Diabetes and obesity are associated with inflammation and activation of the immune system with infiltration of adipose tissue by macrophages. This is mainly studied in adipose tissue, with limited information to clarify immune-skeletal muscle interactions in these conditions. We show that exposure of L6 rat skeletal muscle cells to saturated fatty acid palmitate results in insulin resistance, activation of inflammatory pathways, upregulation of pro-inflammatory cytokine and chemokine gene expression and secretion. We identified monocyte chemoattractant protein-1 [MCP-1] as the main factor responsible for macrophage attraction, as blocking it reduced macrophage migration to muscle cells. When macrophages are exposed to palmitate, a similar response ensues with production of macrophage chemoattractants and activation of inflammatory pathways and gene expression profiles, and secretion of multiple cytokines. Our work identifies MCP-1 chemokine produced in response to palmitate treatment by both muscle cells and macrophages and provides a potential link in immune-metabolic crosstalk in diabetogenic environment.

Globotriaosylceramide (Gb3) is a resistance factor against human immunodeficiency virus (HIV) infection, but its expression has not been studied on human CD4+ T-cells. It was proposed that CD4+ T-cells may express Gb3 upon in vitro stimulation. To examine this, the optimal method for surface-expressed Gb3 detection was determined by comparing reagents, which showed that natural ligand (VT1B) and rat IgM monoclonal antibody (38-13) were the best methods. Using these, stimulated cells upregulated Gb3 in subsets of CD4+ T-cells, including T regulatory and NKT cell phenotypes, although the expression remained less than 2 percent of total cells. An enrichment method confirmed this. Examination of total Gb3 revealed that stimulated CD4+ T-cells without surface-expressed Gb3 did not express intracellular Gb3. Based on these results, it is concluded that CD4+ T-cells do not express Gb3 at significant levels under the conditions examined and, thus, may not have potential for resistance to HIV infection.

Cytokinesis is the final separation of daughter cells after division. Membrane trafficking increases the surface area of dividing cells and may deliver cargo needed for division. The Drosophila PI4-kinase Fwd is required for spermatocyte cytokinesis and likely acts, in part, by mediating Rab11-dependent trafficking to the furrow. To further understand the mechanism of action of Fwd, I attempted to place fwd in a pathway with other cytokinesis genes encoding Rab11, phosphatidylinositol transfer protein and a subunit of the exocyst. I also investigated a potential role for the Rab11 interacting protein dRip11 in cytokinesis. My results suggest that Rab11, like Fwd, is required for cell integrity during cytokinesis and that the Rab11 interacting protein Nuf is an important candidate to investigate along with dRip11 as a relevant Fwd/Rab11 effector during this highly conserved process.

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.

Diabetes and obesity are associated with inflammation and activation of the immune system with infiltration of adipose tissue by macrophages. This is mainly studied in adipose tissue, with limited information to clarify immune-skeletal muscle interactions in these conditions. We show that exposure of L6 rat skeletal muscle cells to saturated fatty acid palmitate results in insulin resistance, activation of inflammatory pathways, upregulation of pro-inflammatory cytokine and chemokine gene expression and secretion. We identified monocyte chemoattractant protein-1 [MCP-1] as the main factor responsible for macrophage attraction, as blocking it reduced macrophage migration to muscle cells. When macrophages are exposed to palmitate, a similar response ensues with production of macrophage chemoattractants and activation of inflammatory pathways and gene expression profiles, and secretion of multiple cytokines. Our work identifies MCP-1 chemokine produced in response to palmitate treatment by both muscle cells and macrophages and provides a potential link in immune-metabolic crosstalk in diabetogenic environment.

Globotriaosylceramide (Gb3) is a resistance factor against human immunodeficiency virus (HIV) infection, but its expression has not been studied on human CD4+ T-cells. It was proposed that CD4+ T-cells may express Gb3 upon in vitro stimulation. To examine this, the optimal method for surface-expressed Gb3 detection was determined by comparing reagents, which showed that natural ligand (VT1B) and rat IgM monoclonal antibody (38-13) were the best methods. Using these, stimulated cells upregulated Gb3 in subsets of CD4+ T-cells, including T regulatory and NKT cell phenotypes, although the expression remained less than 2 percent of total cells. An enrichment method confirmed this. Examination of total Gb3 revealed that stimulated CD4+ T-cells without surface-expressed Gb3 did not express intracellular Gb3. Based on these results, it is concluded that CD4+ T-cells do not express Gb3 at significant levels under the conditions examined and, thus, may not have potential for resistance to HIV infection.

Cytokinesis is the final separation of daughter cells after division. Membrane trafficking increases the surface area of dividing cells and may deliver cargo needed for division. The Drosophila PI4-kinase Fwd is required for spermatocyte cytokinesis and likely acts, in part, by mediating Rab11-dependent trafficking to the furrow. To further understand the mechanism of action of Fwd, I attempted to place fwd in a pathway with other cytokinesis genes encoding Rab11, phosphatidylinositol transfer protein and a subunit of the exocyst. I also investigated a potential role for the Rab11 interacting protein dRip11 in cytokinesis. My results suggest that Rab11, like Fwd, is required for cell integrity during cytokinesis and that the Rab11 interacting protein Nuf is an important candidate to investigate along with dRip11 as a relevant Fwd/Rab11 effector during this highly conserved process.

The term “stem cell” is often broadly applied to a range of cell types that are relatively undifferentiated and have some capacity for proliferation. In this thesis, I employ a strict definition of stem cells as cells that are capable of both self-renewal and multilineage differentiation. These properties are tested in single precursor cells from the forebrain and its derivative, the retina, using clonal assays. Poor survival is a common problem in single cell cultures, and I show that low oxygen dramatically improves viability in neural stem cells clonally derived from mouse embryonic stem cells, as well as in cultured forebrain neural stem cells. Caspase-dependent and apoptosis-inducing factor-dependent cell death pathways were found to be differentially influenced in low oxygen culture of early, primitive and later, definitive neural stem cells. I isolate precursors from 2 separate regions of the adult mouse forebrain, the lateral ventricle and the hippocampus and argue that only cells resident in the lateral ventricle can be classified as stem cells while the hippocampus contains restricted progenitor cells. Unlike neural stem cells, the very existence of retinal precursors in the adult mammal is controversial. I investigate methods to prospectively identify a rare stem cell population in the pigmented ciliary epithelium of the adult mouse eye and show that, although this population intrinsically gives rise to all retinal cell types, cells can be directed specifically towards a photoreceptor fate by the addition of exogenous factors to the culture media. Pigmentation of retinal stem cells is used as a convenient marker to isolate a retinal stem cell from human embryonic stem cells differentiating under conditions known to promote neural differentiation. Retinal stem cells derived from human embryonic stem cells have highly similar properties to those directly isolated from the eye, and their progeny can similarly be driven to differentiate into photoreceptors. The findings presented in this thesis help to define intrinsic properties of adult neural and retinal precursors and provide a basis for manipulating these cells, potentially for future use in clinical applications.

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.