Browsing by Author "Jonathan M. Horowitz, Committee Member"

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    The Cardiac Response to Reovirus Infection
    (2009-08-03) Li, Lianna; Barbara Sherry, Committee Chair; Frank Scholle, Committee Member; James L. Stephenson, Jr., Committee Member; Jonathan M. Horowitz, Committee Member
    LI, LIANNA. The Cardiac Response to Reovirus Infection. (Under the direction of Dr. Barbara Sherry). Viral myocarditis is a common disease in humans. Interferon-β (IFN-β) has been identified as critical for protection against viral myocarditis in mouse models, and IFN-α or -β treatment is beneficial in the treatment of human viral myocarditis. IFN-β expression and its antiviral effects are cell-type specific in murine cardiac myocytes and fibroblasts. However, expression and function of individual IFN-α subtypes in cardiac cells has not previously been investigated. Therefore, IFN-α subtype expression and antiviral effects were studied in reovirus-infected murine primary cardiac myocyte and cardiac fibroblast cultures. In order to quantify the thirteen highly conserved IFN-α subtype, a quantitative Real-Time PCR assay was developed. Results demonstrated that IFN-α induction by reovirus T3D in cardiac cells is both subtype- and cell type-specific, and that some individual IFN-α subtypes are likely important in the antiviral cardiac response. In brief, reovirus T3D induced five IFN-α subtypes in primary cultures of cardiac myocytes and fibroblasts: IFN-α1, -α2, -α4, -α5, and -α8/6. The levels of IFN-α expression were both higher and spanned a greater range in cardiac myocytes than in fibroblasts. Viral induction of IFN-α1, -α2, -α5, and -α8/6 required IFN-α/β signaling in both cell types, while induction of IFN-β and -α4 was more dependent on IFN signaling in myocytes than fibroblasts. Murine IFN-α1, -α2, -α4, or -α5 treatment induced IRF7 and ISG56 in both cardiac cell types, however induction was always greater in cardiac fibroblasts than in cardiac myocytes. Finally, each IFN-α subtype inhibited reovirus T3D replication in both cell types, but protection was subtype-specific. To discover novel proteins or protein post-translational modifications involved in the IFN pathway or displaying antiviral effects against viral myocarditis, a proteomics tool, two-dimensional difference gel electrophoresis (2D-DIGE) coupled with MALDI-TOF-TOF, was used to investigate the reovirus-induced proteome changes in murine primary cardiac myocyte cultures. Results demonstrated that the 2D-DIGE technique is quantitative and reproducible. Whole proteome changes based on differentially expressed proteins were clustered according to viral pathogenic phenotypes and induction of IFN. One hundred and twenty-four differentially expressed proteins were identified, including those involved in calcium signaling, ERK/ MAPK signaling, protein ubiquitination, mitochondrial dysfunction, oxidative stress, amino acid metabolism, and other pathways. Interestingly, 2D-DIGE results and additional studies demonstrated that heat shock protein Hsp25 is modulated differentially by myocarditic and non-myocarditic reoviruses, and suggested that it may play a role in the cardiac antiviral response. This is the eighth virus family found to modulate Hsp25 or its human homolog, Hsp27, suggesting that Hsp25/27 participation in the antiviral response may be widespread. However, results here provide the first evidence for a virus-induced decrease in Hsp25/27, and suggest that viruses may have evolved a mechanism to subvert this protective response, as they have for IFN.
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    Identifying Transcription Factor Targets and Studying Human Complex Disease Genes
    (2009-04-13) Wang, Tianyuan; Elizabeth R. Hauser, Committee Co-Chair; Jonathan M. Horowitz, Committee Member; David McK. Bird, Committee Member; Steffen Heber, Committee Co-Chair; Jeffrey L. Thorne, Committee Member
    Transcription factors (TFs) have been characterized as mediators of human complex disease processes. The target genes of TFs also may be associated with disease. Identification of potential TF targets could further our understanding of gene-gene interactions underlying complex disease. We focused on two TFs, USF1 and ZNF217, because of their biological importance, especially their known genetic association with coronary artery disease (CAD), and the availability of chromatin immunoprecipitation microarray (ChIP-chip) results. First, we used USF1 ChIP-chip data as a training dataset to develop and evaluate several kernel logistic regression prediction models. Our most accurate predictor significantly outperformed standard PWM-based prediction methods. This novel prediction method enables a more accurate and efficient genome-scale identification of USF1 binding and associated target genes. Second, the results from independent linkage and gene expression studies suggest that ZNF217 also may be a candidate gene for CAD. We further investigated the role of ZNF217 for CAD in three independent CAD samples with different phenotypes. Our association studies of ZNF217 identified three SNPs having consistent association with CAD in three samples. Aorta expression profiling indicated that the proportion of the aorta with raised lesions was also positively correlated to ZNF217 expression. The combined evidence suggests that ZNF217 is a novel susceptibility gene for CAD. Finally, we applied our previously developed TF binding site (TFBS) prediction method to ZNF217. The performance of the prediction models of ZNF217 and USF1 are very similar. We demonstrated that our TFBS prediction method can be extended to other TFs. In summary, the results of this dissertation research are (1) evaluation of two TFs, USF1 and ZNF217, as susceptibility factors for CAD; (2) development of a generalized method for TFBS prediction; (3) prediction of TFBSs and target genes of two TFs, and identification of SNPs within TFBSs. This research allows for the development of study design to access TF based interactions in genetic susceptibility to human complex disease.