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Thesis (Ph.D.)--Indiana University, Dept. of Biology, 2005. / Source: Dissertation Abstracts International, Volume: 67-01, Section: B, page: 0106. Adviser: Mark Estelle. "Title from dissertation home page (viewed Feb. 9, 2007)."

The microRNA (miRNA) and RNA interference (RNAi) pathways have recently emerged as an important aspect of virus-host cell interaction. This interaction can occur in several different ways and may favor either the virus or the host cell. Plants and invertebrates use RNAi as a first line of defense against virus infection by cleaving long, double-stranded viral transcripts into small interfering RNAs. However, it remains to be determined whether mammalian cells also initiate a similar response to infection. Here we present evidence that mammalian cells in fact do not induce an antiviral RNAi defense in response to infection by primate retroviruses. Viruses may also interact with host cells by encoding miRNAs to regulate either cellular or viral gene expression. Here we demonstrate that herpes simplex virus type 1 (HSV-1) encodes at least five miRNAs which are primarily expressed during latency. Two of these miRNAs modulate expression of viral genes required for productive replication. We hypothesize that down regulation of these viral genes by these latency associated miRNAs allows HSV-1 to establish and maintain the latent state. / Dissertation

<p>Angiogenesis, the production of new vessels from pre-existing vasculature, is a complex biological process that is dependent on a series of regulated events, including endothelial cell (EC) proliferation, migration, survival, and capillary morphogenesis (tube formation). These events are required for angiogenesis to occur properly and the steps are regulated by a variety of vascular growth factors and their receptors. Tie2, an endothelial receptor tyrosine kinase (RTK), is required for embryonic and postnatal angiogenesis. Studies have demonstrated that Tie2 is proteolytically cleaved, producing a 75 kDa soluble receptor fragment (sTie2). However, the mechanisms and function of sTie2 have not been elucidated. Here, we investigated signaling pathways and effector molecule(s) responsible for Tie2 cleavage. Additionally, we investigated the role of other growth factors and conditions on the degree of Tie2 cleavage. Finally, we examined sTie2 levels in peripheral artery disease, a human model of ischemic disease. We demonstrated that Tie2 cleavage is VEGF- and PI3K/Akt-dependent and sTie2 can bind Ang1 and Ang2 and prevent ligand-mediated Tie2 activation and downstream cellular responses. Also, ADAM15 cleaves Tie2 in a hypoxia-dependent manner and this response was also observed to be VEGF-mediated. With respect to peripheral artery disease, sTie2 levels were only significantly elevated in the most angiogenically compromised group (critical limb ischemia) of patients. These data shed light on the mechanism and function of Tie2 cleavage and suggest a role for sTie2 in mediating the angiogenic process.</p> / Dissertation

<p>Endoglin, an endothelial cell specific transforming growth factor-beta (TGF-beta) superfamily co-receptor, has an essential role in angiogenesis, with endoglin null mice having an embryonic lethal phenotype due to defects in angiogenesis and mutations in endoglin resulting in the vascular disease hereditary hemorrhagic telangiectasia type I. While endoglin is thought to regulate TGF-beta superfamily signaling in endothelial cells through regulating the balance between two TGF-beta responsive pathways, the ALK5/Smad2/3 pathway and the ALK1/Smad1/5/8 pathway, the mechanism by which endoglin regulates angiogenesis has not been defined. Recently, overexpression of wild type endoglin has been demonstrated to increase ALK1 signaling, supporting a role for endoglin as an important regulator of the ALK1 pathway. Here we investigate the role of the cytoplasmic domain of endoglin and its phosphorylation by TGF-beta superfamily receptors in regulating endoglin function in endothelial cells. We demonstrate that the cytoplasmic domain of endoglin is basally phosphorylated by ALK5, primarily on serines 646 and 649, in endothelial cells. This basal phosphorylation primes and is necessary for subsequent phosphorylation of endoglin by ALK1. Functionally, the loss of phosphorylation at serine 646 resulted in a loss of endoglin mediated inhibition of Smad1/5/8 signaling and endothelial cell migration. Taken together these results support endoglin phosphorylation by ALK5 as an important mechanism for regulating TGF-beta superfamily signaling and migration in endothelial cells.</p> / Dissertation

<p>Telomeres are DNA-protein structures that protect eukaryotic chromosome ends from illegitimate recombination and degradation. Telomeres become shortened with each cell division unless telomerase, a reverse transcriptase, is activated. In addition to playing a protective role of chromosome ends, telomeres and telomere binding proteins are also essential for regulating telomere length and telomerase access. The mammalian protein POT1 binds to telomeric single-stranded DNA (ssDNA), protecting chromosome ends from being detected as sites of DNA damage and negatively regulating telomere length. POT1 is composed of an N-terminal ssDNA-binding domain and a C-terminal protein-interaction domain. With regard to the latter, POT1 heterodimerizes with the protein TPP1 to foster binding to telomeric ssDNA in vitro and binds the telomeric double-stranded DNA (dsDNA) binding protein TRF2. I sought to determine which of these functions--ssDNA, TPP1, or TRF2 binding--was required for POT1-mediated telomere localization, protection, and length regulation. Using separation-of-function POT1 mutants deficient in one of POT1's three functions, I found that binding to TRF2 fosters robust loading of POT1 onto telomeric chromatin and regulates telomere length, but is dispensable in the protection of telomeres. Although it remains unclear what role TPP1 binding plays in telomere length regulation, I found that the telomeric ssDNA-binding activity and binding to TPP1 are required in cis for POT1-mediated protection of telomeres, possibly by excluding RPA from telomeres.</p> / Dissertation

<p>Mercury is a toxic metal that can exist in multiple chemical forms, all of which are toxic to humans. Despite years of research, only a fragmented understanding of the molecular mechanisms of toxicity exists. Furthermore, it is not known to what extent different mercury species act similarly or dissimilarly at the molecular level. The objective of this study was to investigate the extent to which inorganic and methylmercury act differently at the molecular level.</p><p>The relative toxicity of mercuric chloride (HgCl2) and methylmercury chloride (MeHg) in C. elegans was determined by testing the effect of mercurial exposure on growth, reproduction and induction of stress-response genes. MeHg was more toxic than HgCl2, though the difference in toxicity between the two mercurials varied by assay. Using approximately sub-, low- and high toxic exposures to both mercurials, microarrays were performed to determine the effects of the HgCl2 and MeHg on transcription. A total of 473 genes were differentially expressed in the three HgCl2 treatments, while a total of 2865 genes were differentially expressed in the three MeHg treatments. Analysis of the microarray data by hierarchical clustering, principal components analysis and a self-organizing map indicated that the transcriptional effects of the two mercurials were vastly different. Gene Ontology analysis and pathway mapping indicated that the two mercurials had very different effects on biological processes as well. </p><p>The biological function of genes up-regulated by mercurials was tested using RNA interference (RNAi). The effect of RNAi and mercury co-exposure on C. elegans growth was tested for 599 genes. Knock-down of 18 of these genes was found to significantly affect growth of C. elegans exposed to mercury. Of these 18 genes, only 2 were found to significantly affect growth in response to both mercurials. </p><p>ABCG2, BACE1, BACE2, CHKA, CHKB, ELOVL3, ELOVL6, GCLC and PARG are human homologs of the genes found to significantly affect growth of C. elegans in mercury exposure. The effect of sub-, low-, and high-toxic treatments of both mercurials on expression of these genes was tested using three cell lines: SK-N-SH, HepG2 and HEK293. Of these 162 cell-gene-mercury-toxicity combinations, there were 36 in which the gene was differentially expressed. In 24 of these, the gene was significantly differentially expressed by only one of the mercurials. The evolutionary conservation of function of these genes in mercury exposure was tested using RNAi. A total of 11 significant gene-mercury interactions were found between the three cell lines, but there was not a cell type-gene combination in which exposure to both mercurials was found to significantly affect cytotoxicity. </p><p>In whole organism and cell culture studies, inorganic and methylmercury were found to have different effects on transcription. In both systems, there was very little overlap in the genes involved in mercury resistance and susceptibility. These data indicate that molecular mechanisms of toxicity differ by mercurial.</p> / Dissertation

Thioredoxin-1 (Trx-1) is a small redox protein that is overexpressed in a number of human cancers. Elevated levels of Trx-1 in tumors is associated with increased cell proliferation, decreased apoptosis, and decreased patient survival. However, the mechanism(s) for the growth stimulating and anti-apoptosis effects of Trx-1 are unknown. We used DNA microarray technology to identify genes whose expression was altered in MCF-7 breast cancer cells stably transfected with wild-type Trx-1 (MCF-7/Trx 9) or a redox inactive mutant Trx-1 (MCF-7/SerB 4) compared to empty-vector transfected cells (MCF-7/neo). The expression of cytochrome P450 1B1 (CYP1B1) mRNA and protein is increased by Trx-1 transfection of MCF-7 human breast cancer cells and decreased by a redox inactive mutant Trx-1. CYP1B1 is a tumor specific CYP which converts 17β-estradiol (E₂) to the carcinogenic 4-hydroxyestradiol (4-OHE₂). The expression of peroxiredoxin 1 (PRDX1) mRNA is increased as a result of Trx-1 overexpression in MCF-7 cells. The peroxiredoxins belong to a conserved family of antioxidant proteins that use thiol groups as reducing equivalents to scavenge oxidants. Transfection of mouse WEHI7.2 thymoma cells with human PRDX1 protects cells from apoptosis induced by H₂O₂. Spermine/spermidine N'-acetyltransferase (SSAT) mRNA expression and enzyme activity is decreased by Trx-1 transfection of MCF-7 human breast cancer cells. SSAT is an important enzyme in the polyamine catabolic pathway. The inhibition of SSAT enzyme activity is associated with decreased putrescine levels in the Trx-1 transfected cells. Therefore, it appears as if the modification of cellular redox signaling brought about by the overexpression of Trx-1 in breast cancer cells induces changes in gene expression that contribute to the transformed phenotype. Trx-1 redirects estrogen metabolism in a more toxic pathway due to the induction of CYP1B1, provides resistance to apoptosis induced by reactive oxygen species via the upregulation of PRDX1, and alters polyamine metabolism by inhibiting the expression of SSAT.

The biological actions of 1α,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) are mediated by the nuclear vitamin D receptor (VDR), which functions as a ligand-dependent transcriptional regulator. We have developed a six-stage molecular model summarizing the VDR transcriptional activation-life cycle, and tested this model using a variety of experimental approaches, including pull-down assays with GST-fusion proteins, as well as assays of the functional activity of VDR and its putative coactivators in transiently transfected mammalian cells. The six stages of the VDR life cycle are: (1) unoccupied VDR binds to a transcriptional corepressor which serves as a chaperone, maintaining the main protein players in close contact in an inactive complex; (2) VDR becomes occupied by 1,25(OH)₂D₃ ligand, enabling the receptor to heterodimerize strongly with a retinoid X receptor (RXR), leading to high affinity DNA binding and recruitment of coactivators with histone acetyl transferase (HAT) activity; (3) coactivator HAT activity promotes chromatin remodeling, rendering the gene promoter free to interact with the transcription preinitiation complex (PIC); (4) dissociation of VDR from the HAT coactivators, followed by association of a second set of coactivators that promote formation of the preinitiation complex (PIC); (5) transcriptional recycling of the liganded receptor and heteropartner to initiate additional rounds of transcription; and (6) ubiquitination and eventual degradation of VDR. Phosphorylation of VDR may influence all six stages. A testable conclusion from our model is that the role of the 1,25(OH)₂D₃ hormonal ligand would be primarily in the transition from stage 1 to stage 2, but the continued presence of 1,25(OH)₂D₃ appears to be necessary also for the progression from stages 3 through 6 to VDR degradation, or alternatively for recycling via stage 5. This characterization of the macromolecular cofactors that transduce the signal of the 1,25(OH)₂D₃ hormone to promote gene expression in vitamin D target tissues should add to our understanding of endocrine control of bone mineral remodeling and of epithelial cell differentiation. The present work also identifies new protein players that are candidates for mutation or dysregulation in the pathophysiology of vitamin D resistant bone disorders (osteoporosis), and in hyperproliferative diseases of vitamin D regulated epithelial tissues such as skin.

The extracellular matrix (ECM) is defined as the many different proteins and secreted substances between cells. The ECM plays a major role in the signaling pathways that stimulate cells to perform many varied functions, ranging from control of gene expression at the cellular level to differentiation and development of tissues, organs, and ultimately the entire organism. A portion of this work describes the identification of the division abnormally delayed gene, which encodes a proteoglycan, that is involved in growth factor reception with important developmental consequences in Drosophila melanogaster. The remainder of this work deals with three Drosophila homologs of vertebrate proteins that may interact with integrins, a family of cell surface receptors for extracellular matrix ligands. The three integrin-interacting proteins are referred to by their vertebrate names, and include CD81, a member of the tetraspanin family, ILK, integrin-linked kinase, and CD98hc, a type II transmembrane glycoprotein which is the heavy chain of a multi-protein complex. In this work, the mutant phenotype of CD98hc is larval lethal and not temperature sensitive. Clonal analyses of CD98hc mutants show no phenotype of mutant clones in the eye. Genetic interactions in adult tissues or interactions affecting larval lethality between CD98hc and Drosophila integrin mutants were not observed, and it remains unclear whether CD98hc physically interacts with Drosophila integrins in tissue culture cells. Since no correlation was seen between the interactions of CD98hc and integrins in vertebrate cells and similar putative interactions in flies, this raises the question as to what role, if any, does CD98hc play as an integrin modulator in this organism.