Thesis (Ph. D.)--York University, 2000. Graduate Programme in Biology. / Typescript. Includes bibliographical references (leaves 153-184). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://wwwlib.umi.com/cr/yorku/fullcit?pNQ67888.

Thesis (M. Sc.)--York University, 2001. / Typescript. Includes bibliographical references (leaves 69-89). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://wwwlib.umi.com/cr/yorku/fullcit?pMQ67745.

published_or_final_version / Paediatrics / Doctoral / Doctor of Philosophy

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

published_or_final_version / Surgery / Master / Master of Philosophy

published_or_final_version / Zoology / Doctoral / Doctor of Philosophy

A major challenge facing the tissue engineering discipline is cartilage tissue repair and engineering, because of the highly specialized structure and limited repair capacity that cartilage possesses. Dental pulp stem cells (DPSCs) were identified about a decade ago as a potential candidate for cell based therapy and tissue engineering applications. The present study aimed to utilize gene therapy with isolated DPSCs to induce chondrogenic transgene expression and chondrogenic lineage differentiation, with the ultimate goal of engineering cartilage tissue-like constructs. We isolated DPSCs from human teeth extracted for orthodontic treatment. We further enriched the isolated population using immunomagnetic bead selection, which increased stem cell markers: Stro-1 and CD146, compared to unselected population. The DPSCs showed the ability to differentiate into the chondrogenic lineage when induced with recombinant hTGFβ-3 and when transduced with hTGFβ-3 transgene. We successfully constructed the recombinant adeno-associated viral vector encoding the human TGFβ-3, and determined the best multiplicity of infection for DPSCs. The transduced DPSCs highly expressed hTGFβ-3 for up to 60 days. Expression of chondrogenic markers; Collagen IIa1, Sox9, and aggrecan was verified by immunohistochemistry and mRNA. We successfully fabricated an electrospun nano-fiber scaffold upon which morphology, proliferation and viability of the DPSCs were examined. DPSCs attached and proliferated on nano-fiber scaffolds demonstrating better viability compared to micro-fiber scaffolds. Transduced cells expressed hTGFβ-3 protein up to 48 days. Cells seeded on nanofiber scaffolds showed higher expression levels compared to micro-fiber scaffolds or culture plate. Scaffolds seeded with DPSCs were implanted in nude mice. Immunohistochemistry for TGFβ-3 DPSCs constructs (n=5/group) showed cartilage-like matrix formation with glucoseaminoglycans as shown by Alcian blue. Immunostaining showed positivity for Collagen IIa1, Sox9 and aggrecan. Semi-thin sections of the transduced DPSCs constructs examined by transmission electron microscopy (TEM) showed chondrocytic cellular and intra-cellular features, as well as extracellular matrix formation (n=2/group). In vivo constructs with the TGFβ-3 DPSCs showed higher collagen type II and Sox9 mRNA expression relative to non-transduced DPSCs constructs (n=5/group). Western blot analysis confirmed this expression pattern on the protein level (n=3/group). Engineered constructs mechanical properties were examined and compared to patellar bovine cartilage to assess functionality (n=5/group). TGFβ-3 transduced DPSCs constructs showed a higher equilibrium elastic modulus compared to nontransduced constructs. Micro-fiber scaffolds constructs showed a higher elastic modulus (0.11 MPa, 18% of bovine cartilage), compared to nano-fiber constructs modulus (0.032 MPa, 6% of bovine cartilage). Nano-fiber based constructs showed a similar Poisson‘s ration to bovine cartilage, while that of micro-fiber scaffolds was lower. As an alternative gene delivery method, electroporation parameters for DPSCs transfection were optimized, and compared to commonly used chemical transfection methods. TGFβ-3 transfected DPSCs showed a significantly higher relative TGFβ-3 mRNA and protein expression compared to non transfected control and to eGFP transfected DPSCs. Transfected DPSCs showed increased relative expression of chondrogenic markers; Collagen II, Sox9 and aggrecan, compared to non transfected DPSCs. Successful chondrogenic differentiation of DPSCs gene therapy with TGFβ-3 transgene, and seeding them on PLLA/PGA scaffolds makes it a potential candidate for cartilage tissue engineering and cell based therapy. / published_or_final_version / Dentistry / Doctoral / Doctor of Philosophy

FGF21 is a stress-inducible hormone predominantly secreted from the liver. FGF21 acts as a downstream target gene of hepatic transcription factor PPARα that plays an obligatory role in mediating metabolic adaptation responses to prolonged fasting. However, the physiological roles of FGF21 in regulating glucose homeostasis during adaptive starvation responses and its underlying mechanisms remain unknown. Furthermore, FGF21 is induced by both acute and chronic exercise training in both rodents and human. The physiological significances and the underlying mechanisms for exercise-induced FGF21 production have not been explored so far. Therefore, this study aims to investigate: 1) the mechanisms whereby FGF21 mediates the role of PPARα activation in modulating hepatic gluconeogenesis in response to prolonged fasting; 2) the physiological roles and mechanisms whereby FGF21 regulates exercise capacity and insulin sensitivity via its actions in the muscle during exercise; 3) the roles of FGF receptor-1 (FGFR1) and co-receptor βKlotho in mediating the metabolic effects of FGF21 during fasting and exercise. Our results show thatFGF21 is induced by fasting in the liver and enters into the brain. Both FGF21knockout (KO) mice and PPARα KO mice exhibit severe hypoglycemia and defective hepatic gluconeogenesis during prolonged fasting, and these changes are accompanied by impaired activation of the hypothalamic-pituitary-adrenal (HPA)axis and blunted release of corticosterone from adrenalgland. Moreover, intracerebroventricular injection of recombinant FGF21 reverses fasting hypoglycemia and impairment in hepatic gluconeogenesis by restoring corticosterone production in both FGF21 KO and PPARα KO mice. These effects are abrogated by blockage of hypothalamic FGFR1or by pharmacological inhibition of ERK1/2in the hypothalamus. In addition,FGF21 acts directly on the hypothalamic neurons to activate FGFR1/βKlotho-ERK1/2-CREBsignaling pathway, thereby leading to the transcriptional activation of corticotropin-releasing hormone (CRH) and subsequent activation of the HPA axis. FGF21 is also induced in the liver by exercise and FGF21 KO mice have decreased exercise capacity associated with lower mitochondrial content and thus lower insulin sensitivity compared to wildtype (WT)mice after exercise training.AMPK-PGC-1α signaling pathway is impaired in the muscle of FGF21 KO mice during exercise.FGF21treatmentincreasesmitochondrial content in myotubes, while inhibition of AMPK and knockdown of FGFR1, βKlotho and PGC-1αblocksthis effect. Moreover, FGF21 regulates genes and proteins involved in different steps of the autophagy process and these alterations are mediated by FGFR1/βKlotho-PGC-1α signaling cascade. Furthermore, replenishment of FGF21 reverses the reduced mitochondrial content and insulin sensitivity by restoring PGC-1α expression and subsequent increase in mitochondrial biogenesis and suppression of autophagy in the muscle of FGF21 KO mice. In conclusion, our results identify FGF21 as a critical hormonal regulator of glucose homeostasis during prolonged fasting, by coupling hepatic PPARα activation to corticosterone release via stimulation of the HPA axis in the brain. Moreover, FGF21 acts in the muscle through two distinctive mechanism to maintain mitochondrial homeostasis during exercise training, involving both biogenesis of new mitochondria and decreased clearance of stressed mitochondria and thus to mediate the beneficial effects of exercise on lipid and glucose metabolism. / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

Choroidal neovascularization (CNV) is a leading threat to severe vision loss, particularly in patients with age-related macular degeneration (AMD). In CNV, newly formed blood vessels sprout from the choroid to the sub-retinal space, where leakage and bleeding of the abnormal vessels lead to photoreceptor death and subsequent vision loss. It is believed that CNV is mediated by growth factors (e.g. vascular endothelial growth factor {VEGF}) produced by the retinal pigment epithelium (RPE) under pathological states (e.g. hypoxia). Current treatments for CNV aiming at countering VEGF only help decrease leakage and inhibit formation of CNV, but none of them is curative and the recurrence rate remains high. In order to find other more powerful potential therapeutic targets, the regulations of VEGF signaling in the pathophysiology of CNV is the focus of numerous translational investigations. Previously, Hypoxia-inducible factor-1 (HIF-1), a crucial transcriptional factor in response to hypoxia, is identified as the master transcriptional factor controlling VEGF expression in the RPE promoting CNV. Alpha-enolase (ENO1), a key glycolytic enzyme, is known to be over expressed in several types of carcinomas also under the regulation of HIF-1. ENO1 has been reported to be closely associated with cancer progression, angiogenesis, and venous invasion. The molecular events of ENO1 in the pathogenesis of promoting angiogenesis are of interest but still barely understood. Recently, the association of ENO1 antibodies with retina has been seen in patients with AMD. We hypothesize that ENO1 expression in the RPE may play a role in the development of CNV, participating in the regulation of VEGF. Hypoxia is an important pathological condition in the formation of CNV. Here, we first determined ENO1 expression and cell death in a human RPE cell line, ARPE-19, under cobalt (II) chloride (CoCl2)-induced hypoxia or anoxia (95% N2, 5% CO2). To further investigate the regulation of ENO1 in CNV, HIF-1α-diminished RPE cells were generated using small interfering RNA (siRNA) and the change of ENO1 expression in response to hypoxic injury was determined. Upon 24 hr of treatment with CoCl2-induced hypoxia or anoxia, the expression of ENO1 and VEGF increased significantly along with HIF-1α in ARPE-19 cells, both of which could in turn be significantly down-regulated by HIF-1α siRNA. Interestingly, cell death remained low in ARPE-19 cells, even after 24 hr of CoCl2-induced hypoxia or anoxia. To further study the role of ENO1 in CNV, we started by investigating the relationship between ENO1 and VEGF. SiRNA was used to knock down the expression of ENO1 in ARPE-19 cells. Upon transfection with the siRNA, ENO1 expression was successfully down-regulated when treated with CoCl2-induced hypoxia. However, VEGF secretions from the ENO1-diminished ARPE-19 cells under CoCl2-induced hypoxia remained unchanged. Double knockdown of ENO1 together with HIF-1α by siRNA also did not help to further suppress VEGF secretion in the hypoxic ARPE-19 cells. Hence, ENO1 was demonstrated to be activated and up-regulated by HIF-1 in RPE cells responding to hypoxia, suggesting a potential role of ENO1 in favoring the formation of CNV, but not through influencing VEGF secretion. / published_or_final_version / Ophthalmology / Master / Master of Philosophy