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Thesis (Ph. D.)--University of Hong Kong, 2008. / Includes bibliographical references (leaf 122-141) Also available in print.

published_or_final_version / abstract / Zoology / Doctoral / Doctor of Philosophy

Thesis (Ph. D.)--Oregon State University, 2010. / Printout. Includes bibliographical references (leaves 102-115). Also available on the World Wide Web.

published_or_final_version / Pathology / Master / Master of Medical Sciences

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

Mammalian skeletal muscle can regenerate after injury and this response is primarily mediated by the satellite cell, a muscle stem cell. Following injury, satellite cells are activated to myoblasts, undergo rapid proliferation, migrate towards the injury site, and subsequently differentiate into myotubes in order to facilitate functional muscle repair. Fibrosis, caused by the secretion of structural extracellular matrix (ECM) proteins such as collagen I and fibronectin, by fibroblasts, impairs complete functional repair of the muscle. In this study, the role of the microenvironment during wound conditions was assessed by analysing the effect of specific extracellular matrix and growth factors on myoblast migration. The role of the Rho/ROCK pathway as a possible mechanism in mediating the effects seen was investigated. In order to analyse wound repair in an in vitro setting, we optimised and improved a wound healing model specifically designed for skeletal muscle repair. To this end we also developed a co-culture assay using primary myoblasts and fibroblasts isolated from the same animal. The studies showed that collagen I and fibronectin both increased myoblast migration in a dose-dependent manner. Decorin displayed opposing effects on cellular movement, significantly increasing collagen I-stimulated, but not fibronectin-stimulated, migration of myoblasts. ROCK inhibitor studies revealed a significant increase in migration on uncoated plates following inhibition with Y-27632 compared to untreated control. When cells were cultured on ECM components (Matrigel, collagen I, or fibronectin), the inhibitory effect of Y-27632 on migration was reduced. Analysis of ROCK and vinculin expression, and localization at the leading front, showed that ROCK inhibition resulted in loosely packed focal adhesion complexes (matrix dependent). A reduced adhesion to the ECM could explain the increased migration rates observed upon inhibition with Y-27632. We also investigated the role of TGF-β and decorin during wound repair, as TGF-β is a known pro-fibrotic agent. TGF-β treatment decreased wound closure rates; however, the addition of decorin with TGF-β significantly increased wound closure. The addition of ECM components, Matrigel and collagen I enhanced the effect seen in response to TGF-β and decorin; however, fibronectin negated this effect, with no increase in migration seen compared to the controls. In conclusion, the importance of extracellular matrix components in regulating myoblast migration and therefore skeletal muscle wound repair was demonstrated. We emphasize that, in order to gain a better understanding of skeletal muscle wound repair, the combination of ECM and growth factors released during wounding need to be utilised in assays which mimic the in vivo environment more closely. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2012.

Many of the anabolic effects of growth hormone (GH) are indirect, occurring through GH-stimulated production of insulin-like growth factor-I (IGF-I) by the liver. As well as being GH regulated, plasma IGF-I concentrations have been demonstrated to be dependent upon protein nutrition, with low protein diets being associated with reduced plasma IGF-I concentrations. This effect cannot be reversed by GH, suggesting that liver sensitivity to GH is impaired. To investigate the mechanisms through which protein supply affects GH sensitivity, primary cultures of ovine hepatocytes were grown in defined media. In a first experiment the media contained various fractions (0.2, 1.0, 5.0) of portal vein amino acid concentrations in fed sheep. In the second 24h incubation period, unstimulated IGF-I secretion was highly sensitive the concentration of amino acids in the media, with significantly greater release of basal IGF-I in 5x compared to either 1x (P<0.05) or 0.2x amino acid containing media. In a second series of experiments the effects of specific amino acid depletions was examined. Methionine depletion of 0.2x portal amino acid concentrations ablated the GH response second 24h of culture without affecting basal IGF-I release. By comparison ³H-leucine incorporation into secreted protein, following 20 hours of culture in defined media was significantly reduced in 0.2x aa (P<0.01) and 1.0x aa (P<0.05) media compared with 5.0x aa media, however secretory protein synthesis was unaffected by methionine depletion to 0.2x portal concentrations. The results suggest that amino acid availability regulates both basal and GH stimulated IGF-I release in ovine hepatocytes. Furthermore reducing methionine concentrations in the culture media to 0.2x portal concentrations diminishes GH response without compromising protein secretion.

Siu, Yeung Tung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 65-75). / Abstracts in English and Chinese. / Title Page --- p.i / Abstract in English --- p.ii / Abstract in Chinese --- p.iii / Acknowledgement --- p.iv / Table of Contents --- p.v-viii / List of Figures --- p.ix / List of Tables --- p.x / Abbreviations --- p.xi-xii / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Growth factors involved in hepatocytes proliferation --- p.1-6 / Chapter 1.1.1 --- Hepatocyte growth factor (HGF) --- p.1 / Chapter 1.1.2 --- Tumor necrosis factor-a (TNF-α) and interleukin-6 (IL-6) --- p.2 / Chapter 1.1.3 --- Epidermal growth factor (EGF) and transforming growth factor-α (TGF-α) --- p.3 / Chapter 1.1.4 --- Other comitogens --- p.4 / Chapter 1.1.5 --- Transforming growth factor-β (TGF-β) --- p.5 / Chapter 1.2 --- Bone marrow stromal cells and hepatocytes proliferation --- p.7-12 / Chapter 1.2.1 --- Role of bone marrow stromal cells in bone marrow --- p.7 / Chapter 1.2.2 --- Bone marrow as a source of hepatic oval cells --- p.8 / Chapter 1.2.3 --- Growth factors secreted by bone marrow stromal cells involved in hepatocytes proliferation --- p.9 / Chapter 1.2.4 --- Endocrine in hepatocytes proliferation --- p.12 / Chapter 1.3 --- Objective of this study --- p.13-15 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Cell cultures --- p.16 / Chapter 2.2 --- Selection of human hepatic cell line for the detection of mitogenic activity --- p.17-18 / Chapter 2.2.1 --- "Enrichment of human hepatic cell lines, Hep 3B, Hep G2, C3A, SK-Hep-1 and Chang cells at G0-G1 phases by serum deprivation" --- p.17 / Chapter 2.2.2 --- "Incubation of serum deprived Hep 3B, Hep G2, C3A, SK- Hep-1 and Chang cells with mitogenic stimuli" --- p.17 / Chapter 2.2.3 --- Cell cycle analysis by flow cytometry using propidium iodide staining --- p.17 / Chapter 2.3 --- "Detection of mitogenic activity of human bone marrow stromal cells on the selected cell line, SK-Hep-1 cells" --- p.18-20 / Chapter 2.3.1 --- Partially growth arrested human SK-Hep-1 cells --- p.18 / Chapter 2.3.2 --- Human bone marrow stromal cells --- p.19 / Chapter 2.3.2.1 --- Bone marrow stromal cellular extract --- p.19 / Chapter 2.3.2.2 --- Total protein assay --- p.19 / Chapter 2.3.3 --- Incubation of SK-Hep-1 cells with bone marrow stromal cellular extracts --- p.20 / Chapter 2.4 --- Characterization of hepatocyte mitogenic activity of bone marrow stromal cellular extract --- p.21-22 / Chapter 2.4.1 --- Dialysis --- p.21 / Chapter 2.4.2 --- Temperature treatment --- p.21 / Chapter 2.4.3 --- Proteolysis --- p.22 / Chapter 2.5 --- Performing a preliminary test on the difference between bone marrow stromal cellular extract and other growth factors --- p.22-26 / Chapter 2.5.1 --- Incubation of SK-Hep-1 cells with bone marrow stromal cellular extract or other growth factors --- p.22 / Chapter 2.5.2 --- Metabolic labeling of SK-Hep-1 cells with [32P]orthophosphate --- p.23 / Chapter 2.5.3 --- Incubation of labeled SK-Hep-1 cells with bone marrow stromal cellular extract or other growth factors --- p.23 / Chapter 2.5.4 --- SK-Hep-1 cells lysate extraction --- p.23 / Chapter 2.5.5 --- Two-dimensional electrophoresis --- p.24 / Chapter 2.5.5.1 --- First dimension isoelectric focusing --- p.24 / Chapter 2.5.5.2 --- Second dimension sodium dodecyl sulfate-polyacrylamide gel electrophoresis --- p.25 / Chapter 2.5.6 --- Amplification of radiolabeled signal by EN3HANCE --- p.25 / Chapter 2.5.7 --- Visualization of autoradiography --- p.26 / Chapter 2.5.8 --- Visualization by silver staining --- p.26 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Selection of human hepatic cell line for the detection of mitogenic activity --- p.27-30 / Chapter 3.1.1 --- "Enrichment of human hepatic cell lines, Hep 3B, Hep G2, C3A, SK-Hep-1 and Chang cells at G0-G1 phases by serum deprivation" --- p.27 / Chapter 3.1.2 --- DNA synthesis of hepatic cell lines in response to 10 % FBS after serum deprivation --- p.29 / Chapter 3.2 --- "Detection of mitogenic activity of human bone marrow stromal cells on the selected cell line, SK-Hep-1 cells" --- p.31-39 / Chapter 3.2.1 --- Cell cycle distribution of partially growth arrested SK-Hep-1 cells in response to mitogens --- p.31 / Chapter 3.2.2 --- Time course on DNA synthesis of partially growth arrested SK-Hep-1 cells in response to FBS and bone marrow stromal cellular extract --- p.36 / Chapter 3.2.3 --- Dose response on DNA synthesis of partially growth arrested SK-Hep-1 cells in response to bone marrow stromal cellular extracts --- p.38 / Chapter 3.3 --- Characterization of hepatocyte mitogenic activity of bone marrow stromal cellular extract --- p.40-44 / Chapter 3.4 --- Performing a preliminary test on the difference between bone marrow stromal cellular extract and other growth factors --- p.45-49 / Chapter 3.4.1 --- Mitogenic response of SK-Hep-1 cells in response to bone marrow stromal cellular extract and other growth factors --- p.45 / Chapter 3.4.2 --- Early intracellular signaling of SK-Hep-1 cells in response to bone marrow stromal cellular extract and other growth factors --- p.47 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- Selection of human hepatic cell line for the detection of mitogenic activity --- p.50 / Chapter 4.2 --- "Mitogenic activity of human bone marrow stromal cells on the selected cell line, SK-Hep-1 cells" --- p.51 / Chapter 4.3 --- Characterization of hepatocyte mitogenic activity of bone marrow stromal cellular extract --- p.52 / Chapter 4.4 --- Performing a preliminary test on the difference between bone marrow stromal cellular extract and other growth factors --- p.53 / Chapter 4.5 --- Possible directions for future investigation --- p.55 / Chapter 4.6 --- Conclusions --- p.56 / Chapter Chapter 5 --- Appendices / Chapter 5.1 --- Reagents and solutiuons --- p.57-64 / Chapter 5.1.1 --- Selection of human hepatic cell line for the detection of mitogenic activity --- p.57 / Chapter 5.1.2 --- "Detection of mitogenic activity of human bone marrow stromal cells on the selected cell line, SK-Hep-1 cells" --- p.59 / Chapter 5.1.3 --- Characterization of hepatocyte mitogenic activity of bone marrow stromal cellular extract --- p.60 / Chapter 5.1.4 --- Performing a preliminary test on the difference between bone marrow stromal cellular extract and other growth factors --- p.61 / Chapter Chapter 6 --- References --- p.65-75

Bone formation and remodeling require continuous generation of osteoprogenitors from bone marrow stromal cells (MSC), which are regulated by local growth factors and hormones with putative roles in mesenchymal proliferation and differentiation. Hepatocyte growth factor (HGF) and its receptor c-Met are widely expressed in MSC and are thought to play a key role in the interactions between cells. 1,25-dihydroxyvitamin D (1,25OHD) is the most active metabolite of vitamin D. 1,25OHD binds to its nuclear/membrane vitamin D receptor (VDR) and generates appropriate biological responses. The purpose of this study was to investigate the regulation of proliferation and differentiation by HGF in human bone marrow-derived stromal cells (hMSC). We examined the impact of HGF on hMSC cell-cycle regulation and the combination effects of HGF and 1,25OHD on hMSC osteogenic differentiation to enhance our knowledge of hMSC regulation. hMSC isolated from bone marrow were plated and grown in DMEM supplemented with 3% FBS incubated at 37C with 5% CO2 in air. HGF treatment of hMSCs reduced the rate of cell proliferation and this result was not due to apoptosis or cell senescence. Real-time RT-PCR and Western blot analysis showed increased gene and protein expression of the cell-cycle inhibitors p53, p21, and p27 after HGF treatment. These results appear to be specific because HGF did not significantly alter the gene expression level of other cell-cycle mediators such as RB, cyclin D1, CDK2, CDK4, or CDK6. Transfection of siRNA specific for cMet, the HGF receptor, eliminated the HGF anti-proliferation effect. cMet siRNA also eliminated the increase in p53, p21, and p27, further supporting a role for these cell-cycle inhibitors in HGF¡¯s regulation of hMSC. These results suggest that treatment of hMSC with HGF slows cell proliferation by increasing the expression of p53, p21, and p27. The reduced rate of cell proliferation did not appear to be due to cell differentiation, because treatment of hMSC with HGF alone did not induce cell differentiation. However, HGF in combination with a known osteogenic differentiation activator, 1,25OHD, significantly increased cell maturation/differentiation compared to 1,25D alone, as indicated by an increase in osteocalcin mRNA (a marker for osteogenic differentiation). Whereas HGF had no effect on 1,25OHD synthesis per se, HGF did induce 1,26OHD receptor (VDR) gene expression. HGF up-regulated the expression of the p63 gene, a member of the p53 family. Knocking down the p63 gene reduced the HGF effect on VDR expression and eliminated the HGF-induced up-regulation of the osteogenic differentiation markers osteopontin (OPN) and bone sialoprotein (BSP). Moreover, the ChIP assay shows that p63 was able to bind to the VDR promoter, possibly explaining the mechanism of p63-mediated VDR up-regulation. These results indicate that HGF can also induce hMSC osteogenic differentiation when combined with 1,25OHD by up-regulating 1,25OHD receptor VDR expression.