Studies show that insulin induced activation and assembly of insulin receptor substrate-1 (IRS1) and phosphatidylinositol-3-kinase (PI3K), within remodelled actin structures is critical for GLUT4 translocation to the cell surface in muscle cells. This study identifies the F-actin binding protein, nexilin, as a novel IRS1 binding partner. Insulin stimulates nexilin to dissociate from IRS1 and interact with actin. Nexilin knockdown has no effect on insulin-stimulated IRS1 tyrosine phosphorylation, but does enhance insulin-stimulated IRS1-PI3K interaction, increasing PIP3 formation, PKB activation and glucose uptake. This study also shows that nexilin overexpression may have an inhibitory effect on PKB phosphorylation and glucose uptake in adipocytes. These findings suggest nexilin is a negative regulator of IRS1 action on PI3K and insulin-stimulated dissociation of IRS1-nexilin allows the formation of IRS1-PI3K complexes in cytoskeletal-membrane compartments. Nexilin also specifically associates with the PH domain of IRS1, and not IRS2, suggesting a mechanism for signaling specificity of these isoforms.

Carboxyl esterases, particularly arylesterases, were predicted from 16 microbial genomes, and then expressed in E. coli. Of the more than 175 cloned genes, 86 were expressed in soluble form. These were screened for activity using a range of both commercial and natural substrates. Forty-eight proteins were active on pNP-acetate at pH 8 whereas 38 proteins did not exhibit any activity towards any substrates. Among the 48 active proteins, 20 proteins showed arylesterase activity. To date, 8 bacterial esterases and 2 archaeal arylesterases were characterized in terms of pH stability and optima, thermal inactivation, solvent stability, and kinetics. To our knowledge there is only one other published report of arylesterases from archaea. The synthetic capability of arylesterases can transform phenolic acids to value-added chemicals. Accordingly, this project provides an arsenal of industrially significant activities that can extend the antioxidant properties of lignin-derived molecules in a broader range of renewable products.

HopZ1a is a type III secreted effector (TTSE) protein from Pseudomonas syringae. The goal of this study was to obtain a 3D crystal structure of HopZ1a to provide insight into its biochemical function. The first objective was to obtain HopZ1a protein that was sufficiently abundant and pure for crystallographic studies. Purification conditions were optimized and multiple constructs of HopZ1a were generated using secondary structure prediction programs as well as structural characteristics inherent to TTSEs. Truncations of HopZ1a from the N- and C-terminus led to a soluble, proteolytically resistant construct, HopZ1a66-261. This protein formed granular precipitates in crystallography screens. These conditions will provide the basis for refinement screens aimed at optimizing the HopZ1a crystallization conditions. Overall, the soluble constructs described in this study will provide invaluable tools for future in vitro functional and structural studies of this important family of type III secreted effector proteins.

C-terminal Binding Protein (CtBP) is a transcriptional co-repressor that plays an important role in mammalian development and tumorigenesis. CtBP is known to interact with Ikaros, an important transcriptional regulator in the pituitary; however CtBP itself has not been examined in this gland. I examined the role of CtBP in pituitary cell growth and survival. Compared to control pituitary GH4 cells, CtBP1-deficient cells exhibit reduced proliferation and de-regulation of genes involved in cell cycle and growth factor signaling. CtBP1-deficient cells were more susceptible to hypoxia-induced apoptosis and showed a reduction in hypoxia-induced Ikaros expression. Interactions between CtBP and Ikaros isoforms were demonstrated in pituitary tumor cell lines. CtBP and Ikaros also bound a common region of the previously characterized Ikaros target, the LDL-R promoter. These results identify oncogenic properties of CtBP1 in the pituitary and set the groundwork for future studies into regulatory roles of CtBP and Ikaros in the pituitary.

Eukaryotic organisms utilize post-translational modifications of highly conserved histone proteins to control gene expression programs. Methylation of lysine 4 on histone H3 (H3K4me) in particular, is thought to be associated with actively transcribed DNA. Paradoxically, recent evidence has suggested that H3K4me has a repressive function as well. Pho23, a member of the highly conserved ING family of tumour suppressor proteins, binds H3K4me and is a component of the gene repressive complex, Rpd3L. My genetic analysis suggests that Pho23 controls transcriptional repression via H3K4me and that Pho23 is itself regulated by the sequence-specific DNA-binding protein Ume6. Moreover, this Ume6-regulated function appears to be governed by Ume6 phosphorylation by Mck1, an evolutionarily conserved kinase. Finally, while Ume6/Pho23 are known to function together with the histone deacteylase Rpd3, my findings suggest the existence of an Rpd3-independent function for Pho23.

Approximately 15% of human cancers utilize a recombination-based mechanism termed Alternative Lengthening of Telomeres (ALT) to maintain the lengths of their telomeres. The Fanconi anaemia protein FANCJ localizes to telomeric foci in human ALT cells, but not in telomerase-positive or primary cells. Telomere-associated FANCJ frequently localizes with FANCD2 and BRCA1, and primarily localizes to ALT-associated PML nuclear bodies. Depletion of FANCJ in human ALT cells causes the loss of BRCA1 at telomeric foci and a decrease in telomeric repeat DNA content primarily as a result of the loss of the brightest telomeric repeat DNA foci. In contrast, depletion of the FANCD2 results in increased telomeric repeat DNA synthesis and this is suppressed upon the codepletion of FANCJ. Together, data from this study suggest that FANCJ is required for telomeric repeat DNA synthesis in human ALT cells, which may or may not be dependent on BRCA1, and FANCD2 restrains this synthesis.

This study examined DNA methylation patterns at promoters of endothelial cell (EC)-enriched genes during differentiation of mouse ES cells towards the EC. We have previously shown that eNOS, CD31, VE-cadherin and vWF, which have an EC-enriched pattern of gene expression are differentially methylated between EC and vascular smooth muscle cells. Given that differential promoter DNA methylation is functionally important we asked when these distinct patterns are established. Using the hanging drop method to differentiate ES cells, followed by FACS, we isolated early (EB-day4 VEGFR2-positive) and late (EB-day7 CD31-positive) endothelial progenitor cells. Though current paradigms suggest that lineage-restricted genes are methylated in ES cells, we show heterogeneous promoter DNA methylation. We show DNA demethylation at the CD31 promoter in EB-day 7 CD31-positive cells. In contrast, the eNOS promoter is still heavily methylated in EB-day 7 CD31 positive cells compared with murine EC where there is no DNA methylation.

Diamond Blackfan Anemia is a rare congenital disease that is primarily characterized by reduced erythroid progenitors. DBA pathogenesis has been associated with genes encoding ribosomal proteins (RPs) which are important in translation. However, this fails to explain why erythropoiesis is specifically disrupted. Our lab previously found that aberrant splicing of the human transcript encoding heme exporter, FLVCR1, is involved in DBA pathogenesis; and that RPS19 implicated in 25% of DBA patients, regulates FLVCR1 transcript splicing. This thesis investigated the role of another DBA associated gene encoding RPS17, in the regulation of FLVCR1 splicing and disrupted erythropoiesis in DBA. My findings further support the role of FLVCR1 aberrant splicing in DBA and provide evidence suggesting that RPS17 may not be a candidate DBA gene. Furthermore, my study implicates a potential role for RPS19 transcript levels in defective erythroid differentiation observed in DBA.

Shiga-like toxin 1 (SLT-1) is produced by Escherichia coli strains like the pathogenic strain O157:H7. These bacterial strains are responsible for worldwide cases of food poisoning and water contamination, and the toxin is a major cause of hemorrhagic colitis and the hemolytic uremia syndrome. SLT-1 is defined as a type II ribosome-inactivating protein (RIP) and belongs to a family of plant and bacterial AB toxins. The A1 chain blocks protein synthesis in eukaryotic cells by depurinating a single adenine base in 28S rRNA. The mechanisms by which the A chain of SLT-1 interacts with the host components to route itself to the cytosol remains largely unknown. This thesis project identified a list of putative cellular proteins that interact with the SLT-1 A1 chain by the use of yeast-2-hybrid (Y2H) screens and HeLa lysate pull down/mass spectrometry analyses. Further assessment of the top 8 host interactors did not yield true interactions.

Pulmonary metastasis is the most frequent cause of osteosarcoma (OS) mortality. The aim of this study was to discover and characterize genetic networks differentially expressed in metastatic OS. Supervised network analysis of OS expression profiles was performed to discover genetic networks differentially activated or organized in metastatic OS. Broad trends among the profiles of metastatic tumours included aberrant activity of intracellular organization and translation networks, as well as disorganization of metabolic networks. The differentially activated PRKCε-RASGRP3-GNB2 network, which interacts with the disorganized DLG2 hub, was additionally found to be differentially expressed among in vitro models of human OS metastasis. PRKCε transcript was more abundant in some metastatic OS tumours; however the difference was not significant overall. In functional studies, PRKCε was not found to be involved in migration of M132 OS cells, but its protein expression was induced in M112 OS cells following IGF-1 stimulation.