Within the phosphatase field, identification of regulatory subunits and associated proteins has proven successful in determining the cellular role and potential substrates of phosphatases. This has been especially valuable for the PPP family of phosphatases due to its complex association with myriad of regulatory subunits, which dictate the activity, localization and substrate specificity of the phosphatases. To identify interactors of protein phosphatase 4 (PP4), I established a sensitive affinity purification coupled with mass spectrometry (AP-MS) approach to generate a high-density PP4 interaction network from the soluble fraction of human cell extracts. Our proteomic approach uncovered previously identified as well as new interactors; some may function as auxiliary regulators and others may serve as potential substrates. One of the interactor identified is a novel cytosolic PP4 regulatory subunit, which we termed PP4R4. PP4R4 displays weak homology to the PP2A A subunit of PP2Ac, but interacts specifically with PP4c and does not function as a scaffolding subunit to bridge other known regulatory subunits of PP4c. Remarkably, the PP4 interaction network revealed significant enrichment for proteins involved in transcription elongation and RNA processing. These interactors associate exclusively with PP4R3-PP4R2-PP4c holoenzyme. Consistent with this finding, PP4R3A possesses characteristics resembling that of splicing and transcription factors. We provided evidence suggesting that depletion of PP4c significantly reduced the transcription-elongation regulated genes, JUN and FOS, and altered the exon inclusion of selective genes. Our results define a high-density interaction network for the mammalian PP4 and uncover a potential role PP4 play in regulating the process of transcription elongation and alternative splicing.

Genes of the Bacillus subtilis infecting bacteriophage SPO1 are classified as early, middle or late genes, based on which sigma factor is used for their transcription. Within the early gene category, there are two subsets of genes, immediate and delayed early (including genes 37-40), which have different temporal expression patterns. Genes 37-40 are shown involved in the shutoff of host macromolecular synthesis. Gene products (gp) 39 and 40 were shown to be lethal when expressed in E. coli cells, through an undetermined mechanism. Gp39 was purified under denaturing conditions, but could not be properly refolded. Genes 2.21, 25.1 and 51 were studied in order to understand how delayed early genes are regulated. Gp51 was partially purified. An additional gene expressed from within gene 51 was discovered. It was determined that SPO1 mutations of genes 2.21 and 25.1 could not be isolated, even when a variety of conditions were tested.

Molecular self-assembly offers unique features for the development of novel supramolecular structures and advanced materials. The inspiration for the creation of these complex structures is often driven by nature, where complex architectures are obtained from simple building block such as amino acids, nucleic acids and lipids. Peptide-based nanostructures constitute a novel route to create well organized self-assembled materials that eventually can be used in a variety of different fields. They can be divided in different classes based on the architecture of the overall molecule. My research is mainly focused on two types of peptide-based structures: peptide amphiphiles, in which an amino acidic sequence is tailed with an hydrophobic molecule, such as palmitic acid, and facially amphiphilic peptides, which is made by proper sequence patterning of hydrophobic and hydrophilic amino acids. In chapter 2 I describe how a peptide amphiphile was designed, synthesized and tested on different types of cancer cell lines, and it proved to be effective in reducing the growth of malignant cells. This peptide works as an inhibitor of the HOX-PBX1 interaction. HOX and PBX I are two transcription factors that are deeply involved in the DNA regulation during embryogenesis and they are found to be highly miss-regulated in several types of cancers. Until now, peptide amphiphiles have been used mostly as drug carriers. This work constitutes one of the first studies in which a peptide amphiphile molecule can work both as a drug and as a drug carrier, simultaneously. 3D-scaffolds for tissue engineering applications are another hot area in which self-assembling peptides are finding several applications. Driven by this motivation, chapter 3, 4 and 5 describe how I designed, synthesized and investigated the properties of a new class of facial peptide amphiphile. These peptides, when dissolved in an aqueous environment and under the right pH and salt conditions, have proven to quickly self-assemble in long and entangled nanofibers that ultimately form stable hydrogels. These hydrogels have potential applications as bio-scaffolds for tissue regeneration, as cells are entrapped in their network. The mechanical properties of these hydrogels can be triggered using different approaches and it is possible to obtain very strong materials, even at very low concentrations of the peptide, such as 1% by weight. In order to be safely used for bio-applications, hydrogel materials must be need to have two main features: biocompatibility and biodegradability. The hydrogels obtained through self-assembling peptides have proven to be both biocompatible and biodegradable, thanks to the introduction of a short amino acid sequence recognized by proteases, which is described in chapter 5. In conclusions, the results collected in this study show how a simple building block, such as a small peptide, can serve as an extremely powerful and flexible tool to design complex self-assembled systems. This is area of research is indeed full of potential that has not yet been completely explored.

This project was concerned with the functional components of mammalian DNA replication origins and how the misuse of a start site for DNA replication at the FMR1 locus might contribute to human Fragile X Syndrome. In the first part of this dissertation, I identified a novel origin of DNA replication near the CGG repeats at the human Fragile X Mental Retardation (FMR1) gene promoter. Expansion of these repeats leads to the epigenetic chromosome modifications that cause Fragile X Syndrome. The experiments described in this dissertation suggest that the position of the FMR1 origin favors contraction of the CGG repeats, thus providing a mechanism to avoid repeat expansion. This model predicts that a change in origin usage accompanies repeat expansion, and I discussed how this could occur. In the second part of this dissertation, I examined the requirement of DNA sequence elements in a mammalian origin to direct DNA replication to start at specific chromosomal sites. In particular, I studied the role of a dinucleotide repeat (DNR) sequence element in the activity of the Chinese hamster dihydrofolate reductase origin beta. The DNR element could be functionally replaced with two different transcriptional elements. This result suggests that DNR shares a functional role with these elements, and we speculate that this role may be to create the proper chromatin environment for recruitment and action of other replication factors to initiate replication.

DJ-1, A NOVEL ANDROGEN RECEPTOR BINDING PROTEIN, ACTIVATES RECEPTOR SIGNALING IN PROSTATE CANCER AND CORRELATES WITH THE DEVELOPMENT OF ANDROGEN-INDEPENDENT DISEASE Jennifer Erin Tillman Dissertation completed under the direction of Dr. Susan Kasper, PhD This research projects focused on investigation of the role of DJ-1 in prostate cancer. In this dissertation, the mechanisms regulating the transition from hormone responsive to hormone refractory prostate cancer were investigated by analyzing androgen and anti-androgen treatment on endogenous AR activity in primary human prostate epithelial cells and established prostate cancer cell lines. We determined that flutamide treatment exhibited agonist activities in cells derived from tumor and non-tumor specimens which contained wild-type AR. After proteomic comparison of these cells to those where flutamide functioned normally as an antagonist, we identified DJ-1, an oncogene and positive regulator of AR. DJ-1 expression increased following flutamide treatment as a result of DJ-1 protein stabilization. To address the function of DJ-1 in prostate, we performed a yeast two-hybrid screen to identify novel DJ-1 binding proteins. The androgen receptor (AR) was identified as a putative DJ-1 binding protein, which was confirmed in the LAPC4 and LNCaP human prostate cancer cell lines. This is the first evidence that DJ-1 directly interacts with AR. We also demonstrate that modulation of DJ-1 expression regulates AR transcriptional activity. Importantly, both the subcellular localization of DJ-1 and the interaction with AR is regulated by androgens and anti-androgens. Additionally, we performed immunohistochemical staining on two human prostate cancer tissue arrays providing the first large scale expression analysis of DJ-1 in prostate. DJ-1 expression does not change with Gleason pattern, but increases after androgen deprivation therapy, indicating that it may be involved in the development of androgen independence. Taken together, we demonstrate that DJ-1 directly interacts with AR, and that this interaction is hormonally regulated. These data provide a novel mechanism for DJ-1 mediated regulation of AR in the progression of prostate cancer to androgen-independence.

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