Browsing by Subject "signaling"
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Item Biological response to 8-oxoguanine base released during DNA base excision repair(2010-01-05) David Noe Saenz; William R. Widger; Tapas K. Hazra; Istvan Boldogh; Darrell H. Carney; Amarnath NatarajanAging and age-associated diseases have been associated with altered cellular signaling, chromatin perturbation, oxidative stress, and DNA damage, especially at the telomere. Despite having evolved a sophisticated surveillance system, oxidative DNA damage still accumulates in the cells of organismal tissues. 8-oxo-7,8-dihydroguanine (8-oxo-Gua) is a compound occurring in cells as a major oxidative lesion due to guanine’s susceptibility to oxidation. A base excision repair pathway initiated by 8-oxoguanine DNA glycosylase (OGG1) excises 8-oxo-Gua lesions from nuclear and mitochondrial DNA, releasing the free oxidized base. It is well-established that failure to repair 8-oxo-Gua can result in mutagenesis and carcinogenesis; however, the cellular response to free 8-oxo-Gua has never been investigated. Initial experiments show that 8-oxo-Gua induced premature senescence in a concentration-dependent manner when added to the medium of human diploid fibroblasts (HDFs), while other oxidized and unmodified bases had no effect. We address a potential cause-effect relationship between free 8-oxo-Gua and the premature senescence of HDFs as a model for aging. We hypothesize that free 8-oxo-Gua released during DNA base excision repair is an endogenous mediator of cellular senescence through oxidative stress and altered cellular signaling. Using cell culture models and advanced cellular and molecular biology methods such as microarray, ROS detection assays, immunoblot, qRT-PCR, shRNA and siRNA technology, microscopy, as well as LC/IDMS for quantification of 8-oxo-Gua to test this hypothesis, we: 1) established that 8-oxo-Gua-induced senescent cells exhibit the morphology, features, and gene expression profile of naturally senescent cells; 2) determined that 8-oxo-Gua activates Ras in an unscheduled, prolonged manner, and that its activation transduces through the Raf-MEK(1/2)-ERK(1/2) MAPK cascade; 3) identified a chemical transformation of 8-oxo-Gua to a hydroperoxide-like form under physiological pH and oxygen atmosphere; and 4) determined 8-oxo-Gua-induced oxidative stress, which arises from the Rac1-Ras-NOX-PLA2-LO pathways, does not have a significant role in the observed senescence.Item Microvascular endothelial response to cocaethylene exposure: morphological and molecular observations(2004-11-02) Danyel Hermes Tacker; Anthony O. Okorodudu, Ph.D.; Robert H. Glew, Ph.D.; Norbert K. Herzog, Ph.D.; M. Tarek Elghetany, M.D.; Kathryn A. Cunningham, Ph.D.; Hal K. Hawkins, M.D., Ph.D.Cocaethylene (CE) is an active metabolite of cocaine and ethanol and is a toxicant of physiological relevance due to the high rate of cocaine and ethanol co-exposure (~80%) in cocaine abusers. It has prolonged action and increased potency on known physiological targets relative to the effect of cocaine. Since pathology in cocaine abusers is typically chronic and systemic, and CE persists in the body three to five times longer than cocaine, a link between CE and systemic disease in cocaine abusers was proposed. Consequently, this dissertation contains the studies that were used to test the hypothesis that the microvascular endothelium is a target tissue that is central in the pathogenic mechanism of cocaine-associated systemic disease, and that endothelial injury after CE exposure would result in dysregulation and altered barrier function due to changes in intracellular second messengers and signaling. To test this hypothesis, an in vitro model of CE exposure in human dermal microvascular endothelial cells (HMEC-1) was developed. Four Aims were designed to compartmentalize various components of the endothelial response to CE. The Aims included an array of methods to address cellular toxicity and dysfunction, including classical cytotoxicity and viability assays (Aim One), microscopic and electrical analyses of monolayer integrity (Aim Two), molecular analysis of second messengers, signaling molecule phosphorylation, and transcription factor DNA binding activity (Aims Three and Four). Aim One experiments demonstrated a lack of overt endothelial cytotoxicity caused by CE. Aim Two morphological analysis of endothelial intercellular borders and barrier integrity showed that CE exposure in the endothelial monolayers resulted in increased permeability, and hence a decrease in barrier integrity. These changes were observed temporally with alterations in cytosolic and total cellular free calcium ion (Aim Three), inositol 1,4,5 trisphosphate, and phosphorylated p38 mitogen-activated protein kinase concentrations, as well as changes in DNA binding activity and dimer composition of nuclear factor-kappaB (Aim Four). The observed changes suggest a distinct alteration of endothelial cell and monolayer function consistent with increased vascular permeability in vivo. Potential pathological outcomes of such effects include inflammation, vasculitis, systemic disease, and organ failure.Item Perlecan regulation of sonic hedgehog signaling: from drosophila to humans(2009-05-15) Hernandez, Ana MariaProstate cancer is the second leading cause of death from cancer in men in the United States. Most men will die of the advanced, metastatic form of the disease. Thus, treatment strategies targeting the metastatic form of the disease are especially needed. Emerging research on metastatic cancer highlights the importance of the microenvironment in cancer progression and metastasis, with an emphasis on deregulated developmental signaling in cancer progression. Research in model organisms has shown that developmental signaling pathways are regulated by various components of the extracellular matrix, including heparan sulfate proteoglycans. In the model system Drosophila, the heparan sulfate proteoglycan Trol is needed for Hhdependent proliferation in quiescent neural stem cells. In collaboration with others, I have shown that the human homolog of Trol, PERLECAN, regulates SONIC HEDGEHOG-dependent proliferation in advanced prostate cancer by two different mechanisms. This makes PERLECAN a potential drug target and biomarker for prostate cancer screening and treatment. My results also validate the discoveries made in Drosophila in the context of human disease. With this validation, I propose and describe the Drosophila Ejaculatory Bulb (EjB) as model for prostate cancer and prostate aging.Item Regulation of Xylella fastidiosa virulence factors by c-di-GMP phosphodiesterases(2012-10-19) Ancona-Contreras, VeronicaXylella fastidiosa is an important bacterial plant pathogen that colonizes the xylem of hundreds of plant species. X. fastidiosa cause Pierce's disease in grapevine by occlusion of the xylem by extensive bacterial colonization, extracellular polysaccharides and the formation of a biofilm. These traits are mediated in a cell-density manner by a cell-to-cell signaling system that transduces a diffusible signaling factor (DSF). This dissertation demonstrates that PD1994, PD1617 and RpfG regulate important traits for bacterial virulence such as cell-cell signaling, biofilm formation and cell aggregation. X. fastidiosa strains harboring mutations in pd1994 (which encodes for a defective GGDEF- EAL-domain protein) and in pd1617 (which encodes for a EAL-domain protein) have increased growth rate, increased biofilm formation, increased plant colonization and decreased cell aggregation. Gene expression analysis of the pd1994 mutant strain showed overexpression of rpfF, which is a DSF synthase, suggesting that PD1994 regulates DSF signaling by repressing rpfF expression. Additionally, the pd1994mutant showed overexpression of pd1617 and rpfG (with EAL and HD-GYP domains respectively, that may be responsible for c-di-GMP turnover), which suggested that this mutant may have low c-di-GMP levels and that PD1994 regulates c-di-GMP turnover by repression of RpfG activity and PD1617 gene expression. X. fastidiosa harboring a mutation on rpfG exhibited decreased biofilm formation while it had no effect in growth or cell aggregation. Together, these results suggest that PD1994, PD1617 and RpfG regulate the DSF regulatory network by controlling the turnover of the second messenger c-di-GMP.