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.

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.

Specialized cells of the innate immune system, such as macrophages, employ lysosomal enzymes, together with cationic peptides and reactive oxygen intermediates, to eliminate invading microorganisms ensnared within phagosomes. The effectiveness of this impressive armamentarium is potentiated by the acid pH generated by the vacuolar-type ATPase (V-ATPase). The determinants of the luminal pH of phagosomes and of the lysosomes they fuse with are not completely understood, but the V-ATPase is known to be electrogenic and net accumulation of protons requires charge compensation. For this reason, counter-ion pathways are thought to serve a central role in the control of acidification. It has generally been assumed that a parallel anion influx accompanies proton pumping to dissipate the voltage that tends to build up. In fact, impaired chloride channel activity in cystic fibrosis has been proposed to underlie the defective phagolysosome acidification and microbial killing reported in lung macrophages. In the first part of this thesis, I devised methods to dialyze the lumen of lysosomes in intact cells, while monitoring lysosomal pH, in order to assess the individual contribution of counter-ions to acidification. Surprisingly, anions were found to be completely dispensable for proton pumping, whereas the presence of permeant cations in the lysosomal lumen was essential. Accordingly, defects in lysosomal anion permeability cannot explain the impaired microbicidal capacity of phagocytes in cystic fibrosis. Even though counter-ion permeation pathways exist, dissipation of the electrical contribution of the V-ATPase may not be complete. If present, a transmembrane potential would alter the rate and extent of proton accumulation in phagosomes and lysosomes. However, no estimates of the voltage across the phagosomes were available. To overcome this deficiency, in the second part of this thesis, I describe a noninvasive procedure to estimate the voltage across the phagosome using fluorescence resonance energy transfer. This novel approach, in combination with organellar pH measurements, demonstrated that proton pumping is not limited by counter-ion permeability.

There is a growing need for bone cells to be used in the generation and repair of bone tissues that have become destroyed as a result of disease (e.g. osteoporosis), injuries and genetic deficiencies. Human umbilical cord blood has been a source of cells that is being investigated for its ability to generate cells for repair of various tissues including bone, which is the focus of this investigation. Similarly cord blood can also be used as a source of progenitors for osteoclasts that might be used for management of other diseases of bone characterized by defects in these cells (e.g. osteopetrosis). Moreover, there are few reliable in vitro models for human osteoclasts and so the ability to develop a cell model for human osteoclasts will also permit more meaningful studies on regulation of these cells than possible in the past. One of the setbacks suffered by researchers in designing therapeutic uses for stem cells is that in laboratory animals tumours can arise from these transplanted and pleuripotential cells. This suggests that stem cells might also have a dysregulated apoptosis pathway, which is critically important to understand before the safe use of stem cells can be assured. In order to increase our understanding of apoptosis in bone cells in particular, studies were done to try to understand the role of harakiri, a pro-apoptotic gene, in the development of osteoblasts and osteoclasts. Therefore, this series of study had two main foci; the development of human bone cells, particularly osteoclasts and osteoblasts, from human umbilical cord blood, and to understand further the mechanisms regulating apoptosis in bone cells. This study showed that osteoblasts could not be derived easily from human cord blood cells while it was possible to generate fully functional osteoclasts, which demonstrated unique properties. Studies done on harakiri deficient mice showed that absence of this gene caused an increase in osteoblast formation and a decrease in osteoclast formation. These findings can be exploited when considering the development of pharmacological agents that might be used in the future to modulate osteoclast cell development, function, and apoptosis.

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.

Cytokinesis, the final step of cell division, is characterized by formation of a cleavage furrow that ingresses to separate the cell into two daughter cells. This process requires rearrangement of the cytoskeleton and addition of membrane to the growing furrow. The phospholipid phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] has been implicated in regulating both actin dynamics and membrane trafficking and, thus, is uniquely poised to coordinate these different processes during cytokinesis. In this study, I show that PtdIns(4,5)P2 is involved in another aspect of cytokinesis: regulation of actomyosin contractility. Perturbing PtdIns(4,5)P2 levels in Drosophila spermatocytes caused constriction to fail and cleavage furrows to regress. Moreover, PtdIns(4,5)P2 hydrolysis is implicated in this process: inhibiting PLC or IP3R or chelating Ca2+ also caused defects in furrow ingression. In addition, I show that PLC and MLCK activities are required for myosin light chain phosphorylation and for proper myosin and actin localization to the cleavage furrow. Thus, I propose a model in which PtdIns(4,5)P2 hydrolysis-dependent Ca2+ release activates MLCK via Ca2+/calmodulin to maintain myosin filaments in the contractile ring and regulate cleavage furrow ingression. Furthermore, I show that PtdIns(4,5)P2 has a role in maintaining contractile ring components in the cleavage furrow that does not depend on PtdIns(4,5)P2 hydrolysis. I conclude that PtdIns(4,5)P2 regulates myosin contractility through a PLC-dependent pathway leading to myosin phosphorylation and is also involved in localizing contractile ring components to the furrow. Thus, PtdIns(4,5)P2 may coordinate signals leading to cytoskeleton rearrangement and actomyosin contractility during cytokinesis.