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Virulence Factor Regulation in Listeria monocytogenesPortman, Jonathan Lewis 09 February 2018 (has links)
<p> <i>Listeria monocytogenes</i> is a Gram-positive intracellular pathogen that is readily amenable to genetic manipulation and for which there are excellent <i>in vitro</i> and <i>in vivo </i> virulence models. These attributes have allowed a thorough examination of the molecular underpinnings of <i>L. monocytogenes</i> pathogenesis, however, there are still a number of major unresolved questions that remain to be answered. For example, it has been known for many years that <i> L. monocytogenes</i> rapidly changes its transcriptional profile upon access to the host cytosol, however the host cues and bacterial components that are involved in driving this change have remained continually unanswered. One large piece of evidence came when the long-sought co-factor for the primary virulence regulator, PrfA, was discovered to be the antioxidant tripeptide, glutathione. Glutathione was demonstrated to play a crucial role in the activation of PrfA <i>in vivo</i>— a finding that has since led to two important discoveries that are described herein. First, the activation of PrfA <i>in vitro</i> requires both exogenous glutathione and a metabolic licensing step that can be recapitulated by a chemically defined synthetic media. Second, glutathione also functions as a post-translational regulator of the pore-forming virulence factor, Listeriolysin O (LLO), by reversibly binding via an S-glutathionylation reaction and preventing membrane association of the LLO monomers. These discoveries elucidate numerous regulatory roles for glutathione during infection and describe how <i>L. monocytogenes </i> is able to sense and respond to critical host compartments to mount a successful infection. </p><p> Upon entry to the host cell cytosol, the facultative intracellular pathogen <i> Listeria monocytogenes</i> coordinates the expression of numerous essential virulence factors by allosteric binding of glutathione (GSH) to the Crp-Fnr family transcriptional regulator, PrfA. Here we report that robust virulence gene expression can be recapitulated by growing bacteria in a synthetic medium (iLSM) containing GSH or other chemical reducing agents. Bacteria grown under these conditions were 45-fold more virulent in an acute murine infection model and conferred greater immunity to a subsequent lethal challenge compared to bacteria grown in conventional media. During cultivation <i>in vitro </i>, PrfA activation was completely dependent on intracellular levels of GSH, as a glutathione synthase mutant (ΔgshF) was activated by exogenous GSH but not reducing agents. PrfA activation was repressed in iLSM supplemented with oligopeptides, but suppression was relieved by stimulation of the stringent response. These data suggest that cytosolic <i>L. monocytogenes</i> interpret a combination of metabolic and redox cues as a signal to initiate robust virulence gene expression <i>in vivo</i>. </p><p> Cholesterol-dependent cytolysins (CDCs) represent a family of homologous pore-forming proteins secreted by many Gram-positive bacterial pathogens. CDCs mediate membrane binding partly through a conserved C-terminal undecapeptide, which contains a single cysteine residue. While mutational changes to other residues in the undecapeptide typically have severe effects, mutating the cysteine residue to alanine has minor effects on overall protein function. Thus, the function of this highly conserved reactive cysteine residue remains largely unknown. We report here that the CDC Listeriolysin O (LLO), secreted by the facultative intracellular pathogen <i>Listeria monocytogenes</i>, was post-translationally modified by a S-glutathionylation at this conserved cysteine residue, and that either endogenously synthesized or exogenously added glutathione was sufficient to form this modification. When recapitulated with purified protein <i>in vitro</i>, this modification completely ablated the activity of LLO, and this inhibitory effect was fully reversible by treatment with reducing agents. A cysteine-to-alanine mutation in LLO rendered the protein completely resistant to inactivation by S-glutathionylation and retained full hemolytic activity. A mutant strain of <i>L. monocytogenes </i> expressing the cysteine-to-alanine variant of LLO was able to infect and replicate within bone marrow-derived macrophages indistinguishably from wild-type <i>in vitro</i>, yet was attenuated 4-6 fold in a competitive murine infection model <i>in vivo</i>. This study suggests that S-glutathionylation may represent a mechanism by which CDC family proteins are post-translationally modified and regulated, and help explain an evolutionary pressure behind the highly conserved undecapeptide cysteine.</p><p>
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Endocytosis-Associated Guanine Nucleotide Exchange Factor Rabgef1 Facilitates the Biogenesis of Outer Segments in Mammalian PhotoreceptorsHargrove, Passley 23 February 2018 (has links)
<p> Rod and cone photoreceptors in the retina are polarized sensory neurons that possess uniquely modified primary cilium, called the outer segment, to capture photons. Circadian-mediated shedding and renewal of outer segment membrane discs requires extensive vesicular transport of protein cargo from the endoplasmic reticulum and Golgi to the base of the cilium. Endocytosis is vesicle transport process of capturing and/or recycling extrinsic components and is shown to occur in retina of early vertebrates, such as <i>Xenopus</i> laevis. In this thesis, I have explored the hypothesis that a critical endocytosis-associated protein Rabgef1 is critical for the genesis of photoreceptor outer segments in the mammalian retina. After demonstrating high expression of Rabgef1 concordant with photoreceptor maturation, I characterized morphology and function of retina from <i>Rabgef1</i>-loss of function (<i>Rabgef1</i><sup>–/–</sup>) mice. Though no gross defect was observed by histology and immunohistochemistry before eye opening (postnatal day 14), transmission electron microscopy demonstrated ultrastructural defects in photoreceptor outer segments by P8. Progressive, yet rapid, photoreceptor degeneration and near-complete ablation of the visual response were evident at and after P15. I show that the outer segment defect noted in <i>Rabgef1</i><sup>–/–</sup> mice was not due to defective ciliogenesis or trafficking of cargo proteins to the cilium. In concordance with other systems, Rabgef1 was enriched in purified endocytic vesicles from the retina and interacted with Rabaptin5, confirming its role in Rab5-mediated endocytosis. Curiously, <i>Rabgef1</i><sup>–/– </sup> photoreceptors accumulated enlarged vesicular/endosomal structures within the inner segment, similar to loss of function mutations in the yeast orthologue of Rabgef1, Vps9p. My studies provide the first evidence of an essential role of Rabgef1-mediated fusion and recycling of endocytic vesicles in the formation and/or renewal of outer segment membrane discs in the mammalian retina. Rabgef1 and other components of the endocytic pathway should therefore be considered as candidates for human retinopathies. </p><p>
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Niche Regulation of Muscle Stem Cell Quiescence by Classical CadherinsGoel, Aviva J. 28 February 2018 (has links)
<p> Many adult stem cells are characterized by prolonged quiescence, promoted by cues from their niche. Upon tissue damage, a coordinated transition to the activated state is necessary for successful repair. Non-physiological breaks in quiescence often lead to stem cell depletion and impaired tissue restoration. Here, I identify cadherin-mediated adhesion and signaling between muscle stem cells (satellite cells; SCs) and their myofiber niche as a mechanism that orchestrates the quiescence-to-activation transition. Conditional removal of N-cadherin and M-cadherin in mice leads to a break in SC quiescence with long-term expansion of a regeneration-proficient SC pool. These SCs have an incomplete disruption of the myofiber-SC adhesive junction, and maintain niche residence and cell polarity, yet show properties of SCs in a state of transition from quiescence towards full activation. Among these properties is nuclear localization of b- catenin, which is necessary for this phenotype. These findings are consistent with the conclusion that injury-induced perturbation of niche adhesive junctions is a first step in the quiescence-to-activation transition. </p><p>
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BinK Domain Functional Characterization in the Regulation of Bioluminescence in Vibrio FischeriSter, Ian M. 14 March 2018 (has links)
<p> Prokaryotes encode a remarkable ability to adapt to niches by sensing environmental cues through signal transduction systems (STSs). Typical STS proteins interact through a phosphorylation relay between histidine (His) and aspartate (Asp) residues within modular domains on sensory kinase and response regulator (RR) proteins to elicit cellular responses. A single point mutation in the sensor kinase BinK (BinK1 R537C) conferred an outstanding ability for the non-native <i>V. fischeri</i> strain MJ11 to successfully colonize <i>Euprymna scolopes</i> by affecting multiple symbiotic phenotypes including luminescence activation. However, the role of BinK in luminescence, the interacting partners, and functional mechanism are unknown. We hypothesized that BinK interacts upstream of an orphaned RR and acts as a canonical sensor kinase using a C-terminal receiver (REC) domain to activate luminescence. Heterologous multi-copy expression of BinK in native <i> V. fischeri</i> strain ES114 demonstrated that BinK does not utilize an orphan RR, but instead interfaces with the LuxU-LuxO node to activate luminescence. Additionally, BinK with a truncated REC domain and a REC domain with an aspartate – alanine substitution abolished luminescence activation where the level of light emitted matched the level of light emitted by a strain harboring the empty vector plasmid, suggesting BinK activates luminescence in a REC-dependent manner using the conserved Asp residue for suspected phosphatase / dephosphorylation activity. Elimination of the kinase / auto-phosphorylation activity of the HisKA domain by incorporating a histidine–glutamine substitution did not alter BinK luminescence activation. Though these findings demonstrate one mechanism by which BinK activates luminescence, it is still not clear how the evolved <i>binK1</i> R537C mutation in the HATPase catalytic domain, a domain important in kinase function, influences REC-dependent dephosphorylation. By using multi-copy expression, BinK1 reduces luminescence and increases <i> qrr1</i> expression, and like BinK, works in a REC-dependent manner. These data suggest that one way BinK1 conferred the jump to symbiosis was through reduced or altered function. Furthermore, this mutation unveiled BinK as another potential regulator in bioluminescence where it is poised to work in a manner similar to quorum sensing activators AinR and LuxQ to activate luminescence.</p><p>
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Genetics and Epigenetics of HPV-Infected Anal CarcinomasIbad-Raja, Aliza 17 March 2018 (has links)
<p> Anal squamous cell carcinoma (SCC) which is strongly associated with human papilloma virus (HPV) infection is a rare cancer but its incidence is increasing throughout the world. Even though it represents just 0.4% of all new cancer cases in the US, the mortality rate is estimated at 14%, which is comparable to both breast and prostate cancer mortality rates. To decrease the high rate of mortality and morbidity of anal cancer there is an enormous need for early detection and prevention strategies. Besides understanding the role of HPV infection, we also need to comprehend the basics of genetics and epigenetics involved in anal cancer progression. With both the highest incidence rate and a lower survival rate among African-American men, we are interested in understanding the relationship of HPV, miRNAs and somatic mutations associated with the African-American population in anal cancers. This was accomplished by (1) identifying and determining HPV genotypes associated with anal condylomas, pre-malignant/dysplastic lesions and malignant anal SCC through type specific genotyping, (2) profiling miRNAs in anal SCC based on gender and type of HPV infection to identify novel biomarkers using Nanostring technology, and (3) by identifying oncogenic mutations associated with anal lesions, transformation and progression using novel next generation sequencing methods. Common HPV genotypes associated with our samples included HPV-11, 16, 6, 32, 35, 51, 58, 59, and 68, of which HPV-32, 51, 59 and 68 are not protected by the current FDA approved nonavalent vaccine. Furthermore, 10 of 800 known human miRNAs were significantly dysregulated in SCC samples; these miRNAs (miR-451a, miR-1185-13p, miR-637, miR-4525a-5p, miR-1275, miR-1303, miR-600, miR-892b, miR-297 and miR-944) target tumor suppressor and oncogenes and potentially play an oncomir role in cancer progression. <i>TP53, PIK3CA, PDGFRA, HRAS</i>, and <i> RET</i> were some of the most frequently found somatic mutations in the sample set and it was observed that the accumulation of mutations begin at the condyloma stage. In conclusion, it was determined that three key factors determine the possible progression of anal cancer and can therefore aid in future development of novel targeted therapy approaches: type of HPV infection, epigenetic factors involving miRNAs, and genetic factors such as ‘driver’ somatic mutations that an individual accumulates over their lifetime.</p><p>
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Processing of Potato Spindle Tuber Viroids (PSTVd) RNAs in Yeast, a Nonconventional HostFriday, Dillon R. 01 February 2018 (has links)
<p> The discovery of viroids in 1971 opened the door to a whole new field of RNA biochemistry. Viroids subsequently became the first of many facets of RNA biochemistry: the first single stranded covalently closed RNA discovered in nature, the first subviral pathogen discovered, and the first pathogen of a eukaryotic system to have its genome sequenced. Viroids are the smallest known agents of infectious disease and they represent the borders of life. They replicate autonomously within their host and since they do not code for their own proteins, they act as scavengers of the host transcriptional machinery. By doing so, viroids find ways of trafficking, localizing, and replicating within their host based on the sequence and structure of the RNA alone. Once in their hosts, viroids are incredibly resilient and can cause economic damage on several commercial crops. Apart from controlling viroids for economic reasons, the more enticing feature of viroid study is the use of viroids as model systems to study essential underlying questions about the evolution of RNA pathogens, and to use viroids as models to study non-coding RNAs. The field of non-coding RNA research has surged within the past decade and viroids are becoming important vehicles to bring insight into this field of study. The study of viroids has been extensive through the years, but several questions remain: What structural conformations do viroids employ to recruit host enzymes, and what are the enzymes that cleave and ligate viroids into mature progeny. To answer some of these questions, we have looked at processing of the potato spindle tuber viroid (PSTVd) RNA in the budding yeast <i> Saccharomyces cerevisiae</i>. We found that one specific construct will process into a mature viroid circle in yeast and we also found that processing in this system is distinct from other plant and non-plant based host systems. This processing is a delicate interplay of ligation and degradation by host machinery. Yeast is a great system to study viroid processing as yeast allows for use of the entire toolbox of temperature-sensitive and knockout protein mutants. By employing yeast, focus can be driven towards the mechanisms of host protein recruitment, viroid processing requirements, and degradation mechanisms from the host. We have ascertained insight into PSTVd processing using yeast. We have found methods to transform and process PSTVd, investigated enzymes that effect processing, and started to establish an <i>in vitro </i> yeast system. Through these studies, we have also developed a method to enrich viroid RNAs from total RNA extractives. This has been vital to assays specific around viroid transcription and cleavage. Overall, this research is further testament that viroids are minimalist scavengers of a very diverse array of cellular transcriptional machinery. They can process in higher eukaryotes (plants) and simple eukaryotes (yeast). They are shown to affect each host in distinct manners using fundamental RNA biology that all organisms share. </p><p>
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Pyruvate Contributes to the Virulence of Staphylococcus aureus by Regulating Toxin Production through the ArlRS Two-Component SystemHarper, Lamia C. 18 April 2018 (has links)
<p> <i>Staphylococcus aureus</i> (<i>S. aureus </i>) is an extremely versatile bacterial pathogen that causes significant disease burden. Its effectiveness in causing disease is due in part to its ability to adapt to diverse host niches. <i>S. aureus</i> senses environmental changes and subsequently adjusts the production of virulence factors needed to initiate and sustain an infection, while combating host immune defenses. Despite the importance of environmental cues on <i> S. aureus</i> pathogenicity, only a limited number of these signals have been investigated in detail for their ability to modulate virulence. During this thesis work, we showed that the central metabolite pyruvate alters the overall metabolic flux of <i>S. aureus</i>, influencing pathways such as central metabolism and amino acid metabolism, and ultimately enhancing the pathogenicity of <i>S. aureus</i>. We demonstrated that pyruvate stimulates production of virulence factors such as the cytolytic leukocidins, and that this induction is responsible for the increased virulence we observe in USA300 LAC, a strain of community acquired methicillin resistant <i> S. aureus</i> (CA-MRSA). Specifically, we showed that an efficient “pyruvate response” requires the ArlRS two-component system, which modulates the Agr quorum sensing system to reduce the production of Rot, a key repressor of toxins. Altogether, these studies further establish a strong relationship between metabolism and virulence and, through activation of intricate but complex regulatory networks identifies pyruvate as an important regulatory signal for the coordination of <i>S. aureus</i> virulence.</p><p>
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Aging, Stress, and Pathogenesis of Parkinson's Disease| Studies Using C. elegansCooper, Jason Fisk 14 April 2018 (has links)
<p> Parkinson’s disease (PD) is an adult onset neurodegenerative disease that is characterized by deficiencies in movement, cognition, and Lewy body neuropathology within the brain. Motor and cognitive deficiencies progressively worsen through the course of disease concurrent with increasing neuropathology and neurodegeneration. Approximately 10–15% of PD patients have a family history of PD with a confirmed genetic cause. Presently PD pathogenesis is incompletely understood and there are no treatments capable of halting or reversing this disease. The extended disease-course and age-dependent nature of PD, especially in genetic cases where a mutation is present from birth, affirm that aging itself is the most important risk factor for disease. We hypothesize that specific cellular changes that occur during the normal process of aging confer susceptibility to disease-causing mutations which, while tolerated at younger ages, contribute to disease with age. Accurate animal models of PD and aging provide the ability to elucidate disease mechanisms and explore novel strategies targeting the aging process. To test the role of aging in PD we utilize the nematode <i>Caenorhabditis elegans</i> because this animal has been used extensively to study animal aging at a cellular level. We confirm that disease phenotypes in genetic <i>C. elegans</i> models of PD such as neurodegeneration, protein aggregation, and mitochondrial deficits are proportional to this organism’s brief lifespan. This indicates that PD progresses according to biological age and not merely to chronological time. As a proof-of-principle we also show that delaying aging by mutation of the gene encoding the insulin-IGF receptor, <i>daf-2</i>, can rescue multiple deficits present in nematode models of PD. Overall we demonstrate that biological aging is a crucial for the development of various PD associated phenotypes and that delaying aging is sufficient to delay these phenotypes. Therefore targeting aging itself may be a sound strategy for the halting or the prevention of PD.</p><p>
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The Molecular Mechanisms of Sex Determination in VertebratesGuo, Lei 19 September 2017 (has links)
<p> Many reptiles display temperature-dependent sex determination (TSD), in which the primary sex is determined by incubation temperatures rather than sex chromosomes. However, temperature is not the only factor that play critical roles in sex determination in the species with TSD. Previous studies in the snapping turtle, a species with TSD, showed that dihydrotestosterone (DHT) induces ovary development at temperatures that normally produce males or mixed sex ratios. In addition, the feminizing effect of DHT was found to be associated with increased expression of the ovary-determining gene <i>Foxl2</i>, suggesting a potential androgen-<i>Foxl2</i> regulatory mechanism. This dissertation aims to clarify the molecular mechanisms underlying TSD in several aspects. First, determine the role of androgen in TSD; second, identify novel thermosensitive genes involved in TSD and lastly, reconstruct gene regulatory networks underlying sex determination. </p><p> To test the hypothetical androgen-<i>Foxl2</i> interaction, I cloned the proximal promoter (1.6 kb) and coding sequence for snapping turtle <i> Foxl2</i> (t<i>Foxl2</i>) in frame with mCherry, a red fluorescent protein. The t<i>Foxl2</i>-mCherry fusion plasmid or mCherry plasmid were stably transfected into mouse KK1 granulosa cells. Although expression of t<i>Foxl2</i>-mCherry was not affected by androgen treatment in KK1 cells, androgen inhibited expression of the endogenous mouse Foxl2 gene, suggesting the androgen-Foxl2 interaction does exist but it differs between species. We also found t<i>Foxl2</i>-mCherry potentiated low dose DHT effects on aromatase expression, which has not been reported in any other studies. </p><p> To identify novel sex-determining genes in TSD, I first <i>de novo </i> assembled and annotated the transcriptome of the snapping turtle using next-generation sequencing (NGS) and then performed RNA-seq analyses on the newly assembled reference transcriptome. With the differential gene expression analyses, I identified 293 thermosensitive genes. Among these genes, I find AEBP2, JARID2, and KDM6B of particular interest because these genes could influence expression of many other genes via epigenetic modifications. </p><p> To further investigate the molecular mechanisms underlying sex determination, I reconstructed gene regulatory networks using an entropy based network reconstructing algorithm—ARACNE with public microarray experiments in mouse gonads. The subsequent hub gene analyses revealed the basic molecular pathways underlying gonadal development and the master regulator analyses identified 110 candidate sex-determining genes including both known sex-determining genes and novel candidate genes. </p><p> My findings demonstrate that androgens can influence expression of key ovarian genes but further studies are needed to understand the androgen signaling in TSD. Furthermore, my study provides a first description of the snapping turtle transcriptome and the effects of temperature on transcriptome-wide patterns of gene expression during the TSP. In addition, hub genes and master regulators identified for mammalian gonad determination will guide the direction of future studies in the field of sex determination. However, additional studies are needed to validate the computational findings.</p><p>
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Reinforced thermoplastics for engineering applicationsQuinn, Neil C. January 1985 (has links)
The effect of mechano-chemically bound polypropylene modifiers on the mechanical performance and thermal-oxidative stability of polypropylene composites has been studied. The mechanical performance of unmodified polypropylene containing silane coupled glass and Rockwool (mineral) fibre was poor by comparison with a similar commercially produced glass reinforced composite; this was attributed to poor fibre-matrix adhesion. Mechano-chemical binding with unsaturated additives was obtained in the presence of a free radical initiator (di-cumyl peroxide). This process was inhibited by stabilisers present in commercial grades of polypropylene composites by chemical bond formation between the chemically bound modifier and the silane coupling agent on the fibre surface, resulting in a dramatic improvement in the mechanical properties, dimensional stability and retention of mechanical performance after immersion in fluids typically found in under-bonnet environments.A feature unique to some of these modifiers was their ability not only to enhance the mechanical properties of polypropylene composites to levels substantially in excess of currently available commercial materials, but their ability to act as effective thermal-oxidative polypropylene stabilisers. The mode of action was shown to be a chain-breaking mechanism and as a result of the high binding levels achieved during melt processing, these modifiers were able to efficiently stabilise polypropylene in the most severe volatilising and solvent-extracting environments, thus giving much better protection to the polymer than currently available commercially stabilised grades of polypropylene.
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