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FOXO3a is a pro-apoptotic transcription factor which shows increased activation in vascular smooth muscle cells (VSMCs) of advanced atherosclerotic plaques, specifically within the intimal layer. Since VSMC apoptosis plays a crucial role in the pathophysiology of atherosclerosis, we investigated the mechanisms underlying FOXO3a-mediated cell death in this particular cell type. We aimed to characterise a novel VSMC system (FOXO3aA3ERTM) and use these cells to validate MMP-13 and TIMP3 as new FOXO3a target genes. Also, we sought to determine the mechanisms of FOXO3aA3ERTM-mediated VSMC apoptosis, particularly regarding MMP-13 and TIMP3, potential MMP-13 substrates in the extracellular matrix and the precise apoptotic signalling involved. Furthermore, we aimed to investigate whether VSMC-specific activation of FOXO3aA3ERTM in mouse affects vascular remodelling during injury and whether this is reliant on MMP-13. Lastly, we aimed to address if endogenous FOXO3a upregulates MMP-13 in mouse and human VSMCs. Our laboratory has created a transgenic rat VSMC line which stably expresses an inducible FOXO3a mutant allele known as FOXO3aA3ERTM and previous microarray experiments identified matrix metalloproteinase 13 (MMP-13) as a potential novel FOXO3a target gene. Initially, we described several key features of the FOXO3aA3ERTM VSMCs used throughout this thesis, and subsequently demonstrated that MMP-13 is a bona fide target whose expression is rapidly upregulated upon FOXO3a activation, leading to markedly higher levels of protein, cleavage and proteolytic capacity. This induction of MMP-13 was responsible for the vast majority of FOXO3a-mediated apoptosis which was accompanied by prominent degradation of fibronectin, a glycoprotein found in the extracellular matrix. However, we could not identify a terminal apoptotic pathway. FOXO3a also downregulated the endogenous MMP inhibitor TIMP3, the recombinant protein of which reduced both MMP-13 proteolysis and FOXO3a-mediated apoptosis. Activation of FOXO3aA3ERTM in the VSMCs of medium and large arteries in mice resulted in heightened expression of MMP-13 in the vessel wall, which contributed to enhanced neointimal formation during carotid ligation. Finally, endogenous FOXO3a activation leads to increased MMP-13 expression in human VSMCs, but not mouse. Overall, we have shown that FOXO3a promotes VSMC apoptosis through MMP-13 both in vitro and in vivo, a novel pathway that has important implications for the pathogenesis and treatment of vascular disease.

The global population is ageing. This is a public health priority as the social and economic challenges are profound. Healthy ageing often associates with exceptional longevity (EL) and centenarians can be seen as exemplars of healthy ageing. Studying centenarians may reveal the underlying mechanisms of the healthy ageing process. Prior heritability studies suggest that genetic, epigenetic, and environmental factors are important in longevity. Therefore, this dissertation aimed to unravel the genetic and epigenetic determinants of EL. Initially, a meta-analysis determined five polymorphisms, ACE rs4340, APOE e2/3/4, FOXO3A rs2802292, KLOTHO KL-VS and IL6 rs1800795, consistently associated with EL, with the pooled effect sizes (odds ratios) ranging from 0.42 (APOE e4) to 1.45 (FOXO3A males). Interestingly, these variants can be linked to pathways that relate to cardiovascular health and metabolic syndrome (MetS). Subsequently, polygenic risk scores (PRSs) were constructed for EL and eight cardiovascular-related traits. These were assessed in exceptionally long-lived individuals (95-106 years) and compared to younger controls (55-65 years). As expected, higher genetic risk for EL was significantly associated with longevity. Only two cardiovascular health PRSs were nominally significant with longevity (HDL cholesterol, triglycerides), with higher PRS associated with EL. A further analysis in 5 cohorts (40-93 years), tested the hypothesis that a high PRS for EL associated with a healthy metabolic profile. In the largest cohort, high EL polygenic risk was associated with lower MetS prevalence and its components. Meta-analyses of replication cohorts showed nominal associations with MetS and 3 components. Prior epigenetic studies of MetS show that there are differentially methylated sites and genes associated with MetS. Two methylation risk scores (MRS) were constructed for MetS based on previous research. As expected, the MetS MRSs associated with MetS in three Australian cohorts, aged 65-104 years. An individual with a high MetS MRS had an increased likelihood (~1.4-3.14) of developing MetS compared to an individual with a lower MetS MRS using methylation data from two platforms. No association was observed between EL and the MetS MRSs. In conclusion, this work drawing from genetic and epigenetic evidence, suggests that cardiovascular and metabolic health is a distinguishing characteristic of attaining EL.

Diabetes is a devastating public health problem affecting millions around the globe. One of the populations most vulnerable to the complications related to this disorder are pregnant women. Diabetes causes miscarriage in 1 of 5 pregnancies and major birth defects in another 10%, statistics that are truly frightening considering that this disorder has reached near pandemic levels in many countries around the world. Though high blood glucose levels have been isolated as the major cause of pregnancy problems among diabetics, the mechanisms by which hyperglycemia threaten the life and health of human embryos still remain to be elucidated. Using embryonic stem cells as a model for pre-implantation embryos, we have begun to examine the effects of hyperglycemia on early stages of pregnancy. By culturing the cells in different glucose concentrations and tracking changes in pluripotency state, cell cycle regulation, and oxidative stress management, we hope to gain further insights into what may be happening during these critical stages of development when the plan for life is being laid out. Specifically, we have examined changes in the expression and localization of CTNNB1 and other proteins it is known to interact with, as CTNNB1 is one of the major regulators of human embryonic development. Specifically, we have determined that hyperglycemia increases levels of nuclear CTNNB1 as well as nuclear levels of the pluripotency inhibitor TCF7L1 and the oxidative stress regulator FoxO3a. Upstream of these transcription factors, we have found that JNK, activated in response to oxidative stress, may be responsible for their increased accumulation in the nucleus. In response to misregulation of these transcription factors that are critical for proper development, embryonic stem cells increase proliferation while initiating the process of differentiation, a possible mechanism for ensuring that these abnormal cells do not contribute to future generation of body tissues. This abnormal differentiation may contribute to gastrulation defects that harm the embryo in such a way that it cannot survive or sustains a major structural defect, thus contributing to diabetic pregnancy complications. Through uncovering mechanisms related to proper maintenance of the stem state and differentiation, we hope to provide knowledge that will inform future treatments to protect diabetic mothers and their unborn children.

Triple negative breast cancer (TNBC) is a highly aggressive cancer, which is known for a high rate of metastasis and poor prognosis. TNBC cells are hypothesized to undergo the cellular processes epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET). The known prognostic markers CD44+ and Ki67 as well as the transcription factor and proposed tumor suppressor FoxO3a are thought to be indicators of EMT and MET activity in cancer cells. In order to better understand these processes an effective cell culture technique to mimic the tumor environment needs to be established. The purpose of this study was to determine if changing the cell culture environment would impact the expression of CD44+, Ki67 and FoxO3a in TNBC cells. The three TNBC cell lines BT-549, MDA-MB-157, and MDA-MB-231 were cultured in either a monolayer (2D) environment or in Matrigel (3D). FoxO3a was knocked down using siRNA to in MDA-MB-231 cells. The cells were immunofluorescently stained to measure the intensity of CD44+, KI67, and FoxO3a expression in both the 2D and 3D cell culture environment. Proliferation and wound healing assays were performed to determine the proliferation and migration of MDA-MB-231 cells. The results indicate the expression of FoxO3a had significantly increased (P

Recent research suggests that [beta]-hydroxy-[beta]-methylbutyrate (HMB), a metabolite of leucine (Leu), increases muscle mass and attenuates muscle damage during resistance training. Although Leu acts as a potent stimulator of protein synthesis, HMB, but not Leu, has been reported to be effective in suppressing proteolysis in skeletal muscle. However, mechanisms for the effects of HMB on cell signaling pathways controlling muscle protein turnover during rest and after endurance exercise are still poorly understood. Furthermore, the effects of HMB on cell signaling pathways controlling protein synthesis and degradation under normal in vivo conditions warrant further investigation. For optimal gains in muscle mass, the appropriate type and amount of protein (PRO) is required for positive protein balance to occur in skeletal muscle. Therefore, this dissertation was designed to determine the effect of HMB, PRO and Leu, individually and in combination, on the regulation of cellular signaling pathways controlling muscle protein turnover during sedentary and post-exercise conditions. Study 1 demonstrated that, compared with HMB and PRO alone, the combination of HMB and PRO was more effective in activating the mTOR signaling pathway, which controls protein synthesis, and inhibiting FOXO3A, a major regulator of the ubiquitin-proteasome proteolytic signaling pathway. Study 2 demonstrated that, compared with its individual components, a novel HMB/PRO/Leu supplement better activated protein-synthetic signals and inhibited proteolytic signals in skeletal muscle, and these effects were better sustained. Finally, Study 3 demonstrated that adding Leu to PRO-enriched mixtures after exercise additively activated protein-synthetic signals in a fiber type-specific manner, and adding HMB clearly inhibited proteolytic signaling proteins. Furthermore, provision of an HMB/PRO/Leu supplement after exercise was found to favorably modulate signaling pathways controlling both protein synthesis and degradation. Taken together, the results of these studies suggest that a novel nutrient supplement, composed of HMB, Leu and PRO, additively enhances the intracellular signaling proteins controlling protein synthesis and attenuates signaling proteins controlling proteolysis in skeletal muscle during sedentary and post-exercise recovery. Therefore, such a supplement may be beneficial for both athletic and therapeutic purposes.

A comprehensive understanding of the molecular signaling pathways that regulate cell growth and proliferation is essential in the realization of new therapeutic options to facilitate early detection and eradication of malignancy. Understanding the transcriptional regulation of the YPEL3 and FHIT genes forms the basis of this dissertation. YPEL3, or Yippee-like 3, is a newly identified p53 target gene that inhibits tumor cell growth and is thus itself, a novel tumor suppressor gene. FHIT, or Fragile histidine triad, is a well known tumor suppressor gene and is regulated at the transcriptional level by another growth inhibitory protein, FOXO3a, a Forkhead box transcription factor.Our laboratory has determined that YPEL3 is a direct transcriptional target of the tumor suppressor gene p53. The first section of this dissertation provides significant experimental evidence to validate this observation. Briefly, YPEL3 was shown to be upregulated downstream of p53 protein stabilization in a microarray screen that explored global gene expression modulation after RNAi-mediated reduction of p53's negative regulators, Hdm2 and HdmX. Genotoxic stress induced by treatment with DNA-damaging agents resulted in stabilization of p53 protein along with elevation of YPEL3 transcript and protein levels. Moreover, there exists a cis-acting p53 response element within the YPEL3 promoter that is bound by p53 in response to this stress. YPEL3 also elicits growth inhibition and decreases in colony formation when expressed in tumor cells. It is apparent that cells which express exogenous YPEL3 are forced into a permanent cell cycle arrest, termed “premature senescence.” Similar growth suppressive phenotypes are typical of many other known p53 target genes. However, most of these genes are associated with the regulation of transient inhibition of cell division or the induction of apoptosis. YPEL3 is unique in its ability to trigger premature cellular senescence. Indeed, YPEL3 stands out among many other p53 targets because it is among the first to play a role in this process.To further understand the mechanism of YPEL3-induced senescence, the second portion of this dissertation focuses on the generation of a three-dimensional model of YPEL3's protein structure. It was hypothesized that predicting YPEL3's protein structure may also aid in understanding the molecular events involved in the induction and maintenance of premature senescence. Identifying structural homology between a predicted model of YPEL3 and other known structures may provide new insights into this process. This would especially be the case if homology existed between the predicted structure of YPEL3 and other proteins that have established functions in stress-responsive or senescence-associated molecular pathways. By using two independent structural prediction algorithms (Rosetta ab initio and I-TASSER), I have been able to estimate a three-dimensional model of the YPEL3 protein which has significant structural homology to a family of Methionine oxidoreductases. These enzymes catalyze redox-mediated antioxidant reactions which dissipate intracellular reactive oxygen species (ROS) through the repair of proteins damaged by ROX-mediated methionine to methionine sulfoxide oxidation. It is even plausible that YPEL3 could be involved in the mediation of cellular senescence in response to severe oxidative stress. If this is the case, the implications of this speculation, linking the oxidative stress response to YPEL3-dependent cellular senescence, are indeed profound. YPEL3 may not only play a role in the molecular biology of cancer but also may be involved in the cellular oxidative stress response as it relates to senescence and aging.In the third part of this dissertation, FHIT, a tumor suppressor gene often deleted in epithelial-derived tumors, was also initially postulated to be regulated by p53 (just as in the case of YPEL3). However, FHIT gene expression was instead found to be modulated by FOXO3a. This gene’s expression was induced coincidentally under similar conditions as p53 such as cell cycle arrest. FHIT is suppressed by the AKT/PI3K pathway upon growth factor-dependent stimulation. These protooncogenic kinases phosphorylate and, thereby prevent FOXO3a from entering the nucleus and activating FHIT gene expression. Thus, FOXO3a is only able to enter the nucleus in the absence of mitogenic stimulation. FHIT transcript levels are elevated in response to the removal of mitogen-containing serum from breast, colon carcinoma, and immortalized mammary epithelial cells. Induction of FHIT gene expression is dependent on decreased PI3K kinase activity and subsequent reduced AKT phosphorylation which occur in the absence of growth factor stimulation. As such, quiescence-dependent elevation of FHIT mRNA can be elicited independently of mitogen removal, by treatment with PI3K inhibitors, or attenuated by ectopic expression of constitutively active AKT during serum deprivation. Furthermore, ectopic expression of FOXO transcription factors result in the upregulation of FHIT mRNA and protein levels. Additionally, nuclear shuttling experiments with a FOXO3a-estrogen receptor fusion protein have demonstrated that FOXO must enter the nucleus to modulate expression of FHIT. Finally, experiments using shRNA targeting FOXO3a have revealed that the modulation of FHIT gene expression, downstream of PI3K inactivation, is dependent on endogenous FOXO3a.In summary, this project began with investigating the regulation of two genes derived from a microarray screen to search for new p53 targets. Validation of YPEL3 as a novel p53 target gene was very straightforward. All assays employed in characterizing YPEL3 revealed that it behaved exactly as a classic p53 transcriptional target. The latter of the two genes, FHIT, however was determined not to be a p53 target but rather a transcriptional target of FOXO3a – another tumor suppressive transcription factor. It is the hope of the author that this information will serve to enhance our basic understanding of the paradoxically both, overwhelmingly complex and elegantly simple molecular dynamics of gene regulation, as it pertains to the biology of cancer.