Browsing by Author "Robert C. Smart, Committee Member"

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Cardiac Cell Type-Specific Differences in the Interferon (IFN) Response, and Reovirus Repression of IFN
(2008-08-08) Zurney, Jennifer Michelle; Barbara Sherry, Committee Chair; Michael Sikes, Committee Member; Ian T.D. Petty, Committee Member; Robert C. Smart, Committee Member
Clustering of Mixed Data Types with Application to Toxicogenomics
(2006-04-25) Bushel, Pierre Robert; Greg C. Gibson, Committee Chair; Russell D. Wolfinger, Committee Member; Spencer V. Muse, Committee Member; Robert C. Smart, Committee Member
DNA microarray analysis provides unprecedented capabilities for simultaneous measurement of genome-wide alterations in transcription levels. Toxicogenomics bridges gene and protein expression analyses with conventional toxicology to elucidate a global view of the toxic outcomes and mechanistic changes elicited by toxicant exposure and environmental stressors to biological systems. Inherent in toxicogenomics data are systematic error, stochastic variation and disparate measurement domains and types which complicate the acquisition of significant, meaningful and broad biological interpretations from analysis of the data. In this dissertation, a classification regimen comprised of analysis of replicate data, outlier diagnostics and gene selection procedures was employed to utilize microarray data for categorization of sub-classes of biological samples exposed to pharmacologic agents. To assess contrasts of centrilobular congestion severity of the rat liver subsequent to exposure with acetaminophen (APAP), microarray data, clinical chemistry evaluations and histopathology observations were integrated in a database and analyzed using mixed linear model approaches. Finally, the k-prototype algorithm with a mixed objective function comprised of the sum of the squared Euclidean distance to measure the dissimilarity of samples based on microarray array and clinical chemistry numeric data features and simple matching to measure the dissimilarity of the samples based on histopathology features with categorical values, was modified (Modk-prototypes) to the specifications of k-means clustering. In addition, the objective function included weighting terms for the microarray, clinical chemistry and histopathology domain data in order to computationally integrate the data as well as constrain the clustering of the APAP-treated samples according to similarity of gene expression and toxicological profiles. Simulated annealing optimization of the Modk (SA-Modk) —prototypes algorithm was used to validate the clustering of the APAP-treated samples. The clusters were vetted for gene expression and toxicological (VETed) k-prototypes features that discerned clusters from one another. The VETed k-prototypes are shown to be ideal for distinguishing between zero, minimal, and moderate levels of necrosis of the hepatocytes and centrilobular region of the rat liver that are end-point representations of the clusters of APAP-treated samples. In this dissertation, chapter 1 is an introduction to general toxicology, microarray gene expression array platforms, experimental designs, preprocessing of the data and gene selection approaches, toxicogenomics as it applies to compound classification and phenotypic anchoring of gene expression, databases and informatics resources for toxicogenomics and clustering of mixed data types. Chapter 2 is dedicated to statistical validation and significance of differentially expressed genes as well as sub-categorization of samples exposed to phenobarbital and peroxisome proliferators clofibrate, gemfibrozil and Wyeth 14, 643. Chapter 3 presents integration of microarray data with clinical chemistry and histopathology data to contrast levels of centrilobular congestion of the rat liver by mixed linear modeling of gene expression ratio values acquired from rats exposed to APAP. Chapter 4 describes the utilization of a modified k (Modk) —prototypes objective function and algorithm, and simulated annealing optimization version of the Modk (SA-Modk)-prototypes objective function, for computational integration of microarray, clinical chemistry and histopathology mixed numeric and categorical data. It also includes partitioning of APAP-treated biological samples into clusters which contain vetted expression and toxicological (VETed) k-prototypes features that distinguish between levels of necrosis of the hepatocytes and centrilobular region of the rat liver. In chapter 5, a conclusion of the research, development and analyses presented in this dissertation is provided.
Monogenic Diabetes and Regulation of the Transcription Factor, HNF1.
(2008-01-23) Wan, Cheng; William L. Miller, Committee Member; Robert C. Smart, Committee Member; Carla Mattos, Committee Member; Robert B. Rose, Committee Chair
Regulation of Sp2 DNA-Binding Activity and trans-Activation
(2005-08-02) Moorefield, Kristopher Scott; Jonathan M. Horowitz, Committee Chair; Robert C. Smart, Committee Member; Spencer V. Muse, Committee Member; Jim W. Mahaffey, Committee Member
Regulated transcription requires the collaboration of a variety of transcription factors, including sequence-specific DNA-binding proteins. The Sp-family of DNA-binding proteins governs the expression of a wide variety of mammalian genes, including housekeeping, cell cycle-regulated, and developmentally regulated genes. A wealth of information has been obtained about the functional and biochemical properties of two Sp-family members, Sp1 and Sp3, whereas studies of other Sp proteins have been limited. The purpose of this research has been to characterize the functional and biochemical properties of Sp2, and to provide insights into mechanisms regulating Sp2 DNA-binding activity and trans-activation. To initiate my analysis of Sp2 I first identified its consensus DNA-binding sequence (5'-GGGCGGGAC-3') using a PCR-based protocol, and determined that Sp2 binds this sequence with high affinity in vitro (225 pM). Despite the incorporation of this consensus sequence within the promoter of a well-characterized Sp-dependent gene, I found that Sp2 was a relatively weak activator of transcription compared with Sp1 and Sp3. To begin to define mechanisms limiting Sp2 function, chimeric proteins carrying portions of Sp2 and Sp1 were created and analyzed. These studies demonstrated that Sp2 DNA-binding activity and trans-activation are each negatively regulated in mammalian cells. In mixing experiments I detected an activity in mammalian extracts that abrogates Sp2 DNA-binding activity. I showed further that an 84 kDa mammalian protein bound to the trans-activation domain of Sp2, but not that of Sp1 or Sp3. Phosphatase treatment revealed that Sp2 DNA-binding activity requires phosphorylation, and phosphoamino acid analysis confirmed that Sp2 is phosphorylated in vivo. Size-exclusion chromatography indicated that Sp2 is specifically phosphorylated in vitro by an activity in fractions carrying high-molecular weight proteins or protein complexes. Indirect immunofluorescence studies indicated that, unlike other Sp proteins, the vast majority of Sp2 localizes to sub-nuclear foci associated with the nuclear matrix. The data reported herein indicates that Sp2 is functionally distinct from other Sp proteins, and provides insight into mechanisms that negatively regulate Sp2-mediated transcription. Taken together, my results suggest that Sp2 may perform a highly specialized role in the regulation of gene expression.
The Role of TAK1 Associated Binding Protein 2 in Tumor Necrosis Factor Signaling and Cell Death
(2010-03-29) Broglie, Peter Michael; Jun Ninomiya-Tsuji, Committee Chair; James C. Bonner, Committee Member; Yoshiaki Tsuji, Committee Member; Robert C. Smart, Committee Member
Tumor Necrosis Factor (TNF) is a key modulator of the innate immune system, aiding in the activation of immune cells as well as killing damaged cells. Dysregulation of the TNF-initiated intracellular signaling pathways can cause a number of pathologic conditions. TNF binds to TNF receptor (TNFR) and initiates signaling by assembling a protein complex on the receptor consisting of Receptor Interacting Protein 1 (RIP1) and TNF-Receptor Associated Factor 2/5 (TRAF2/5). The RIP1-TRAF complex ultimately activates the IÎºB kinase (IKK)-NF-ÎºB pathway as well as mitogen activated protein kinase (MAPK)-AP-1 pathway, and transcriptionally activates various anti-inflammatory genes. A key step in this signaling pathway is the activation of the MAPK kinase kinase family member TGF-Î² Activated Kinase 1 (TAK1) through which the IKK and MAPK pathways are activated. Activation of TAK1 occurs within 3-5 minutes in TNF signaling and is quickly deactivated by the protein phosphatase PP6, which is important to avoid prolonged signaling. It has been demonstrated that RIP1 is ubiquitinated and this polyubiquitin chain is important for assembly of the signaling complex consisting of RIP1, TAK1, TAB2 and IKK. However, the mechanism by which PP6 is recruited to TAK1 has not yet been determined. In this study, we show that the TAK1 associated binding protein TAB2 is critical in the deactivation of TAK1 by localizing TAK1 and PP6 in close proximity. We found that, in TAB2 knockout fibroblasts, TAK1 is not deactivated in a timely manner as it is in TAB2 wild-type cells. We found that the interaction between TAK1 and PP6 is disrupted in TAB2 knockout fibroblasts. We demonstrated that PP6 was normally associated with a polyubiquitin chain, and the TAB2-polyubiquitin complex was associated with PP6. Our results have revealed that a polyubiquitin chain is important not only for the assembly of the activation complex but also of the deactivation complex for TAK1 in TNF signaling pathway, and that TAB2 participates in these complex as an indispensable adaptor. We have observed that, following prolonged exposure to TNF, TAB2 knockout fibroblasts underwent cell death. We next investigated the role of TAB2 in TNF-induced cell death. Treatment with caspase inhibitors only further sensitized cells to TNF-induced cell death, indicating that TAB2 knockout cells were not dying through an apoptotic mechanism. Treating cells with a specific necroptosis inhibitor, Necrostatin-1, we found that this cell death hypersensitivity was completely rescued, suggesting that TAB2 deficiency activates TNF-induced necroptosis. Necroptosis is an active form of necrotic cell death that is induced when apoptosis is inhibited. It has been known that a complex consist of RIP1 and a RIP1 related kinase, RIP3, plays key roles in the activation of the necroptotic pathway. However, the mechanism by which the RIP1-RIP3 is activated has not been determined. We found that TAB2 deficiency caused prolonged activation of TAK1 resulting in association of TAK1 with RIP1 in a prolonged manner. TAK1 could activate RIP1 and RIP3 kinase activity. Our results in this study demonstrate that TAB2 regulation of TAK1 is critical in the prevention of TNF-induced necroptosis. In summary, TAB2 is a dual role TAK1 adaptor protein that mediates both activation and deactivation of TAK1. The TAB2-mediated deactivation of TAK1 is particularly important, because impairment of this deactivation results in necroptosis leading to dysregulated inflammatory conditions.
TAK1 is a Central Mediator of NOD2 Signaling and is Essential for Intestinal Epithelial Cell Protection against Chemical-induced Colitis.
(2009-12-19) Kim, Jae-Young; Robert C. Smart, Committee Member; Yoshiaki Tsuji, Committee Member; Jun Ninomiya-Tsuji, Committee Chair; Christian Jobin, Committee Member
Innate immunity is the first line of defense against invasive microbial pathogens. It is triggered by several families of pattern-recognition receptors (PRRs), which activate cellular responses including activation of NF-ÃŽÂºB and mitogen-activated protein kinases (MAPKs) signaling, and subsequently the induction of proinflammatory genes. There are two groups of PRRs, toll-like receptors (TLRs) and nod-like receptors (NLRs). In contrast to TLRs that recognize microbes via plasma membrane or endosomal receptors, NLRs induce innate immune response by detecting bacterial components released into the cytosol. Nucleotide oligomerization domain 2 (NOD2) is the best characterized member of the NLR family. Upon NOD2 stimulation by muramyl dipeptide (MDP), a NOD2-specific ligand originating from the bacterial cell wall, it associates with an adaptor molecule, RIP-like interacting CLARP kinase (RICK, also called Rip2) and activates both NF-ÃŽÂºB and MAPKs signaling pathways, which lead to inflammatory gene expression. However, the molecular mechanism by which NOD2-RICK complex activates its downstream signals remains elusive. Here, we report that TGF ÃŽÂ²-activated kinase 1 (TAK1) is a central mediator of NOD2 signaling in epidermal cells. TAK1 belongs to MAPKKK family and plays an essential role in activating NF-ÃŽÂºB and MAPKs signaling in tumor necrosis factor (TNF), interleukin-1 (IL-1), and TLR signaling pathways. In this study, we found that MDP-induced proinflammatory gene expression as well as the activation of NOD2 downstream signals including NF-ÃŽÂºB, c-Jun N-terminal kinase (JNK) and p38 were completely abolished in TAK1-deficient keratinocytes, indicating that TAK1 is essential for eliciting NOD2-induced innate immune responses. NOD2 and its downstream adaptor, RICK, associated with and activated TAK1. Endogenous TAK1-RICK interaction was enhanced by MDP, suggesting that NOD2-RICK-TAK1 complex is induced in an MDP-dependent manner. We have previously reported that intestinal epithelium-specific TAK1 deletion mice showed severe inflammation and mortality at postnatal day 1 due to TNF-induced epithelial cell death. In this study, we investigated TNF-independent role of TAK1 by utilizing mice harboring double deletion of TNF receptor 1 (TNFR1) and intestinal epithelium-specific deletion of TAK1 (TNFR1KO/TAK1IEKO). To study the role of TAK1 in the intestinal epithelial barrier, the mice were subjected to acute colitis by administration of dextran sulfate sodium (DSS). We found that loss of TAK1 significantly augments DSS-induced experimental colitis. DSS-induced weight loss, intestinal damage and inflammatory markers were significantly increased in TNFR1KO/TAK1IEKO mice compared to the TNFR1KO control mice. Following DSS exposure, apoptosis was strongly induced and epithelial cell proliferation was decreased in the TAK1-deficient intestinal epithelium. These results suggest that epithelial-derived TAK1 signaling is important for cytoprotection and tissue repair after injury. Finally, we showed that TAK1 is essential for IL-1- and bacterial components-induced expression of cytoprotective factors including IL-6 and cycloxygenase 2 (COX2). Collectively, we propose that homeostatic cytokines and microbes-mediated intestinal epithelial TAK1 signaling is pivotal for protecting the intestinal epithelium against injury by promoting cell survival and maintaining proliferation. Taken together, this study demonstrates that TAK1 is an essential mediator of NOD2 signaling in epidermal cells, and also suggests that bacterial components- and cytokine-induced TAK1 signaling both play a pivotal role in preventing intestinal inflammation by maintaining the integrity of the intestinal epithelium.