TGF-beta activation has been inhibited with Troglitazone, a synthetic PPARgamma ligand (Peng, Liu et al. 2006). The fibrotic response in the collecting ducts of the nephron, particularly in terms of EMT, has only recently been established (Ivanova, Butt et al. 2008). The purpose of this study was to characterize a fibrotic response in the collecting duct with TGF-beta and to assess PPARgamma's role in the phenomenon. We hypothesized PPARgamma activation will have a protective effect in the collecting duct, preventing TGF-beta mediated EMT. TGF-beta was unable to initiate a fibrotic response in the IMCD-K2 & M1 collecting duct cell lines. In contrast, Troglitazone caused morphological changes and decreases in E-cadherin, alpha-catenin & beta-catenin, as well as an increase in fibronectin. These effects were not reversed with PPARgamma antagonists or a GSK-3beta blocker. Troglitazone induced a transformation in M1 & IMCD-K2 cells independently of PPARgamma.

Aromatase is responsible for the conversion of androgens to estrogens in the brain and gonadal tissues. Teleosts have been characterized as having high levels of brain estrogen biosynthesis. Little is known about the effects of estrogens on brain function in teleosts. The main objective of this study was to assess the effects of fadrozole, a powerful aromatase inhibitor, on gene expression in the zebrafish brain. A fadrozole exposure leads to a decrease in estrogens and an increase in androgens. In the telencephalon, 235 genes were identified by Affymetrix GeneChip analysis as being differentially regulated. Real-time RT-PCR and in situ hybridization were used to validate the data obtained from the microarrays. Collectively, the results provide a better understanding of the effects of aromatase inhibitors on gene expression and also shed light on the underlying effects of sex hormone variation and their importance in the brain of teleosts.

Retinal detachments cause photoreceptor apoptosis. XIAP (X-linked inhibitor of apoptosis) inhibits caspases-3, -7, and -9, which prevents the apoptotic cascade. This study evaluates XIAP gene therapy as a means to provide photoreceptor neuroprotection following retinal detachment. Subretinal injections of virally-delivered XIAP or green fluorescent protein (GFP; injection control) were performed in rats. Two weeks later, retinal detachments were created at the viral injection site. Eyes were harvested 24 hours post-detachment to analyze caspase activity and at 3 days and 2 months for histological analysis. Caspase assays indicated rises in caspase-3 and -9 activities in detached GFP-treated retinas, whereas XIAP-treated retinas behaved comparably to attached controls. Three day TUNEL analysis showed less apoptosis in XIAP-treated detachments. Two month histology confirmed preservation of photoreceptors in XIAP-treated detachments, whereas GFP-treated detached retinas had deteriorated significantly. The results suggest that XIAP confers structural photoreceptor neuroprotection for at least two months following retinal detachment.

The X-linked Inhibitor of Apoptosis Protein (XIAP) counters diverse apoptotic pathways through inhibition of caspases. The levels of XIAP protein can be increased translationally in response to pathophysiological stress, elevating the apoptotic threshold of cells. Here, I have characterized two XIAP mRNA isoforms, which differ only in their 5' untranslated regions. When global translation is attenuated during pathophysiological stress, the translation of one XIAP mRNA isoform is dramatically increased through the action of an Internal Ribosome Entry Site (IRES). In contrast, the second XIAP mRNA isoform supports cap-dependent translation of Xiap, but does not contain a functional IRES element. The physiological relevance and contribution of the XIAP mRNA variants to XIAP protein levels have been examined using RNA Interference, in conjunction with a cellular stress model. The distinct translational activities of these two transcripts under cellular stress suggest a model for a dual mode of XIAP regulation, which may be a common mechanism.

Our bodies go through cycles of feeding and fasting, and in order to control blood glucose levels, will respond by releasing hormones such as glucagon and insulin. After feeding, insulin is released from the pancreatic beta cells to reduce blood glucose levels. When our bodies are unable to do this the result is hyperglycemia, leading to diabetes, of which there are two types. Type I diabetes is an autoimmune disease where the body's T cells attack and destroy the insulin producing beta cells, rendering these patients insulin-dependent. Type 2 diabetes however, results from chronic high blood glucose levels. This will lead to insulin resistance and eventually beta cell apoptosis. Recently it was observed that the transcription factor cAMP Response Element Binding (CREB) protein is required for beta cell survival. Expression of a dominant-negative CREB under the control of the beta cell specific rat insulin promoter leads to progressive reduction of beta cell mass and diabetes in mice. Interestingly, the same signals that lead to insulin secretion, cAMP and Ca2+, are required for activation of CREB. Transducers of Regulated CREB activity (TORCs) were recently identified as CREB coactivators that are responsible for the synergistic activation of CREB target genes by cAMP and Ca2+. Under resting conditions phosphorylated TORCs are sequestered in the cytoplasm by 14-3-3 proteins. After stimulation with cAMP and Ca2+ TORCs become dephosphorylated and enter the nucleus, where they bind to the bZIP domain of CREB to activate transcription. In the absence of these stimuli, TORC2 is phosphorylated by the salt inducible kinase 2 (SIK2) at S171. Treatment of 293 cells with cAMP leads to the phosphorylation of SIK2 and therefore the dissociation of this kinase from TORC2. Additionally, the calcium responsive phosphatase calcineurin (Cn) in response to calcium stimuli dephosphorylates TORC2 leading to dissociation of 14-3-3 proteins and hence TORC2 nuclear entry. Interestingly while 293 cells require no more than a cAMP stimulus for TORC2 nuclear entry, beta cells remain in the cytoplasm following the same treatment. Additionally, only after treatment with cAMP and calcium does TORC2 lose 14-3-3 binding and enter the nucleus of beta cells to activate CREB. My hypothesis was that there remained additional regulatory phosphorylation site(s) that governed TORC2: 14-3-3 binding and therefore nuclear entry of TORC2 in beta cells. Work in this thesis reveals that s275 is the remaining phosphorylation site that governs 14-3-3 binding and is regulated by glucose/Ca2+ stimuli in beta cells. This evidence provides an explanation for the synergistic activation of TORC2 and therefore CREB in response to cAMP and Ca2+ . Additionally the potential kinase responsible for phosphorylation of this site was identified to be MARK2, a member of the AMPK family of kinases. I have identified an additional phosphorylation site in TORC2, S275 that is involved in 14-3-3 binding. This serine, in addition to S171 must be dephosphorylated to induce TORC2 nuclear entry and subsequent CREB activation. I was also able to show that the phosphorylation of this site is responsive to glucose in beta cells and in primary mouse islets. The goal of this thesis was to identify these remaining sites in order to uncover the regulatory mechanisms of TORC2-dependent CREB activation in response to cAMP and Ca2+ in the insulin producing beta cells.

The basic biological processes between the yeast Saccharomyces cerevisiae and mammals are highly conserved. Yeast posses many genes that are implicated in human diseases and have been successfully used as a model for the study of neurodegeneration. Platelet-Activating Factor (C16:0 PAF) causes neuronal cell death independent of its receptor and has been implicated in Alzheimer's disease. I hypothesized that yeast could be used as a model system for deciphering PAF receptor-independent signalling and have utilized genome-wide chemical genomic screening in yeast to further characterize the molecular mechanism of PAF toxicity. Two complementary screens implicate PAF in many cellular processes, some of which parallel results obtained in mammalian studies. I have found that PAF challenge is cytotoxic, delays cell cycle progression, and affects actin stability leading to spindle misorientation and bi-nucleate mother cells.

The mature retina consists of six neuronal and one glial cell type that are derived from a pool of mulipotent progenitor cells (RPC). The decision to remain as a multipotent progenitor or to specify a particular retinal cell lineage and differentiate are governed by cell intrinsic and extrinsic factors. Sonic hedgehog (Shh) is a secreted lipoprotein that is motigenic for RPCs and influences cell fate decisions. Suppressor of fused (Sufu) is an intracellular antagonist of the pathway; however, its role in regulating Shh signaling and influencing cell fate decisions in RPCs are unknown. Here, I demonstrate that Sufu antagonizes the Hh pathway in RPCs both in vitro and in vivo. Surprisingly, Sufu was required to maintain early RPC identity and multipotency. Conditional deletion of Sufu in early RPCs resulted in the down-regulation of transcription factors required to maintain RPC identity and multipotency as well as transcription factors required to specify all seven retinal cell types.Sufu-null RPCs were incapable of differentiating into the normal complement of retinal cell types and instead differentiated into restricted subsets of interneurons. These data demonstrate that Sufu antagonizes the Hh pathway in RPCs and provides novel evidence that Sufu is required for proper progenitor cell behavior.

Placental growth and development depend on highly regulated extravillous trophoblast (EVT) function, such as proliferation, migration and invasion. NUMB has been reported to playa role in cell differentiation and proliferation. An established cell line (HTR8/SVneo) was used as a model to study the role of NUMB in invasive extravillous trophoblast cell migration and survival in the human placenta. Immunofluorescence showed the presence of NUMB at the plasma membrane and the cytoplasmic region in HTR8/Svneo cells. NUMB isoform 1 significantly increased EVT migration while NUMB knockdown decreased the rate of migration as compared to the negative control. Overexpression of Numb 1,3,4 and 8 did not influence cellular viability whereas Numb 2 overexpression decreased trophoblast viability significantly. We have demonstrated for the first time that Numb is expressed in human invasive extravillous trophoblasts and that its role is isoform-dependent. Dysregulation of NUMB expression/function may playa role in placental pathologies such as preeclampsia and IUGR.

Angiotensin-converting enzyme 2 (ACE2) converts angiotensin II to angiotensin- (1-7) (Ang-(1-7)). The role of ACE2 and Ang-(1-7) in the progression of chronic kidney disease (CKD) is unclear. We studied the effects of ACE2 inhibition and Ang-(1-7) in the 5/6 nephrectomy (5/6 Nx) mouse model of CKD and sought to generate kidney-specific ACE2 transgenic mice. We hypothesized that pharmacological inhibition of ACE2 would accelerate kidney injury in 5/6 Nx mice whereas administration of Ang-(1-7) might be renoprotective. Four weeks after 5/6 Nx, kidney cortex ACE2 protein expression and enzymatic activity were decreased, compared to Sham-operated mice. In 5/6 Nx mice, ACE2 inhibition significantly increased urine albumin excretion, an effect reversed by Ang II AT1 receptor antagonism. In contrast, Ang-(1-7) did not affect CKD progression. Finally, using a plasmid vector containing the human ACE2 gene linked to the nephrin promoter, transgenic mice were generated that selectively overexpress human ACE2 in the glomerular podocyte. These data suggest that kidney ACE2 is down-regulated in the 5/6 Nx mouse and that further inhibition of ACE2 increases albuminuria via an AT1 receptor-dependent mechanism. The generation of ACE2 transgenic mice will allow for further delineation of the physiological role of ACE2 in the kidney.

Laminar shear stress is atheroprotective mainly due to its effect on endothelial cell morphology. Endothelial cells subjected to laminar flow elongate, align in the direction of flow and form robust stress fibers which align along with microtubules parallel to the direction of flow. We investigated the role of the actin/MRTF-A/SRF pathway in mediating the endothelial remodeling response. We hypothesize that MRTF-A is required for shear induced cytoskeletal remodeling to occur. We were able to show that MRTF-A translocates to the nucleus within 3 hours of shear stress application. The duration of nuclear translocation was short term lasting only 6 hours. After 6 hours of flow, MRTF-A was predominantly cytoplasmic. The inhibition of ROCK abolished shear induced MRTF-A nuclear translocation and cytoskeletal remodeling. The inhibition of PI-3 kinase, myosin II and GSK-3beta attenuated shear induced MRTF-A nuclear translocation and cytoskeletal remodeling whereas the inhibition of Rac1 and MEK had no effect. Shear induced MRTF-A nuclear translocation and the cytoskeletal remodeling response appear to be largely ROCK dependent. In addition, we investigated the requirement of MRTF-A for cell migration. HUVECs infected with adenovirus expressing a dominant negative form of MRTF-A were unable to migrate in a scratch wound assay. Moreover, HUVECs expressing DN MRTF-A were unable to form a capillary network consisting of dispersed cells in a matrigel assay but formed networks where cells remained in clumps.