Phagocytosis is an innate immune response that is paramount in the clearance of pathogenic particles. Recognition of target particles by phagocytic receptors expressed on phagocytes induces modifications in the underlying actin cytoskeleton to form pseudopods that encircle and internalize the target particle into a membrane bound organelle called the phagosome. The nascent phagosome undergoes a maturation sequence that is characterized by substantial remodeling of the membrane and its luminal contents through interactions with components of the endocytic pathway, culminating in an acidic and hydrolytic organelle capable of digesting and elminating pathogens. Phagosome maturation is a complicated pathway that involves many protein and lipid signaling molecules. Several factors that influence phagosome maturation particularly the participation of pH, lysosome-associated membrane proteins-1 and –2, cholesterol, in addition to the survival and escape mechanisms used by, Burkholderica cenocepacia were explored. All three tenets are essential for phagosome maturation, although each factor has different mechanistic consequences. Acidification alters Rab5 activation, while ablation of LAMPs and accumulation of cholesterol interferes with various aspects of Rab 7 turnover in phagosomes and/or endosome membranes. Moreover, Burkholderia cenocepacia, an intracellular pathogen, inactivates Rab7 on phagosome membranes from within the vacuole lumen. Herein, mechanisms that govern phagosome maturation are explored and several molecules are added to the long list of essential players in this complicated pathway.

Plant form and structure is remarkably diverse encompassing a myriad of shapes and sizes, and is a result of coordinated instances of cell growth and division. This varied form and structure represents adaptive strategies enabling plants to exploit and endure their environment maximizing fitness. Common to both cell growth and division processes, is the requirement of growth material and effectors via the secretory system. The secretory system is a dynamic pathway of organelles connected via vesicular traffic, responsible for protein modification and delivery. Challenges in studying secretion are related to its' dynamic flow between organelles, and the prevalence of redundancy and lethality, which cause difficulties in interpretation and genetic modulation. To further our understanding of the secretory process, we employed a chemical genetic approach to identify small molecule probes of secretion by first screening for inhibitors of cell expansion, followed by a microscopy based screen of GFP marker lines for perturbagens of subcellular structures. Small molecules offer the advantages of temporal and spatial application, have been shown to overcome redundancy and can be varied in concentration to control the severity of effects. We identified twenty-five small molecule probes of secretion and pursued two chemicals with striking properties, polarazine and eroonazole. Polarazine was identified as a cell division inhibitor, affecting phragmoplast structure and function, while eroonazole remodels the endoplasmic reticulum into small vesicles and is implicated in auxin signaling. This approach shows the utility of small molecule probes in furthering our understanding of secretory processes.

The GATA transcription factors consist of six family members that bind the consensus DNA binding element W-GATA-R, and are poorly characterized in the central nervous system (CNS). In this thesis we identify GATA4 to be expressed in the neurons and glia of normal murine and human embryonic and adult CNS with significant loss in Glioblastoma Multiforme (GBM). GBM is the most common and lethal primary brain tumour and exhibits multiple molecular aberrations. Here we report that loss of the transcription factor GATA4, a negative regulator of normal astrocyte proliferation, is a driver in glioma formation and fulfills the hallmarks of a tumour suppressor gene. Although GATA4 was expressed in normal brain, loss of GATA4 was observed in GBM operative samples and was a negative survival prognostic marker. GATA4 loss occurred through promoter hypermethylation or novel somatic mutations. Loss of GATA4 in normal human astrocytes promoted high-grade astrocytoma formation, in cooperation with other relevant genetic alterations such as activated Ras or loss of TP53. Loss of GATA4 with activated Ras in normal astrocytes promoted a progenitor like phenotype, formation of neurospheres and the ability to differentiate into astrocytes, neurons and oligodendrocytes. Re-expression of GATA4 in human GBM cell lines, primary cultures and brain tumour initiating cells suppressed tumour growth in vitro and in vivo through direct activation of the cell cycle inhibitor P21CIP1, independent of TP53. Re-expression of GATA4 also conferred sensitivity of GBM cells to temozolomide, a DNA alkylating agent currently used in GBM therapy. This sensitivity was independent of MGMT, the DNA repair enzyme often implicated in temozolomide resistance. Instead GATA4 reduced expression of APNG, a DNA repair enzyme poorly characterized in GBM mediated temozolomide resistance. Identification and validation of GATA4 as a tumour suppressor gene and its downstream targets in GBM may yield promising novel therapeutic strategies.

The Hedgehog (Hh) signaling pathway has received attention regarding its important role in embryonic development, however the mechanism by which pathway regulators, such as Suppressor of fused (Sufu), Kinesin family member 7 (Kif7), and primary cilium, mediate Hh signaling transduction is not entirely understood. The work presented here examines the roles of Sufu and Kif7 in regulating Hh signaling in growth plate chondrocytes, as well as how they mediate parathyroid hormone-like hormone (Pthlh) signaling during chondrocyte development. I show here that Sufu and Kif7 are essential regulators of Indian hedgehog (Ihh) signaling. While Sufu negatively regulates Gli transcription factors, Kif7 functions both positively and negatively in chondrocytes. Kif7 plays a role in Sufu protein degradation and the exclusion of Sufu-Gli complexes from the primary cilium. Importantly, halving the dosage of Sufu restores normal Hh pathway activity and chondrocyte development in Kif7-null mice, demonstrating that the positive role of Kif7 is to restrict the inhibitory function of Sufu. Furthermore, Kif7 exerts inhibitory function on Gli transcriptional activity in chondrocytes when Sufu function is absent. Therefore, Kif7 regulates the activity of Gli transcription factors through both Sufu-dependent and Sufu-independent mechanisms. I show that Sufu is crucial for mediating the negative effect of Pthlh on Gli transcriptional activity and chondrocyte hypertrophic differentiation, whereas Kif7 and primary cilium are dispensable in this process. Although primary cilium is required for Hh ligand-mediated activation of Gli transcription, Pthlh negatively controls Gli transcriptional activity in a cilia-independent manner. The results of this work provide insight into how Hh signaling is regulated by Sufu and Kif7 in the context of primary cilium, but also suggest Sufu serves as an important link between Ihh and Pthlh signaling during growth plate chondrocyte development.

Amphiphysin (Amph) is a multi-domain protein that has been implicated in synaptic vesicle (SV) endocytosis. In vertebrates, Amph1 associates with SVs and binds to known endocytic proteins, such as dynamin and clathrin. Overexpression of the vertebrate Amph1 SH3 domain is sufficient to inhibit SV endocytosis in the lamprey synapse. However, these in vitro and overexpression studies may not reflect Amph function in vivo. To investigate Amph function in vivo, I used Drosophila melanogaster as a model organism. I discovered that Drosophila Amph was broadly expressed throughout all developmental stages and was also highly expressed in specialized membranes such as the postsynaptic membrane at the larval neuromuscular junction and the t-tubule membranes of muscles. amph mutants were viable and had normal synaptic transmission, results that were inconsistent with a role for Amph in SV endocytosis. However, amph mutants had impaired locomotion, which may reflect a defect in the t-tubule network, a membrane system that is specialized to couple muscle membrane excitation to muscle contraction. To further explore this idea, I undertook a structure-function approach to ask if different Amph functional domains could rescue the t-tubule and locomotory defects observed in amph mutants. Partial rescue was observed for most constructs, suggesting that Amph function was dependent on more than one domain. To further elucidate how Amph functions at the t-tubule network, I used different in vitro methods to investigate novel protein partners for Amph. A GST pull-down approach identified actin as a potential Amph partner, consistent with studies in yeast. However, I could not confirm a direct interaction between Amph and actin in Drosophila. Another candidate partner was the actin-nucleating protein, Wiskott Aldrich Syndrome Protein, WASP. Although WASP and Amph could be coimmunoprecipitated in vitro, WASP was not expressed at the t-tubule membrane, and Wasp mutants had normal t-tubule morphology. Clearly, Amph is essential for normal t-tubule morphology and future work is needed to further define the function of Amph at the t-tubule network.

A pancreatic stem cell could provide the tissue necessary for widespread β-cell transplantation therapy for diabetes. It is disputed whether pancreatic stem cells or β-cell replication are responsible for maintenance and regeneration of endocrine cells. Evidence presented here shows that pancreatic stem cells express insulin and produce multiple endocrine, exocrine and neural cells in vitro and in vivo. The human pancreas also contains stem cells that produce functional β-cells capable of reducing blood sugar levels in a diabetic mouse. Initial studies of pancreatic stem cells grown clonally in vitro indicated that they produced large numbers of neurons, suggesting they may be derived from the neural crest. Evidence shows that there are at least two distinct developmental origins for stem cells in the pancreas; one from the pancreatic lineage that produces endocrine and exocrine cells and one from the neural crest lineage that produces neurons and Schwann cells. Furthermore, pancreatic stem cells require the developmental transcription factor, Pax6, for endocrine cell formation suggesting they are using expected differentiation pathways. There is an interesting evolutionary connection between pancreatic β-cells and neurons which was applied to the derivation of pancreatic stem cells from human embryonic stem cells by using a clonal neural stem cell assay. These pancreatic stem cells express pancreatic and neural markers, self-renew and differentiate into insulin-expressing cells. The overexpression of SOX17 in these cells increases stem cell formation and self-renewal but inhibits differentiation. Overall I will show that there is a genuine stem cell in the adult mammalian pancreas capable of producing functional β-cells, that this stem cell is derived from the pancreatic developmental lineage but the pancreas also contains stem cells from the neural crest lineage, and that the neural stem cell assays that have identified these adult stem cells can be applied to the derivation of a pancreatic stem cell from hESCs.

The tumour suppressor p27kip1 (p27) is a potent inhibitor of cell growth and proliferation. We identified NF-κB activator, IKKα, as a novel interacting partner of p27 in a protein microarray screen. Both the IKKα and IKKβ components of the IKK complex were mapped to the C-terminal domain of p27. To investigate the physiological function of the p27-IKK interaction, we employed a well-established model of LPS-induced sepsis which is known to activate the IKK/NF-κB pathway. Lentivirally-mediated overexpression of p27 blocked LPS activation of NF-κB. Furthermore, in LPS-injected animals transduced with TAT-p27, a significant improvement in the left ventricular function of the heart was observed. TAT-p27 treatment was also shown to attenuate the endotoxin effect and significantly improve survival compared to both saline and TAT-LacZ controls. Our results indicate that p27 attenuates inflammation, possibly through inhibiting the IKK-dependent activation of NF-κB, thus supporting a novel link between both cell cycle regulation and inflammation.

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.

Endoplasmic reticulum (ER) stress has been implicated in pancreatic beta-cell loss contributing to diabetes mellitus, however the molecular mechanisms of ER stress-induced apoptosis are unclear. In the first project of this thesis, the contribution of ER stress in proinflammatory cytokine-mediated beta-cell dysfunction and apoptosis is examined. Although exogenous cytokine treatment did induce unfolded protein response (UPR) genes, increased chaperone capacity had no effect on apoptosis induction, insulin biosynthesis and insulin secretion. Thus, ER stress is most likely not an important pathway in cytokine toxicity under our experimental system. The second project develops a pathophysiological model of ER stress based on the mutant misfolded insulin of the Akita mouse. Microarray analysis was conducted and we observed early induction of ER chaperone and ER-associated degradation (ERAD) genes, followed by a large increase in pro-apoptotic genes with mutant insulin expression. A detailed analysis of the ER stress response in this system is presented.

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.