression of atherosclerosis by increasing macrophage numbers and oxidized lipid accumulation. IL-1 is a pro-inflammatory mediator in acute and chronic inflammation, and a powerful inducer of the immune response. Elevated levels of IL1-1β, its body-circulating form, have been found in patients with cardiovascular disease, and polymorphisms in genes encoding IL-1β have been associated with premature onset of atherosclerosis. NFκB reporter luciferase assay showed that HEK293 cells overexpressing both ELMO1 and BAI1 displayed a much greater inflammatory response following infection compared to HEK 293 vector control cells. These findings suggest a novel role for ELMO1 in cytosolic sensing and the induction of inflammatory cytokines linked to inflammatory diseases.
Abstract Title : The Effect of PHLPP2 Removal on Cardiac Hypertrophy
Abstract : Crucial cellular events such as death, growth, metabolism, proliferation, and hypertrophy are regulated by phosphorylation and dephosphorylation of proteins. PH domain leucine-rich repeat protein phosphatase (PHLPP) is a serine/threonine phosphatase that has been shown to directly dephosphorylate several members of the AGC family of kinases. Preliminary data suggests that removal of PHLPP2 in neonatal rat ventricular myocytes (NRVMs) activates fetal gene re-expression at baseline and potentiates phenylephrine (PE) induced gene expression by 2 folds over siControl. Since we have previously published that removal of PHLPP2 in NRVMs does not affect Akt phosphorylation as previously reported in other cells, we hypothesize that PHLPP2 must have the ability to repress cellular hypertrophy through the dephosphorylation of other unknown targets. G-protein coupled receptor kinase 5 (GRK5), an AGC kinase, has been shown to regulate cardiac hypertrophy through de-repression of gene transcription and by directly binding DNA. Upon stimulation of NRVMs with the hypertrophic agonist phenylephrine (PE), GRK5 translocates to the nucleus to regulate hypertrophic gene expression. We found that removal of PHLPP2 increased GRK5 translocation to the nucleus at baseline and following PE treatment. Immunoprecipitation experiments confirm an interaction between PHLPP2 and GRK5. Our preliminary data is the first to uncover GRK5 as a novel PHLPP2 target in cardiomyocytes. Since little is known about the non-canonical regulation of GRK5, understanding whether phosphorylation and localization is regulated within the cardiomyocyte by PHLPP has potential for new therapeutic targets in the treatment of cardiac hypertrophy and failure.