Thesis (M. Phil.)--University of Hong Kong, 2002. / Includes bibliographical references (leaves 146-161).

This study aimed to characterize the in-situ mechanical property and morphology of individual collagen fibril in osteoarthritic (OA) cartilage using indentation-type atomic force microscopy (IT-AFM). The specimens with intact articular cartilage (AC), mild to severe degenerated OA cartilage were collected with informed consent from the postmenopausal women who underwent hip or knee arthroplasty. The fresh specimens were cryo-sectioned by layers with 50m thick for each from the articular surface to calcified cartilage, and then processed for AFM imaging and nanoindentation test. For each layer, a total of twenty collagen fibrils were randomly selected for testing. AFM tips with the nominal radius less than 10 nm were employed for probing the individual collagen fibril, and the obtained cantilever deflection signal and displacement were recorded for calculating its elastic modulus. Besides AFM nanoindentation, AFM and scanning electron microscopy (SEM) images, haematoxylin & eosin (H&E) staining and micro-indentation were performed on AC to study the changes of ultrastructure and composition between intact AC and OA cartilage. Results showed that an intact AC exhibited a gradation in elastic modulus of collagen fibrils from surface region (2.65±0.31GPa) to bottom region (3.70±0.44GPa). It was noted in the initial stage of OA cartilage that the coefficient of variation for mechanical properties of collagen fibers, ranging from 25~48%, significantly increased as compared with intact one (12%). The thickened and stiffened collagen fibrils initially occurred at either surface region (3.11±0.91GPa) or bottom region (5.64±1.10GPa) with OA progression. Besides thickens, alteration of D-periodic banding patterns of collagen fibrils was observed. It was echoed by fibrotic changes of surface region and tidemark irregularities. On the contrast, the micromechanical properties of cartilage decreased while AC suffered from OA. This result revealed the different approachs of nano and micro-mechanical properties changes in AC. In summary, the alteration of mechanical properties of collagen fibrils started from calcified cartilage as well as articular surface during OA onset, and the low compliance of thickened collagen fibrils deteriorated along disease progression. This study also reveals that the outstanding ability by AFM, in investigating the structure and mechanical properties of collagen fibrils and AC in nanometer scale, is impressive and this nanotechnological instrument is worth to be expected in further development for clinical use. / published_or_final_version / Mechanical Engineering / Master / Master of Philosophy

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

Despite advances in investigating functional aspects of osteoblast (OB) differentiation, especially studies on how bone proteins are deposited and mineralized, there has been little research on the intracellular trafficking of bone proteins during OB differentiation. Collagen synthesis and secretion is the major function of OBs and is markedly upregulated upon ascorbic acid (AA) stimulation, significantly more so than in fibroblast cells. Understanding the mechanism by which collagen is mobilized in specialized OB cells is important for both basic cell biology and bone disease studies. Cellular organelles and vesicles in the exocytic and endocytic pathways have a distinctive spatial distribution and their trafficking is aided by many molecules, Rab GTPases being a master regulator. In this work, I identified the Rab GTPases that are upregulated during OB differentiation using microarray analysis, namely Rab1, Rab3d, and Rab27b, and investigated their role in regulating the trafficking of collagen from the site of synthesis in the ER to the Golgi and ultimately to the plasma membrane (PM) utilizing their dominant negative (DN) expression. The experimental halting of biosynthetic trafficking by these mutant Rabs initiated proteasome-mediated degradation of procollagen and ceased global protein translation. Acute expression of Rab1 and Rab3d DN constructs resulted in impaired ER to Golgi trafficking of procollagen. Similar expression of Rab27b DN constructs resulted in dispersed collagen vesicles which may represent failed secretory vesicles sequestered in the cytosol. A significant and strong reduction in extracellular collagen levels also was observed showing roles of Rab1, Rab3d and Rab27b in the specific function of these major collagen producing cells in the body. I further observed that a fraction of procollagen colocalized with lysosomes which was markedly increased when procollagen was experimentally misfolded. Lysosomes, essential organelles for intracellular degradation, are generally sequestered near the cell centre to receive vesicles with contents targeted for destruction. During AA-induced differentiation of OB cells, I saw a marked increase in total degradative lysosome organelles in addition to an enhanced endocytic rate. Interestingly, lysosomes were dispersed toward the cell periphery in differentiating OBs without being secreted. This required intact microtubules for long range transport and was kinesin motor-dependent but did not involve cytosolic acidification. Moreover, impairment of lysosome dispersion markedly reduced AA-induced OB differentiation. Taken together, this study provides an important general mechanism for cell secretion phenomena that may ultimately lead to clinical targets for treatments of diseases driven by aberrant collagen processing and secretion including Osteogenesis Imperfecta (OI).

Despite advances in investigating functional aspects of osteoblast (OB) differentiation, especially studies on how bone proteins are deposited and mineralized, there has been little research on the intracellular trafficking of bone proteins during OB differentiation. Collagen synthesis and secretion is the major function of OBs and is markedly upregulated upon ascorbic acid (AA) stimulation, significantly more so than in fibroblast cells. Understanding the mechanism by which collagen is mobilized in specialized OB cells is important for both basic cell biology and bone disease studies. Cellular organelles and vesicles in the exocytic and endocytic pathways have a distinctive spatial distribution and their trafficking is aided by many molecules, Rab GTPases being a master regulator. In this work, I identified the Rab GTPases that are upregulated during OB differentiation using microarray analysis, namely Rab1, Rab3d, and Rab27b, and investigated their role in regulating the trafficking of collagen from the site of synthesis in the ER to the Golgi and ultimately to the plasma membrane (PM) utilizing their dominant negative (DN) expression. The experimental halting of biosynthetic trafficking by these mutant Rabs initiated proteasome-mediated degradation of procollagen and ceased global protein translation. Acute expression of Rab1 and Rab3d DN constructs resulted in impaired ER to Golgi trafficking of procollagen. Similar expression of Rab27b DN constructs resulted in dispersed collagen vesicles which may represent failed secretory vesicles sequestered in the cytosol. A significant and strong reduction in extracellular collagen levels also was observed showing roles of Rab1, Rab3d and Rab27b in the specific function of these major collagen producing cells in the body. I further observed that a fraction of procollagen colocalized with lysosomes which was markedly increased when procollagen was experimentally misfolded. Lysosomes, essential organelles for intracellular degradation, are generally sequestered near the cell centre to receive vesicles with contents targeted for destruction. During AA-induced differentiation of OB cells, I saw a marked increase in total degradative lysosome organelles in addition to an enhanced endocytic rate. Interestingly, lysosomes were dispersed toward the cell periphery in differentiating OBs without being secreted. This required intact microtubules for long range transport and was kinesin motor-dependent but did not involve cytosolic acidification. Moreover, impairment of lysosome dispersion markedly reduced AA-induced OB differentiation. Taken together, this study provides an important general mechanism for cell secretion phenomena that may ultimately lead to clinical targets for treatments of diseases driven by aberrant collagen processing and secretion including Osteogenesis Imperfecta (OI).

Bibliography: leaves 117-132 / 132 leaves, [25] leaves of plates : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 1985

Thesis (M. Phil.)--University of Hong Kong, 2008. / Also available in print.

Thesis (M. Phil.)--University of Hong Kong, 2007. / Also available in print.

Thesis (M.S.)--Missouri State University, 2008. / "May 2008." Includes bibliographical references (leaves 44-51). Also available online.

Thesis (Ph. D.)--Ohio State University, 2008. / Title from first page of PDF file. Includes bibliographical references (p. 114-127).