Thesis (M.Sc.)--York University, 2008. Graduate Programme in Biology. / Typescript. Includes bibliographical references (leaves 105-106). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:MR38818

The proteins and molecular machinery mediating the release of cortical granule (CG) contents from fertilized embryos is not completely understood. The process of vesicle fusion involves linking chaperones prior to vesicle to membrane contact. Rab3A, a member of a low-molecular weight GTP-binding protein superfamily has been detected in mouse embryos from the unfertilized meiotic II stage to the 2-cell. It is believed to positively regulate the final step of CG exocytosis by binding to Rabphillin, calcium ions (Ca2+), and phospholipids. SNAP23 a member of soluble NSF [N-ethylmaleimidesensitive factor] attachment protein receptors (SNAREs) binds together with parts of the Rab3A-rabphilin3A complex and is believed to be involved in the Ca2+-dependent exocytosis of non-neuronal systems. In this study we observed the mRNA expression for SNAP23 and Rab3A in pre-Meiotic I, post-Meiotic I unfertilized eggs (pre-MI UFE and post-MI UFE), and fertilized eggs (FE) utilizing RT-PCR. The products were analyzed in 2% agarose gel stained with ethidium bromide. Density analysis using a globin external standard showed that the levels of mRNA transcripts declined from the UFE to the FE in both genes, SNAP23 and Rab3A. Immunofluorescence was used for the detection and localization of Rab3A protein within the pre-MI and post-MI UFE and FE mouse egg. Eggs were stained with anti-Rab3A primary antibody and lens culinaris agglutinin (LCA) conjugated to FITC. Rab3A showed punctate staining in pre- and post-MI UFEs on small vesicles assumed to be CGs and in FEs on vesicles of a larger size. Uniform cytoplasmic expression was also seen, throughout the cells cortical and subcortical regions in each stage (pre- and post-MI UFEs and FEs), but with decreasing intensity as the eggs matured. This cytoplasmic stain may represent inactive Rab3A in the cytosol. The LCA stain showed punctate expression of cortical granules with localization within the cortical region and the plasma membrane. The addition of information on SNAP23 and Rab3A will aid in the process of studying CG exocytosis as well as in understanding the temporal and spatial development pathways involved in stimulating the cortical reaction. / Department of Biology

Statins are widely used to treat hypercholesterolemia. Statins inhibit cholesterol biosynthesis, thereby activating genes involved in cholesterol homeostasis, which are under the control of the Sterol Regulatory Element (SRE). Statins also have cholesterol-independent beneficial cardiovascular effects mediated through the phosphoinositide 3-kinase (PI3-K) / Akt signaling pathway and by inhibition of protein prenylation. Because statins inhibit the synthesis of isoprenoids, they can act by inhibiting the small signaling GTPases Ras and Rho, which require post-translational prenylation to become membrane-anchored and functional. We showed that simvastatin-mediated inhibition of protein prenylation does not appear to play a role in activation of SRE transcriptional activity in HepG2 cells. We also found that when isoprenoids were replenished, basal phospho-Akt decreased, suggesting that inhibition of prenylation by simvastatin mediates Akt phosphorylation. Future studies will be needed to investigate the role that inhibition of protein prenylation plays in the activation of the PI3-K/Akt pathway by simvastatin. / Department of Biology

The Ras signaling cascade is a vital component in the processes that mediate cell survival, growth, differentiation and transformation through activation of MAP kinase (mitogen-activated protein kinase). The recent discovery of a new scaffold of the Ras signaling pathway, Kinase Suppressor of Ras (KSR), is found to be a positive effector of Ras signaling which further contributes to proliferation and transformation in the ERK/MAPK pathway. This thesis describes the roles of Ras and Kinase Suppressor of Ras 1 (KSR-1) in regulating the expression of tumor promoting genes such as urokinase plasminogen activator (uPA) in the development and progression of breast cancer in vitro and in vivo. Ras and KSR increase the proliferative capacity and migration of MDAMB-231 human breast cancer cells in vitro. In contrast, Ras and KSR decrease the invasiveness of MDA-MB-231 human breast cancer cells in vitro. Furthermore, uPA gene expression levels do not correlate with uPA protein expression levels suggesting a possible mutation induced by KSR and/or Ras. In vivo studies reveal that Ras and KSR increase tumor volume in mice, as well as more advanced osteolytic bone metastases. Collectively, these results indicate that Ras and KSR play significant roles in breast cancer development and metastasis.

Thesis (Ph. D.)--University of Washington, 1997. / Vita. Includes bibliographical references (leaves [111]-126).

Thesis (Ph. D.)--University of Washington, 1997. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

The questions outlined in this thesis dissertation were proposed in order to provide insight regarding the mechanism by which the Drosophila Son of sevenless (dSOS) protein activates Ras. Ras proteins are GTP-binding proteins which bind guanine nucleotides very tightly and cycle between the inactive GDP-bound state and the active GTP-bound state. To address the mechanism by which the dSOS proteins activates Ras, a structure-function analysis of the dSOS protein was performed using truncation and deletion mutants of dSOS. In vivo Ras activation experiments using transiently transfected cells revealed that the NH2-terminal domain of dSOS is required in order for the catalytic domain of dSOS to exhibit exchange activity in cultured mammalian cells. The COOH-terminal GRB2 (Growth Factor Receptor Binding Protein) binding domain on the otherhand was insufficient to confer Ras exchange activity to the dSOS catalytic domain. Further analysis of the NH2-terminal domain of the dSOS protein demonstrated that the function of promoting catalytic domain activity could be localized by mutational analysis to the pleckstrin (PH) and DBL (Diffuse B-cell Lymphoma) homology sequences. Fractionation studies of cells transiently transfected with various dSOS mutant proteins demonstrated that the NH2-terminus of dSOS is also necessary for membrane association. These findings suggested that the model proposing that the recruitment of SOS via the adaptor protein GRB2 to the membrane is the main mechanism by which SOS activates Ras is unlikely to be the only mechanism by which SOS can activate Ras. From our data, a model can be proposed which postulates that SOS can activate Ras as a consequence of at least two steps. One step involves the SOS/GRB2 interaction and the second step involves the NH2-terminal domain of SOS associating with unidentified cellular elements.

No description available.

Ras is a central player in signal transduction that mediates cellular proliferation and differentiation. Recent evidence has shown that lipid and non-lipid modified domains participate in Ras traffic and that plasma membrane association is mediated by vectorial vesicular transport from the endomembrane system. ERp57, an ER chaperone, has been shown to specifically bind farnesylated Hras but not non-farnesylated Hras. The objective of this study was to determine if ERp57 participates in Ras trafficking and function. First, the effect of ERp57 knock down by siRNA technology on Hras function was studied; there was a reduction in ERp57 cellular levels that led to a decrease of active ras. Second, specific anti-ERp57 antibodies were delivered into 3T3 cells expressing GFP-ras chimeras to observe the effect on intracellular trafficking. Anti-ERp57 antibodies blocked Hras plasma membrane localization but not Kras suggesting that ERp57 may be involved in Hras intracellular trafficking and function.

In experimental membranous nephropathy, complement C5b-9-induced glomerular epithelial cell (GEC) injury leads to breakdown of glomerular peimselectivity and proteinuria. This study addresses mechanisms that limit complement-mediated injury, focusing on Ras. Complement-mediated injury was attenuated in cultured GEC expressing a constitutively active form of Ras (V12Ras), compared with Neo (control) GEC. V12Ras GEC showed constitutive activation of phosphatidylinositol 3-kinase and extracellular signal-regulated kinase pathways, but inhibition of these pathways did not reverse the protective effect of Ras. V12Ras GEC showed smaller and rounder morphology, decreased F- to G-actin ratio, decreased activity of the Rho GTPase, Rac, and decreased Src activity. In V12Ras GEC, disruption or stabilization of the F-actin cytoskeleton reversed the protective effect of V12Ras on complement-mediated injury. Thus, the protective effect of V12Ras may be dependent on remodeling of the actin cytoskeleton. Furthermore, the reduction of Src activity due to Ras activation may alter the equilibrium in activities of Rho GTPases, a family of proteins known regulate the actin cytoskeleton. Activation of Ras signaling is a novel pathway to consider in developing strategies for cytoprotection in complement-mediated injury.