Dissertation (Ph.D.)--University of South Florida, 2007. / Title from PDF of title page. Document formatted into pages; contains 168 pages. Includes vita. Includes bibliographical references.

Dissertation (Ph.D.)--University of South Florida, 2007. / Includes vita. Includes bibliographical references. Also available online.

Thesis (M.Sc.)--Memorial University of Newfoundland, Faculty of Medicine, 1999. / Typescript. Bibliography: leaves 145-163.

Thesis (M.S.)--University of Michigan, 2003. / Includes bibliographical references.

by Yeung Hiu Yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 158-171). / Abstracts in English and Chinese. / ACKNOWLEDGEMENT --- p.i / ABBREVIATIONS --- p.ii / ABSTRACT (English & Chinese) --- p.iii / TABLE OF CONTENT --- p.viii / INDEX FOR FIGURES --- p.xii / INDEX FOR TABLES --- p.xvi / Chapter 1. --- INTRODUCTION --- p.2 / Chapter 1.1. --- History of Distraction Osteogenesis --- p.3 / Chapter 1.2. --- Clinical Application of Distraction Osteogenesis --- p.5 / Chapter 1.2.1. --- Limb-Lengthening --- p.5 / Chapter 1.2.2. --- Correction of Deformities and Non-Unions --- p.5 / Chapter 1.2.3. --- Bone Transport --- p.6 / Chapter 1.2.4. --- Reconstruction of the mandible --- p.7 / Chapter 1.3. --- Bone-specific Alkaline Phosphatase (BALP) --- p.8 / Chapter 1.4. --- Osteocalcin --- p.9 / Chapter 1.5. --- Bone Growth Factors --- p.11 / Chapter 1.6. --- Fibroblast Growth Factors (FGFs) --- p.12 / Chapter 1.6.1. --- Acidic Fibroblast Growth Factor (aFGF) --- p.13 / Chapter 1.6.2. --- Basic Fibroblast Growth Factor (bFGF) --- p.14 / Chapter 1.7. --- Transforming Growth Factor-pi (TGF-β1) --- p.16 / Chapter 1.8. --- Fracture Healing --- p.18 / Chapter 1.8.1. --- Histology --- p.18 / Chapter 1.8.2. --- Growth Factor Expression --- p.18 / Chapter 1.9. --- Distraction Osteogenesis --- p.19 / Chapter 1.9.1. --- Histology --- p.19 / Chapter 1.9.2. --- Growth Factor Expression --- p.20 / Chapter 1.10. --- Aim of the Study --- p.21 / Chapter 2. --- METHODOLOGY --- p.23 / Chapter 2.1. --- Animal Model --- p.23 / Chapter 2.1.1. --- Source of Animal --- p.23 / Chapter 2.1.2. --- Animal Operation --- p.23 / Chapter 2.1.3. --- Fracture Healing Model --- p.24 / Chapter 2.1.4. --- Distraction Osteogenesis Model --- p.24 / Chapter 2.2. --- Sample Collection --- p.25 / Chapter 2.2.1. --- Tissue Sample Collection and Preparation --- p.25 / Chapter 2.2.1.1. --- Test for the Complete Decalcification of the Calluses --- p.26 / Chapter 2.2.2. --- Blood Sample Collection and Storage --- p.26 / Chapter 2.3. --- Bone Mineral Density Measurement of the Distracted Callus and the Fracture Callus --- p.27 / Chapter 2.3.1. --- Fracture Healing Group --- p.27 / Chapter 2.3.2. --- Distraction Osteogenesis Group --- p.28 / Chapter 2.4. --- Serum Bone Specific Alkaline Phosphatase (BALP) Activity --- p.28 / Chapter 2.4.1. --- Wheat Germ Lectin (WGL) Precipitation of BALP --- p.28 / Chapter 2.4.1.1. --- Reagent --- p.28 / Chapter 2.4.1.2. --- Preparation and Measurement of Samples --- p.29 / Chapter 2.4.1.3. --- Auto-analyzer Setup --- p.30 / Chapter 2.5. --- Quantification of the Osteocalcin in Serum --- p.30 / Chapter 2.5.1. --- Reagent and Sample Preparation --- p.31 / Chapter 2.5.2. --- Detection Procedures --- p.31 / Chapter 2.6. --- Localization of the Growth Factors in Distraction Osteogenesis and Fracture Healing --- p.32 / Chapter 2.6.1. --- Immunohistochemistry of the Growth Factors --- p.33 / Chapter 2.6.1.1. --- Reagents and Solution Preparation --- p.33 / Chapter 2.6.1.2. --- Experimental Procedure --- p.36 / Chapter 2.6.1.3. --- Evaluation of Immunohistochmical Staining Results --- p.37 / Chapter 2.6.2. --- Verification of the Primary Antibody Used in the Study --- p.37 / Chapter 2.6.2.1. --- Tissue Preparation --- p.37 / Chapter 2.6.2.2. --- Antibody to Acidic Fibroblast Growth Factor (aFGF) --- p.38 / Chapter 2.6.2.2.1. --- Immunohistochemistry of Goat Brain and Growth Plate --- p.38 / Chapter 2.6.2.2.2. --- Dot Blot --- p.38 / Chapter 2.6.2.2.2.1. --- Materials and Reagents --- p.38 / Chapter 2.6.2.2.2.2. --- Procedures --- p.39 / Chapter 2.6.2.2.3. --- Sodium Dodecylsulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.41 / Chapter 2.6.2.2.3.1. --- Materials and Reagents --- p.41 / Chapter 2.6.2.2.3.2. --- Procedures --- p.42 / Chapter 2.6.2.2.4. --- Western Blotting --- p.43 / Chapter 2.6.2.2.4.1. --- Materials and Reagents --- p.43 / Chapter 2.6.2.2.4.2. --- Procedures --- p.44 / Chapter 2.6.2.3. --- Antibody to Basic Fibroblast Growth Factor --- p.45 / Chapter 2.6.2.4. --- Antibody to Transforming Growth Factor-β1 --- p.45 / Chapter 3. --- RESULTS --- p.53 / Chapter 3.1. --- Animal Model --- p.53 / Chapter 3.1.1. --- Fracture Healing Animal Model --- p.53 / Chapter 3.1.1.1. --- Radiography of Fracture Healing --- p.53 / Chapter 3.1.2. --- Distraction Osteogenesis Animal Model --- p.54 / Chapter 3.1.2.1. --- Gross Morphology of Distraction Osteogenesis --- p.54 / Chapter 3.1.2.2. --- Radiography of Distraction Osteogenesis --- p.55 / Chapter 3.2. --- Bone Mineral Density (BMD) Measurement --- p.56 / Chapter 3.2.1. --- In Fracture Healing --- p.56 / Chapter 3.2.2. --- Distraction Osteogenesis --- p.57 / Chapter 3.3. --- Bone-specific Alkaline Phosphatase Activity in Goat Serum --- p.59 / Chapter 3.3.1 --- ", Fracture Healing" --- p.59 / Chapter 3.3.2. --- Distraction Osteogenesis --- p.59 / Chapter 3.4. --- Serum Osteocalcin Measurement --- p.60 / Chapter 3.4.1. --- Fracture Healing --- p.60 / Chapter 3.4.2. --- Distraction Osteogenesis --- p.60 / Chapter 3.5. --- Histology --- p.61 / Chapter 3.5.1. --- Fracture Healing --- p.61 / Chapter 3.5.2. --- Distraction Osteogenesis --- p.64 / Chapter 3.6. --- Verification of Primary Antibody Used in the Study --- p.67 / Chapter 3.6.1. --- Antibody to Acidic Fibroblast Growth Factor --- p.67 / Chapter 3.6.1.1. --- Dot Blot --- p.67 / Chapter 3.6.1.2. --- Western Blotting --- p.68 / Chapter 3.6.1.3. --- Immunohistochemistry of Goat Brain and Growth Plate --- p.68 / Chapter 3.6.2. --- Antibody to Basic Fibroblast Growth Factor --- p.69 / Chapter 3.6.2.1. --- Dot Blot --- p.69 / Chapter 3.6.2.2. --- Immunohistochemistry of Goat Brain and Growth Plate --- p.69 / Chapter 3.6.3. --- Antibody to Transforming Growth Factor-β1 --- p.70 / Chapter 3.6.3.1. --- Western Blotting --- p.70 / Chapter 3.6.3.2. --- Immunohistochemistry of Growth Plate --- p.70 / Chapter 3.7. --- Localization of Growth Factors in Fracture Healing and Distraction Osteogenesis --- p.70 / Chapter 3.7.1. --- Acidic Fibroblast Growth Factor --- p.71 / Chapter 3.7.1.1. --- Fracture Healing --- p.71 / Chapter 3.7.1.2. --- Distraction Osteogenesis --- p.72 / Chapter 3.7.2. --- Basic Fibroblast Growth Factor --- p.73 / Chapter 3.7.2.1. --- Fracture Healing --- p.73 / Chapter 3.7.2.2. --- Distraction Osteogenesis --- p.74 / Chapter 3.7.3. --- Transforming Growth Factor-β1 --- p.75 / Chapter 3.7.3.1. --- Fracture Healing --- p.75 / Chapter 3.7.3.2. --- Distraction Osteogenesis --- p.76 / Chapter 4. --- DISCUSSION --- p.142 / Chapter 4.1. --- The Biochemical Events in Fracture Healing --- p.142 / Chapter 4.2. --- The Biochemical Events in Distraction Osteogenesis --- p.147 / Chapter 4.3. --- Limitations of the present study --- p.153 / Chapter 4.4. --- Future Study --- p.154 / Chapter 5. --- CONCLUSION --- p.156 / BIBLIOGRAPHY --- p.158

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

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Cartilage development relies on the coordinated presentation of biological signals to direct chondrocyte morphology and function. This is largely controlled by perlecan, a heparan sulfate proteoglycan (HSPG). Understanding the role of perlecan and its pendant glycosaminoglycan chains (GAG) in cartilage development is essential for advances in tissue engineered cartilage replacement strategies. Perlecan was immunolocalised to the pericellular matrix of prehypertrophic and hypertrophic chondrocytes in human fetal feet. Human fetal chondrocytes were isolated and cultured in 3-dimensional (3D) scaffolds for a period of 4 weeks. Their chondrogenic phenotype, based on extracellular matrix (ECM) components, was assessed and compared to 2D cultures. Chondrocyte perlecan was immunopurified from human fetal chondrocytes grown in vitro and fetal cartilage tissue and characterised using a combination of antibody-based techniques (ELISA, Western blotting) and gel electrophoresis. The biological function of chondrocyte perlecan was determined by its ability to form ternary complexes with fibroblast growth factors (FGF) and their receptors (FGFR) using an antibody-based technique as well as a cell proliferation assay using cells expressing FGFR isotypes. Perelcan was restricted to the prehypertrophic and hypertrophic zones of cartilage. This zonal organisation of chondrocytes and chondrogenic properties, determined by their morphology and PG deposition, was recapitulated in the 3D constructs while 2D cultures displayed dedifferentiated chondrocytes. Exogenous FGF2 promoted chondrocyte proliferation, while FGF18 stimulated the synthesis of perlecan, reflecting chondrocyte hypertrophy. Chondrocyte perlecan (630kDa) contained HS, chondroitin sulfate (CS) and keratan sulfate (KS) chains. Chondrocyte perlecan formed HS dependent ternary complexes with FGF2-FGFR1c and FGF18-FGFR3c, while FGF18-FGFR3c binding to perlecan protein core was also observed. Binding of FGF18-FGFR3c to chondrocyte perlecan HS was more promiscuous than FGF2-FGFR1c. Furthermore, chondrocyte perlecan HS mediated biological activity with FGF18 via FGFR3c, which was modulated by mammalian heparanase, while no biological activity was elicited by FGF2-FGFR1c. The findings underline how perlecan and its GAGs interact with FGF and FGFR in a spatio-temporal manner to promote signalling, effecting chondrocyte behaviour and morphology in cartilage development. This insight can be utilised in tissue engineering to improve the development of biologically functional cartilage replacements.

Cartilage development relies on the coordinated presentation of biological signals to direct chondrocyte morphology and function. This is largely controlled by perlecan, a heparan sulfate proteoglycan (HSPG). Understanding the role of perlecan and its pendant glycosaminoglycan chains (GAG) in cartilage development is essential for advances in tissue engineered cartilage replacement strategies. Perlecan was immunolocalised to the pericellular matrix of prehypertrophic and hypertrophic chondrocytes in human fetal feet. Human fetal chondrocytes were isolated and cultured in 3-dimensional (3D) scaffolds for a period of 4 weeks. Their chondrogenic phenotype, based on extracellular matrix (ECM) components, was assessed and compared to 2D cultures. Chondrocyte perlecan was immunopurified from human fetal chondrocytes grown in vitro and fetal cartilage tissue and characterised using a combination of antibody-based techniques (ELISA, Western blotting) and gel electrophoresis. The biological function of chondrocyte perlecan was determined by its ability to form ternary complexes with fibroblast growth factors (FGF) and their receptors (FGFR) using an antibody-based technique as well as a cell proliferation assay using cells expressing FGFR isotypes. Perelcan was restricted to the prehypertrophic and hypertrophic zones of cartilage. This zonal organisation of chondrocytes and chondrogenic properties, determined by their morphology and PG deposition, was recapitulated in the 3D constructs while 2D cultures displayed dedifferentiated chondrocytes. Exogenous FGF2 promoted chondrocyte proliferation, while FGF18 stimulated the synthesis of perlecan, reflecting chondrocyte hypertrophy. Chondrocyte perlecan (630kDa) contained HS, chondroitin sulfate (CS) and keratan sulfate (KS) chains. Chondrocyte perlecan formed HS dependent ternary complexes with FGF2-FGFR1c and FGF18-FGFR3c, while FGF18-FGFR3c binding to perlecan protein core was also observed. Binding of FGF18-FGFR3c to chondrocyte perlecan HS was more promiscuous than FGF2-FGFR1c. Furthermore, chondrocyte perlecan HS mediated biological activity with FGF18 via FGFR3c, which was modulated by mammalian heparanase, while no biological activity was elicited by FGF2-FGFR1c. The findings underline how perlecan and its GAGs interact with FGF and FGFR in a spatio-temporal manner to promote signalling, effecting chondrocyte behaviour and morphology in cartilage development. This insight can be utilised in tissue engineering to improve the development of biologically functional cartilage replacements.