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Importance of the Pre-\(NH_2\)-Terminal Domain of HSV-1 DNA Polymerase for Viral ReplicationTerrell, Shariya Louise 06 February 2014 (has links)
The catalytic subunit of the herpes simplex virus 1 DNA polymerase (HSV-1 Pol) has been extensively studied; however, its full complement of functional domains has yet to be characterized. The previously uncharacterized pre-NH2-terminal domain (residues 1-140) within HSV-1 Pol is unique to the herpesvirus Pol family. We sought to investigate the importance of this domain for viral replication in cell culture and an animal model of infection. We evaluated the enzymatic activity of purified pre-NH2-terminal Pol mutant proteins in which conserved residues had been deleted or substituted. Subsequently, the corresponding pol mutant viruses were engineered for viral genetic analyses. We found that the extreme N-terminal 51 residues were not required for wild type 5’-3’ polymerase activity in vitro. Interestingly, the extreme N-terminal 42 residues were dispensable for viral replication in cell culture while a conserved motif at residues 44-49 was necessary for efficient viral DNA synthesis and production of infectious virus. Viral replication proteins have proven to be particularly important in the context of acute and latent infections in animals. Characterization of pol mutant virus replication in a mouse ocular model of infection revealed that the extreme N-terminal 42 residues were not required for viral replication and reactivation from latency. The conserved motif, however, was shown to be required for robust acute ganglionic replication and efficient latency establishment. We hypothesized that the conserved motif at residues 44-49 mediates a protein- protein interaction that positively impacts viral DNA synthesis during infection. Specific protein candidates were evaluated using purified proteins in vitro, and proteins that coprecipitated with wild type and mutant polymerases from infected cell lysates were analyzed. To date, we have yet to identify a protein whose binding was disrupted as a result of the mutation. Ultimately, we have established a role for the pre-NH2-terminal domain of HSV-1 Pol during viral replication that is distinct from 5’-3’ polymerase activity. The conserved motif mediates a function that is required for efficient viral DNA synthesis in cell culture and is of even greater importance for acute ganglionic replication in mice. The mechanism of action more than likely reflects a conserved mechanism for herpesvirus replication.
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Identification and Characterization of Novel Drug Resistance Loci in Plasmodium falciparumVan Tyne, Daria Natalie 15 March 2013 (has links)
Malaria has plagued mankind for millennia. Antimalarial drug use over the last century has generated highly drug-resistant parasites, which amplify the burden of this disease and pose a serious obstacle to control efforts. This dissertation is motivated by the simple fact that malaria parasites have become resistant to nearly every antimalarial drug that has ever been used, yet the precise genetic mechanisms of parasite drug resistance remain largely unknown. Our work pairs genomics-age technologies with molecular biology, genetics and molecular epidemiology in order to identify and characterize novel genes that contribute to drug resistance in P. falciparum. In the Introduction, we highlight relevant opportunities and challenges in trying to identify and understand the genetic basis of malaria drug resistance as it emerges to currently used therapies. In Chapter One, we demonstrate how genome-wide association studies (GWAS) can be applied to P. falciparum in order to identify novel drug resistance loci. Functional follow-up revealed that overexpression of the novel candidate gene PF10_0355 made parasites more resistant to the drugs halofantrine, lumefantrine and mefloquine. These findings show that PF10_0355 plays a role in parasite drug response, as well as provides validation of our GWAS approach. In Chapter Two, we further characterize PF10_0355 and show that modulation of the gene by either knockout or allelic replacement changes parasite drug sensitivity. Furthermore, we show that moderate changes in drug response measured in the short-term can have dramatic effects when parasites are competed with one another under drug pressure. In Chapter Three, we use an overexpression approach to functionally follow up other novel drug resistance genes generated by GWAS in P. falciparum. We find that overexpression is a useful way to begin to screen candidate drug resistance loci in the malaria parasite. In Chapter Four, we use a DAPI-based ex vivo drug assay to monitor drug resistance among parasites circulating in Thiés, Senegal. In the future, we will look for genetic markers of parasite drug resistance in this population by GWAS. Finally, in the Discussion we present an essay about malaria evolution and eradication written for non-specialists. Our hope is that the work presented in this dissertation furthers understanding of drug resistance in the malaria parasite, both within and beyond the malaria research community.
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Secretory Mechanisms of aP2: an Adipokine Integrating Adipose Depots with MetabolismErikci Ertunc, Meric January 2014 (has links)
Adipose Fatty Acid Binding Protein 4 (FABP4) or aP2, plays an important role in several immunometabolic pathologies such as type 2 diabetes, atherosclerosis, fatty liver disease, asthma, and cancer. Long considered to be a cytosolic protein, aP2 has recently been detected in conditioned media of adipocytes. Interestingly, there is a growing body of literature showing association of increased circulating levels of aP2 with cardiovascular disease and metabolic syndrome. Our lab has discovered a role for aP2 secreted from adipocytes in regulating liver glucose output and blood glucose levels in diabetes. The emerging biology of this novel adipokine makes it critical to understand the route and mechanisms that lead to its secretion.
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Identification of Adenyl Cyclase Activity in a Disease Resistance Protein in Arabidopsis thalianaHussein, Rana 11 1900 (has links)
Cyclic nucleotide, cAMP, is an important signaling molecule in animals and plants. However, in plants the enzymes that synthesize this second messenger, adenyl cyclases (ACs), remain elusive. Given the physiological importance of cAMP in signaling, particularly in response to biotic and abiotic stresses, it is thus important to identify and characterize ACs in higher plants. Using computational approaches, a disease resistance protein from Arabidopsis thaliana, At3g04220 was found to have an AC catalytic center motif. In an attempt to prove that this candidate has adenyl cyclases activity in vitro, the coding sequence of the putative AC catalytic domain of this protein was cloned and expressed in E. coli and the recombinant protein was purified. The nucleotide cyclase activity of the recombinant protein was examined using cyclic nucleotide enzyme immunoassays. In parallel, the expression of At3g04220 was measured in leaves under three different stress conditions in order to determine under which conditions the disease resistance protein could function. Results show that the purified recombinant protein has Mn2+ dependent AC activity in vitro, and the expression analysis supports a role for At3g04220 and cAMP in plant defense.
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Visualization and exploration of transcriptomics dataGehlenborg, Nils January 2010 (has links)
No description available.
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Studies on SET and MYND domain proteins in DrosophilaThompson, Elizabeth Claire January 2008 (has links)
No description available.
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Coordinated regulation of the snail family of transcription factors by the notch and tgf-0 pathways during heart developmentNiessen, Kyle 05 1900 (has links)
The Notch and TGF13 signaling pathways have been shown to play important roles in regulating endothelial-to-mesenchymal transition (EndMT) during cardiac morphogenesis. EndMT is the process by which endocardial cells of the atrioventricular canal and the outflow tract repress endothelial cell phenotype and upregulate mesenchymal cell phenotype. EndMT is initiated by inductive signals emanating from the overlying myocardium and inter-endothelial signals and generate the cells that form the heart valves and atrioventricular septum. The Notch and TGFf3 pathway are thought to act in parallel to modulate endothelial phenotype and promote EndMT. Vascular endothelial (VE) cadherin is a key regulator of cardiac endothelial cell phenotype and must be downregulated during EndMT. Accordingly, VE-cadherin expression remains stabilized in the atrioventricular canal and outflow tract of Notchl-deficient mouse embryos, while activation of the Notch or TGFP pathways results in decreased VE-cadherin expression in endothelial cells. However, the downstream target gene(s) that are involved in regulating endothelial cell phenotype and VE-cadherin expression remain largely unknown.
In this thesis the transcriptional repressor Slug is demonstrated to be expressed by the mesenchymal cells and a subset of endocardial cells of the atrioventricular canal and outflowtract during cardiac morphogenesis. Slug is demonstrated to be required for cardiac development through its role in regulating EndMT in the cardiac cushion. Data presented in Chapter 6 further suggests that Slug-deficiency in the mouse is compensated for by a increase in Snail expression after embryonic day (E) 9.5, which restores EndMT in the cardiac cushions. Additionally, the Notch pathway, via CSL, directly binds and regulates expression of the Slug promoter, while a close Slug family member, Snail is regulated by the TGFB pathway in endothelial cells. While Notch does not directly regulate Snail expression, Notch and TGFB act synergistically to regulate Snail expression in endothelial cells. It is further demonstrated that Slug is required for Notch mediated EndMT, binds to and represses the VE-cadherin promoter, and induces a motile phenotype. Collectively the data demonstrate that Notch signaling directly regulates Slug, but not Snail, expression and that the combined expression of Slug and Snail are required for cardiac cushion morphogenesis.
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Development and demonstration of a quantitative PCR based method to enumerate copepod nauplii in field samplesJungbluth, Michelle J. 08 May 2013 (has links)
<p> Copepod nauplii are important members of the marine planktonic community, and they can be the most abundant component of the microzooplankton. Despite the importance of copepod early life history stages to food web dynamics and carbon flux in the sea, there is a paucity of information about their ecology due to challenges in identifying nauplii to species, and in sampling them quantitatively. I report here on the development and optimization of a new molecular method that uses quantitative PCR (qPCR) to identify and estimate the abundances of nauplii of a common coastal copepod, <i>Parvocalanus crassirostris,</i> in field samples. The following experiments were performed towards this goal: I surveyed the genetic diversity of copepods in the study region, optimized sample treatment for qPCR, developed a size fractionation protocol to separate life stages of the target species, quantified the mitochondrial cytochrome C oxidase subunit I (mtCOI) gene copies in each <i> P. crassirostris</i> life stage, tested the effect of food levels on mtCOI copy number in nauplii, and compared direct counts to qPCR estimates of the target species to validate the qPCR method. The number of mtCOI gene copies in each developmental stage of this species was found to increase by ∼1.5 orders of magnitude from early nauplii to adult. Food level experiments suggested that mtCOI copy number may be influenced by feeding environment in late naupliar stages. In validation experiments, qPCR estimates were 68 to 130% of the number estimated from direct counts. Both methods had a coefficient of variation of approximately 16%, indicating similar precision across methods. As a field test of the method, daily samples were collected in southern Kane‘ohe Bay and used to quantify the density <i>P. crassirostris</i> nauplii over a 13-day period in the summer of 2011. The average density of <i> P. crassirostris</i> nauplii in developmental stages NII - NV was found to be 1.5×10<sup>3</sup> individuals m<sup>-3</sup> over the 13-day period. The qPCR-based method developed here will enable future studies on naupliar ecology in the field, including investigation of food web, population, and community dynamics.</p>
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Microspherule Protein Msp58 and Ubiquitin Ligase EDD Form a Stable Complex that Regulates Cell ProliferationBenavides, Mario 22 June 2013 (has links)
<p> A complex molecular network is put into place at specific phases of the cell cycle to prevent unscheduled cell division that could result in malignant cell growth. Emerging evidence shows that still uncharacterized proteins play crucial functions at those cell cycle transition points. Nuclear protein Msp58 and EDD E3 ubiquitin ligase have been implicated in different aspects of cell proliferation and reported to be abnormally expressed in numerous types of cancers. The molecular mechanisms underlying Msp58 and EDD functions, however, are not well understood. The work presented here shows that Msp58 and EDD form a stable protein complex that regulates cell viability and proliferation. Interestingly, knockdown of EDD by RNA interference leads to a significant accumulation of Msp58 protein, which suggests that EDD serves as a negative regulator of Msp58. In addition, our in vivo ubiquitination assays and analyses of various cell lines treated with translational and proteasomal inhibitors demonstrate that Msp58 is regulated post-translationally by the ubiquitin-proteasome pathway. These results imply that EDD ligase activity is involved in this regulatory process. Using flow cytometry analyses and biochemical characterization of Msp58 and/or EDD depleted cells, we show that the Msp58-EDD complex plays important roles in cell cycle progression via the control of cyclin gene expression. In particular, silencing Msp58 and/or EDD alters the protein levels of cyclins B, D and E. Taken together, our data suggest that a set of the biological roles attributed to Msp58 and EDD may be executed in the context of the complex that they form, thereby revealing a novel molecular mechanism for these two proteins to accomplish their functions.</p>
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The cloning and characterization of the novel genes ENV10 and ENV11 in S. cerevisiaeOliveira, Lisa Ann K. 09 August 2013 (has links)
<p> Through the use of a novel immunodetection assay, our lab identified a series of mutants that internally accumulate the precursor form of the vacuolar hydrolase carboxypeptidase Y (CPY), a phenotype that suggests a defect at the late e&barbelow;n&barbelow;dosome to v&barbelow;acuole (<i> ENV</i>) interface of the biosynthetic pathway. This study focuses on two of the novel genes identified: <i>ENV10</i> and <i>ENV11 </i> and is the first to establish the cellular localization of Env10p in the endoplasmic reticulum. Assays of vacuole and lipid droplet morphology, as well as growth characterization under various stressors demonstrate Env10p has a role in vacuolar protein trafficking and endomembrane system integrity and may operate in a parallel or compensatory manner to Env9p. This study also confirms that Env11p localizes to the nucleus in a saturable fashion where it may be involved in transcriptional regulation of genes involved in vacuole events in conjunction with Vid22p and Tbf1p.</p>
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