Nonhomologous end-joining (NHEJ) is a major DNA double-strand break (DSB) repair pathway. NHEJ is initiated through DSB recognition by the DNA end-binding heterodimer, Ku, while end-joining is accomplished by the XRCC4-DNA ligase IV (X4L4) complex. This thesis reports that APLF (Aprataxin and Polynucleotide kinase-Like Factor), an endo/exonuclease with a forkhead-associated (FHA) domain and two unique zinc fingers (ZF), interacts with both Ku and X4L4. The APLF-X4L4 interaction is FHA- and phospho-dependent, and is mediated by CK2 phosphorylation of XRCC4 in vitro. APLF binds Ku independently of the FHA and ZF domains, and complexes with Ku at DNA ends. APLF undergoes ionizing radiation induced ATM-dependent hyperphosphorylation and ATM phosphorylates APLF in vitro. Downregulation of APLF is associated with defective NHEJ and impaired DSB repair kinetics. These results suggest that APLF is an ATM target that is involved in NHEJ and facilitates DSB repair, likely via interactions with Ku and X4L4.

Wnt signalling is involved throughout development and is inappropriately activated in a variety of human cancers. In the canonical pathway, secreted Wnt proteins induce the stabilization of b-catenin. Drosophila Naked (Nkd), or Nkd1 and Nkd2 in vertebrates, is believed to antagonize canonical Wnt signalling through an interaction with Dishevelled (Dvl). Analysis of a high-throughput protein-protein interaction screen conducted in our lab led to the identification of novel Nkd1 interacting proteins, including Nkd1/2 and Axin1. Mapping of Nkd1 regions required for these novel interactions and functional studies, including transcriptional and siRNA mediated knockdown assays, were performed to examine the role of Nkd1 in canonical Wnt signalling. Previous work suggests that Nkd1 functions only through Dvl, but this work suggests that Nkd1 acts via a more complex mechanism. In addition to serving as an antagonist to regulate the canonical Wnt pathway, we propose that Nkd1 may also act positively to promote signalling.

Hyperosmotic stress represents a major threat to cellular integrity and may lead to cell death via apoptosis. Accordingly, each cell reacts to hyperosmolarity with a set of functional and structural compensatory responses. Recently it has been shown that the mitochondria remodel during hyperosmotic stress. Although changes in mitochondrial dynamics could be crucial for both adaptation and apoptosis, hyperosmolarity-induced mitochondrial remodeling has not been characterized. We found that hyperosmotic stress translocates dynamin like protein 1 (DLP-1) to the mitochondria and induces DLP-1 mediated, F-actin-modulated, Rac-dependent fragmentation of these organelles in LLC-PK1 cells. Downregulation of DLP-1 mitigates the activation of the osmotic response element and increases the susceptibility of tubular cells to hyperosmotically-induced apoptosis, suggesting that DLP-1 (or mitochondrial fragmentation) may have a protective role during osmotic stress. The hyperosmolarity-triggered remodeling of the mitochondrion represents a hitherto unrecognized response to osmotic shock, which may have significant impact on adaptation and apoptosis.

A DNA palindrome is a sequence of DNA followed by an exact inverted copy of itself. Palindromes are associated with gross chromosomal instability in mammalian cells. This may be related to their ability to extrude a double-stranded cruciform structure. In mammalian cells, palindromes have been shown to undergo centre-directed rearrangements resulting in a central region of asymmetry. This process occurs via a mechanism termed â centre break palindrome revisionâ . In this thesis, I have investigated palindrome revision in mammalian cells using two existing assays. In the first, performed by transfection of an extrachromosomal palindromic dimer, I have shown that joining of palindrome-mediated double-strand breaks does not depend solely on NHEJ and instead relies heavily on an alternate end-joining pathway. Using the second assay, the Line78 mouse model which contains a 15.4kb transgenic palindrome, I have shown that small modifications near the centre of the palindrome prevent these centre-directed rearrangements possibly by inhibiting cruciform formation.

Autophagy, a cellular degradative pathway, plays a key role in protecting the cytosol from bacterial colonization, but the mechanisms of bacterial recognition by this pathway are unclear. Autophagy is also known to degrade cargo tagged by ubiquitinated proteins, including aggregates of misfolded proteins, and peroxisomes. Autophagy of ubiquitinated cargo requires p62, an adaptor protein with multiple protein-protein interaction domains. Previous studies demonstrated that the intracellular bacterial pathogen S. typhimurium is targeted by autophagy during infection of host cells. Here I show that p62 is recruited to S. typhimurium targeted by autophagy, and that the recruitment of p62 is associated with ubiquitinated proteins localized to the bacteria. Expression of p62 is required for efficient autophagy of bacteria, and restriction of their intracellular replication. My study demonstrates that the surveillance of misfolded proteins and bacteria occurs via a conserved pathway and reveals a novel function of p62 in innate immunity.

Metabolic channelling occurs when two enzymes that act on a common substrate pass that intermediate directly from one active site to the next without allowing it to diffuse into the surrounding aqueous medium. In this study, properties of channelling are investigated through the use of computational models and cell simulation tools. The effects of enzyme kinetics and thermodynamics on channelling are explored with the emphasis on validating the hypothesized roles of metabolic channelling in living cells. These simulations identify situations in which channelling can induce acceleration of reaction velocities and reduction in the free concentration of intermediate metabolites. Databases of biological information, including metabolic, thermodynamic, toxicity, inhibitory, gene fusion and physical protein interaction data are used to predict examples of potentially channelled enzyme pairs. The predictions are used both to support the hypothesized evolutionary motivations for channelling, and to propose potential enzyme interactions that may be worthy of future investigation.

The pathogenic subgroup C feline leukemia virus (C virus) arises in infected cats by mutations in the envelope gene (env) of subgroup A FeLV (A virus) that switches the host receptor used for infection. To better understand C virus emergence and potential FeLV variants that may arise, I characterized FeLV Env sequence isolated from the primary FY981 FeLV isolate derived from an anemic cat. I show that the FY981 pseudotype virus is capable of using both the A virus receptor THTR1, and the C virus receptor FLVCR1 for infection, consistent with the FY981 Env being a hybrid A virus/C virus Env. Furthermore, I propose that pathogenic C virus arise in infected cats through FeLV variants or intermediates that are multi-tropic in their receptor use.

Insulin resistance associated with obesity has been suggested to contribute to an increased risk and poor prognosis for breast cancer. In this study, a HER2/Neu transgenic mouse model of breast cancer was used to assess how obesity-induced insulin resistance and hyperinsulinemia can influence the development and progression of breast cancer. We investigated the effect of a high-fat diet and found a tumor-promoting effect in the absence of overt insulin resistance. In contrast, a high-fat combined with fructose diet induced significant hyperinsulinemia but no tumor promoting or growth effect was observed. Treatment with the anti-diabetic, insulin-lowering agent metformin led to a delay in tumor onset in mice on control diet, but this effect was abrogated by the high-fat fructose diet. These data indicate that the effects and potential interactions of insulin, nutrition and drugs on breast cancer development and progression are complex and require further study.

In the budding yeast Saccharomyces cerevisiae, the G1-specific cyclin-dependent kinases (Cdks) Cln1-, Cln2-Cdc28 and Pcl1-, Pcl2-Pho85 are essential for ensuring that DNA replication and cell division are properly linked to cell polarity and bud morphogenesis. However, like most genes in S. cerevisiae, individual cyclin genes are not required for viability, and the phenotypes associated with deletion of any single cyclin gene tend to be subtle. My goal was to dissect the cellular roles of the G1 cyclins by systematically identifying their genetic interactions. To do this, I conducted Synthetic Genetic Array (SGA) screens using strains deleted for different combinations of cyclin genes. The results of screens with strains deleted for the G1 cyclin pairs, CLN1, CLN2, or PCL1, PCL2, confirmed a role for these cyclins in cell polarity regulation and identified novel G1 Cdk substrates, which I examined in more detail. One cell polarity regulator that showed an interesting pattern of genetic interactions with G1 cyclins was BNI1, which encodes a yeast formin protein. Overexpression of BNI1 caused an Synthetic Dosage Lethal interaction in the absence of both G1 cyclin pairs while its deletion caused synthetic lethality specifically in the absence of PCL1, PCL2. Consistent with these genetic interactions, phosphorylation of Bni1 was partially dependent on CLN1, CLN2. It has been proposed that Bni1 is regulated by intramolecular interactions. In an effort to discover how phosphorylation might affect Bni1 function, I developed assays to test for intramolecular interactions. In my experiments I found no evidence that Bni1 is regulated by intramolecular binding, as was proposed from parallels with its mouse homolog mDia1. I also found that deletion of BNI4, which encodes an adaptor protein that targets several proteins to the bud neck, results in severe growth defects in the absence of the Cdc28 cyclins Cln1 and Cln2, and overexpression of BNI4 was toxic in yeast cells lacking the Pho85 cyclins Pcl1 and Pcl2. I discovered that Bni4 was phosphorylated by Pcl1- and Pcl2-Pho85 in vitro and that phosphorylation of Bni4 was dependent on PCL1 and PCL2 in vivo. Further analysis showed that phosphorylation of Bni4 by Pcl-Pho85 is necessary for its localization to bud neck, and the bud neck structure can be disrupted by overexpressing BNI4 in pcl1pcl2 mutant cells. I propose that if Bni4 cannot be regulated by phosphorylation, it may titrate away an essential component that resides at the bud neck, thus causing catastrophic morphogenesis defects. The relationship between G1 Cdk activity and the polarity regulator Bni4 serves as a bridge to link the cell cycle machine to the regulation of cell.

Erythropoietin (EPO) is the primary cytokine regulator of erythropoiesis. Fundamental to this action is the ability of EPO to bind the EPO receptor (EPO-R), and activate the primary associated tyrosine kinase, JAK2. The critical importance of EPO, EPO-R and JAK2 to erythropoiesis is demonstrated by the fatal embryonic anemia that develops upon EPO, EPO-R or JAK2 deletion. Positive regulation of intracellular signal transduction pathways downstream of EPO-R and JAK2 are well documented, but less is known about negative regulation of EPO-R signaling. Two distinct experimental strategies were utilized to examine a subset of the negative regulatory mechanisms underlying EPO-R signaling and erythropoiesis. Mice deficient in the E3 ubiquitin ligase, Cbl, were generated previously and displayed elevated platelet numbers, expansion of splenic red pulp and splenomegaly, suggesting that Cbl-/- mice have defects in megakaryocyte/erythrocyte progenitors or more committed cells of each lineage. Our studies illustrated that genetic ablation of Cbl resulted in elevated total numbers of Burst Forming Unit-Erythroid and Colony Forming Unit-Erythroid, but decreased bone marrow-derived late erythroblasts. Cbl-deficient late erythroblasts displayed elevated apoptosis, as well as increased expression of Foxo3a and increased mRNA levels of the pro-apoptotic genes, Bim and FasL. These studies implicate Cbl as an important negative regulator of multiple facets of erythroid signaling. The discovery that EPO-R is ubiquitinated and degraded by the proteasome and lysosome led us to examine the role of EPO-R ubiquitination on signal transduction and proliferation. Lysine mutagenesis of EPO-R showed that K348, K388 and K428 were the primary ubiquitin acceptor sites when EPO-R mutants were expressed in HEK 293T cells. BaF3 cells expressing an EPO-R deficient in cytoplasmic lysines displayed diminished EPO-mediated EPO-R, JAK2, PKB and STAT5 phosphorylation and could not proliferate in response to EPO. The membrane proximal lysines of EPO-R, K256 and K276, were necessary for proliferation at physiologic EPO concentrations but were not required at saturating EPO concentrations. Single lysine EPO-R add-back mutants restored signaling and proliferation to BaF3 cells at physiologically elevated EPO concentrations, signifying that EPO-R lysines finely mediate EPO-dependent proliferation and signal transduction. These analyses demonstrate a positive regulatory role for lysines in signal transduction and proliferation.