<p><strong> </strong>Fibrotic cataracts such as anterior subcapsular cataract (ASC) are induced by transforming growth factor beta (TGFβ). The mechanism which governs TGFβ-mediated ASC has not been elucidated. What is known is that TGFβ initiates the conversion of lens epithelial cells (LECs) to myofibroblast-like cells, through a process known as epithelial to mesenchymal transition (EMT). TGFβ-induced EMT leading to ASC has been associated with the upregulation of two matrix metalloproteinases (MMPs), MMP2 and MMP9. However, roles for either of these MMPs have yet to be established in ASC. To determine the involvement of MMP2 and MMP9 I used synthetic inhibitors in conjunction with an established <em>ex vivo </em>rat lens model initiated by TGFβ. The results demonstrated that co-culturing rat lenses with TGFβ and the matrix metalloproteinase inhibitor (MMPI), GM6001 or an MMPI specific for MMP2/9 suppressed ASC. Additionally, studies conducted on the conditioned media from these treatments revealed that TGFβ induces the cleavage of E-cadherin ectodomain which is suppressed by coculturing with either MMPI. To further delineate a role for MMP9 <em>in vivo</em>, ASC formation was examined in two models of lens specific TGFβ overexpression in the absence of functional MMP9. Adenoviral delivery of TGFβ to the anterior chamber of the eye in the absence of functional MMP9 resulted in complete suppression of ASC. Similarly, lens specific TGFβ overexpression in the absence of MMP9 suppressed ASC in 75% of mouse lenses. Additional studies determined that connective tissue growth factor is able to mediate ASC, albeit to a lesser degree than TGFβ.</p> / Doctor of Philosophy (PhD)

<p>There is increasing evidence that prostate tumours are organized as a hierarchy with rare cancer stem cells (CSCs) implicated in initiating and maintaining the tumour. However, prospective prostate cancer stem cells (PCSCs) have not been thoroughly characterized from primary tissue specimens. Using the DU145 cell line, PCSCs have been propagated as non-adherent spheres <em>in vitro</em>. Approximately 1.25% of monolayer DU145 cells formed primary spheres while 26% of sphere cells formed subsequent spheres; a measure of PCSC self-renewal capacity. Spheres are enriched for cells expressing prostate basal and luminal cytokeratins and CSC markers (CD44, CD24, integrin alpha2beta1). PCSCs initiate xenograft tumours with enhanced capacity compared to monolayer cells. While epidermal growth factor (EGF) promoted PCSC propagation, basic fibroblast growth factor (bFGF) inhibited these events. Activation of EGF receptor (EGFR) signalling, following EGF treatment or expression of constitutively-active EGFR (EGFRvIII), increased sphere formation. Conversely, attenuation of EGFR signalling inhibited PCSC self-renewal. Consistent with the MEK-ERK pathway being a major target of EGFR signalling, the MEK-ERK pathway contributes to EGFR-facilitated PCSC propagation. Inhibition of ERK activation following MEK inhibitor treatment, expression of dominant-negative MEK1(K97M), or knockdown of ERK1 or ERK2 reduced PCSC propagation. Therefore, EGFR signalling promotes PCSC self-renewal by activating the MEK-ERK pathway.</p> <p>SOX2 is an essential transcription factor for stem cells, however, its role in PCSCs remains unclear. SOX2 protein is upregulated in PCSCs propagated as spheres, and its expression is regulated by EGFR signalling. EGFR activation, following EGF treatment or expression of constitutively-active EGFRvIII, increased SOX2 expression and PCSC self-renewal, while being attenuated by EGFR inhibitor treatment. Ectopic SOX2 expression enhanced EGF-induced PCSC self-renewal, while SOX2 knockdown renders PCSCs non-responsive to EGF-induced self-renewal and reduced their anchorage-independent growth. Furthermore, SOX2 expression is associated with the ability of PCSCs to form aggressive xenograft tumours. Collectively, SOX2 regulates EGFR-mediated PCSC self-renewal.</p> / Doctor of Philosophy (PhD)

Anti-tumor therapeutic strategies based on combinations of chemotherapeutic agents with a death inducing ligand such as TNF-α Related Apoptosis Inducing Ligand (TRAIL), are directed towards selective and effective cancer cell apoptosis and enhanced therapeutic response. We previously demonstrated that proteasome inhibition sensitizes TRAIL resistant prostate cancer cells to TRAIL-mediated apoptosis via stabilization of the active p18 subunit of initiator caspase-8. The present study investigated the functional link between caspase-8 and the proteasome, by analyzing the impact of caspase-8 ubiquitination and proteasomal degradation on the outcomes of the extrinsic apoptosis pathway in cancer cells. Caspase-8 ubiquitination status was assessed by polyubiquitin immunoprecipitation (IP) and fluorescent microscopy. Apoptosis induction in response to death receptor stimuli or proteasome inhibitor was evaluated using the Annexin V/Propidium iodide staining (PI). To determine the consequences of proteasome inhibition on caspase-8 stability, trafficking, and activity following death receptor activation, we used the TRAIL-resistant human prostate cancer LNCaP cells, and the caspase-8 deficient Neuroblastoma 7 (NB7) cells, as cellular models for reconstituting the non-cleavable mutant forms of caspase-8. Our findings demonstrate that the non-cleavable forms of caspase-8 are capable of inducing apoptosis comparably to wild-type caspase-8 upon treatment with proteasome inhibitor and GST-TRAIL. Furthermore, caspase-8 processing into its active subunits preceded caspase-8 polyubiquitination, implicating caspase-8 processing as a potential regulatory mechanism, rather than a requirement for caspase-8 activation in apoptosis induction. The mechanistic control of caspase-8 by ubiquitination in cancer cells may have significant significance in bypassing mechanisms of therapeutic resistance in human tumors and optimization of anti-cancer treatment strategies in human tumors and optimization of anti-cancer treatment strategies.

All human chromosomes end with protective structures called telomeres, which consist of thousands of double-stranded TTAGGG repeats and end in a 3’ guanine-rich overhang. These structures shorten normally during each round of replication, and extremely short telomeres along with telomere dysfunction are thought to contribute to the development of aging and cancer. Although many proteins have roles in telomere maintenance, WRN, which is a 3’ to 5’ helicase that is deficient in the premature aging disorder Werner’s syndrome, has been proposed to play multiple roles at telomeres. In this study, I focus on the effect of telomeric sequences and/or structures formed during DNA replication or recombination and how WRN functions at these sites. This study suggests that WRN may promote proper replication of telomeres by accurately aligning telomeric sequences during replication fork regression, potentially the first step in responding to a blockage, such as DNA damage. However, even in the presence of WRN, replication of telomeric sequences is difficult, possibly due to the ability of G-rich sequences to form secondary structures such as G-quadruplexes. I demonstrate that the translesion polymerase pol η, as well as a variety of other polymerases, is unable to synthesize past an intramolecular G-quadruplex formed from telomeric sequence on the template strand. Furthermore, in physiological salt concentrations, WRN favors binding and unwinding a structure that mimics a strand invasion intermediate over other similar structures especially when it possesses G-telomeric sequence. In addition, WRN promotes unwinding of these structures in a direction that would promote additional annealing and strand invasion, supporting a role for WRN in promoting telomeric recombination and formation of a T-loop, a proposed protective structure specific to telomeres. Overall, the data suggest that telomeres may pose problems in replication due to the G-rich, repeating nature of the structures, while WRN may aid in promoting proper replication at these and other replication blocks. Furthermore, WRN may play a role in promoting additional formation of T-loops and other telomeric recombination, thus supporting the relationship of WRN, telomere maintenance, and potentially development of certain aging characteristics.

Estrogens, such as the biologically active 17-b estradiol (E2) have many actions in the male and female brain. Not only does E2 regulate reproductive behavior in adults, it organizes and activates the brains of younger animals in a sex-specific manner. In addition, many human studies have shown E2 to provide protection against a variety of neurological disorders, including stoke. These studies have been controversial and depend largely on the type and timing of hormone replacement. Animal studies are much less controversial and clearly demonstrate a neuroprotective role for E2 following ischemic brain injury. Because much of E2 neuroprotection requires sex steroid hormone receptors, it is essential to understand expression patterns of these receptors. For the current studies, I evaluated estrogen receptor alpha (ER α), estrogen receptor beta (ER β) and androgen receptor (AR) expression in the cortex. It is known that these receptors change in expression at several times in an animal’s life span including during early postnatal development and following ischemic brain injury. Here I used an in vitro cortical explant model to further examine how these receptors change both during development and following injury. This in vitro model is important because it provides a way to investigate changes in receptor expression pattern in the cortex without input from other brain regions. In addition to characterizing this model, I also evaluated the contribution of E2 to changes in receptor expression and on cell death following injury in the explants. To begin to decipher mechanisms for E2 mediated neuroprotection, I added antagonist for each of the receptors before and after injury. In each these experiments, I also examined potential sex differences by separating the female and male brains before I cultured the explants. Overall, these experiments showed that cortical explants are a good in vitro model. Here we found that E2 was protective in female, but not male cortical explants following injury. However, the exact mechanisms of E2-mediated neuroprotection are still to be deciphered.

Human immunodeficiency virus type 1 (HIV-1) is known to cause a spectrum of neurological, behavioral and motor deficits collectively termed as HIV-1 associated neurocognitive impairments (HAND). Opiates augment HIV-related CNS complications through both direct and indirect mechanisms that disrupt glial and neuronal function. All CNS macroglia and neurons derive from neural progenitor cells (NPCs) during development, and NPCs in the adult brain contribute to repair processes. Since disruptions in NPC function are known to impact CNS populations and brain function in a number of disease/injury conditions, we determined whether HIV ± opiate exposure affected the maturation and fate of human NPCs (hNPCs). As hNPC infection by HIV has occasionally been reported, we also reexamined this question, and parsed between effects due directly to hNPC infection by HIV, or to hNPC dysfunction caused by the infective milieu. Multiple approaches confirmed the infection of hNPCs by R5 tropic (CCR5 utilizing) HIVBaL, and demonstrated that active infection could be sequentially transferred to naïve hNPCs. Exposure to supernatant from HIVBaL-infected cells (HIVsup) reduced hNPC proliferation and led to premature differentiation into astrocytes and neurons. Morphine co-exposure prolonged hNPC infection and exacerbated functional effects of HIVsup. Neither purified virions nor UV-inactivated HIVsup altered proliferation, indicating that this effect did not require infection. Gene array analysis and RT-qPCR with immunoblot validation suggested that Cdk5 signaling was involved in HIV-morphine interactions. siRNA-mediated knockdown of Cdk5 expression attenuated the effect of HIV-1 and morphine on hNPC proliferation and MAP2 differentiation, but also increased hNPC death. Furthermore, in an attempt to understand the role of mu-opioid receptor (MOR) splice variants on the interactive effect of HIV-1 and morphine on hNPCs, we found that both MOR-1 and MOR-1K are differentially regulated by HIV-1 in these cells. This suggests that these splice variants may have differential actions in the response of hNPCs to HIV-1 and morphine co-exposure. Given the overlap of Cdk5 and MOR signaling, it is likely that MOR-1K and/or MOR-1 converge with Cdk5 in the mechanism underlying HIV-1 and morphine interaction in hNPCs. Overall, dysregulation of hNPC functions by the infectious environment may create cell population imbalances that contribute to CNS deficits in both adult and pediatric patients. Additionally, infected hNPCs may pass virus to their progeny, and serve as an unappreciated viral reservoir. The recent epidemic of opiate/heroin abuse highlights the clinical importance of HIV and opiate interactions.

Ataxia telangiectasia (AT), caused by mutations in the gene encoding ataxia telangiectasia mutated kinase (ATM), is a rare autosomal recessive disorder. AT individuals exhibit neuronal degeneration and are predisposed to cancer. Carriers of this disorder are predisposed to cancer and ischemic heart disease. Heart disease, mostly due to myocardial infarction (MI), is a leading cause of death in the US. Following MI, release of catecholamines in the heart stimulates β- adrenergic receptors (β-AR). Our lab has shown that β-AR stimulation increases ATM expression in the heart and myocytes, and ATM plays an important role in β-AR-stimulated myocardial remodeling with effects on function, fibrosis and apoptosis. Using wild-type (WT) and ATM heterozygous knockout (hKO) mice, this study investigated the role of ATM in the inflammatory, proliferative and maturation phases of infarct healing post-MI. During the inflammatory phase, 1 and 3 days post-MI, a deficiency of ATM resulted in decreased left ventricular dilation as measured by echocardiography. It decreased the number of neutrophils and macrophages in the heart 1 day post-MI. Myocardial fibrosis, expression of alpha-smooth muscle actin (α-sma) and apoptosis were higher in the infarct region of ATM deficient hearts. Akt activation (anti-apoptotic) was lower, while Bax expression (pro-apoptotic) was higher in the infarct region of ATM deficient hearts. During the proliferative phase, 7 days post-MI, ATM deficiency attenuated cardiac dysfunction as measured by echocardiography. ATM deficient hearts exhibited increased fibrosis and expression of α-sma in the infarct region with increased myocyte apoptosis in the border area. During the maturation phase, 14 and 28 days post-MI, ATM deficiency resulted in exaggerated cardiac function. It associated with increased fibrosis, expression of α-sma and decreased cardiac cell apoptosis in the infarct region 28 days post-MI. Myocyte hypertrophy was greater in the non-infarct region during ATM deficiency. ATM deficiency decreased expression of p16 (marker of cell senescence) and activation of proapoptotic protein, GSK-3β. Thus, ATM modulates the remodeling processes of the heart including function, fibrosis, apoptosis and hypertrophy post-MI. ATM (1) delays the inflammatory response post-MI, (2) decreases dilative remodeling during inflammatory and proliferative phases and (3) exaggerates dysfunction during the maturation phase.

Erbin belongs to the LAP (leucine-rich repeat and PDZ domain) family of scaffolding proteins that play important roles in orchestrating cell signaling. Here, we show that Erbin functions as a tumor suppressor in colon cancer. Analysis of Erbin expression in patient specimens reveals that Erbin is downregulated at both mRNA and protein levels in tumor tissues. Functionally, knockdown of Erbin disrupts epithelial cell polarity and increases cell proliferation in 3D culture. In addition, silencing Erbin results in an increase in the amplitude and duration of signaling through Akt and RAS/RAF pathways. Moreover, Erbin-loss induces epithelial-mesenchymal transition (EMT), which coincides with a significant increase in cell migration and invasion. Erbin interacts with KSR1 and displaces it from the RAF/MEK/ERK complex to prevent signaling propagation. Furthermore, genetic deletion of Erbin in Apc knockout mice promotes tumorigenesis and significantly reduces survival. Tumor organoids derived from Erbin/Apc double knockout mice have increased tumor initiation potential along with increased Wnt target gene expression as seen by qPCR. Collectively, the studies within this dissertation identify Erbin as a negative regulator of EMT and tumor progression by directly suppressing Akt and RAS/RAF signaling in vivo.

The C type natriuretic peptide receptor (NPRC) also known as NPR3 is a widely expressed single transmembrane-spanning protein. NPRC functions as a homodimer at the cell surface for the metabolic clearance of a broad range of natriuretic peptides from circulation. The intracellular domain of NPRC is coupled to inhibitory G proteins and is involved in mediating signal transduction. In order to further elucidate the role of NPRC in signal transduction a proteomic approach was taken to identify putative protein binding partners for NPRC in different cell-types. An interrogation of the molecular association between NPRC and its identified protein binding partner(s) was carried out in different cell types to identify the specific interacting domains. The physiological role of the association between NPRC and its protein binding partner(s) were investigated in situ. Furthermore NPRC is subject to post translation modifications including glycosylation and phosphorylation. Although evidence suggests NPRC is phosphory ated on serine residues the specific amino acid residues that are phosphorylated and the kinases responsible for their phosphorylation has yet to be determined. A recombinant GST-NPRC fusion protein polyclonal NPRC antibody kinase prediction algorithm and several phosphospecific and substrate motif antibodies were utilized to characterize the phosphorylation state of NPRC in vitro.

The neuron-derived orphan receptor 1 (NOR1) belongs to the NR4A nuclear receptor subfamily. As an immediate early response gene, NOR1 is rapidly induced by a broad spectrum of physiological and pathological signals. Functional studies demonstrate NOR1 as a constitutively active ligand-independent nuclear receptor whose transcriptional activity is dependent on both expression level and posttranslational modifications. To date, an increasing number of studies have demonstrated a pivotal role of NOR1 in the transcriptional control of metabolism and the development of cardiovascular diseases. In this dissertation, we demonstrate NOR1 expression in endothelial cells and sub-endothelial cells of human atherosclerotic lesions. In response to inflammatory stimuli, NOR1 expression is rapidly induced in endothelial cells through an NF-κB-dependent signaling pathway. Functional studies reveal that NOR1 increases monocyte adhesion by inducing the expression of adhesion molecules VCAM-1 and ICAM-1 in endothelial cells. Transient transfection and chromatin immunoprecipitation assays identify VCAM-1 as a bona fide NOR1 target gene in endothelial cells. Finally, we demonstrate that NOR1-deficiency reduces hypercholesterolemia-induced atherosclerosis formation in apoE-/- mice by decreasing the macrophage content of the lesion. In smooth muscle cells (SMC), NOR1 was previously established as a cAMP response element binding protein (CREB) target gene in response to platelet-derived growth factor (PDGF) stimulation. CREB phosphorylation and subsequent binding of phosphorylated CREB to the NOR1 promoter play a critical role in inducing NOR1 expression. In this dissertation, we further demonstrate that histone deacetylase (HDAC) inhibition potentiates and sustains PDGF-induced NOR1 mRNA and protein expression in SMC. This augmented NOR1 expression is associated with increased phosphorylation of CREB, recruitment of phosphorylated CREB to the NOR1 promoter, and trans-activation of the NOR1 promoter. Additionally, HDAC inhibition also increases NOR1 protein half-life in SMC. Collectively, these findings identify a novel pathway in endothelial cells underlying monocyte adhesion and expand our knowledge of the epigenetic mechanisms orchestrating NOR1 expression in SMC. Finally, we establish a previously unrecognized atherogenic role of NOR1 in positively regulating monocyte recruitment to the vascular wall.