Nogo is well established for its inhibitory action on axon regeneration in the adult central nervous system. It binds to the Nogo receptor (NgR) through an extracellular active site on the protein-Nogo-66. Although it is reported that Nogo is widely expressed in the developing brain, its exact function during development of the nervous system is unclear. / The contribution of Nogo on patterning the axon routing at the optic chiasm of mouse embryo was investigated in this thesis. Using immunocytochemical staining, Nogo protein was localized on the Miller glial cells in the retina and at the optic disk. A few migrating retinal neurons also expressed Nogo. In the chiasm, Nogo was localized exclusively on the radial glia, which generate a midline domain where turning of uncrossed axons occurs. In vitro study showed expression of NgR on retinal neurites and growth cones, and neurite outgrowth from both dorsal nasal (contralaterally projecting) and ventral temporal (ipsilaterally projecting) retina was inhibited by Nogo. In the pathway, NgR expression was regionally regulated. NgR was obvious in the optic stalk and the optic tract, but not in the chiasm. Blocking Nogo function with NEP1-40, a peptide antagonist of NgR, in brain slice culture of the pathway produced significant reduction in the uncrossed projection, but had no effect on axon crossing at the midline. Furthermore, the age related fiber arrangement in the optic tract was abolished after disturbing of Nogo function. Similar abnormalities were observed in slices treated with Nogo blocking antibody. In vitro studies showed that NEP1-40 rescued the inhibition of Nogo to the retinal neurites. The downregulation of NgR at the chiasm was supported by in vitro assays showing significant reduction of receptor expression on dorsal nasal but not ventral temporal growth cones when they encountered the chiasm, thus generating a differential inhibition to ventral temporal neurites. / These results provide evidences that Nogo is a guidance molecule during the development of CNS. Interaction of Nogo and its receptor plays important role for patterning the axon divergence in the mouse optic pathway and the age related fiber order in the optic tract. / Wang Jun. / "September 2006." / Adviser: Sun-On Chan. / Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1474. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 130-142). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

促红细胞生成素（EPO）和EPO受体（EPOR）是调节红细胞生成所必需的细胞因子。越来越多证据表明，EPO/EPOR在非造血器官包括心脏、大脑和骨骼的发育和再生中发挥重要作用。但目前人们对EPO/EPOR在骨骼发育和再生中的机制知之甚少。最近一些研究表明，系统性或局部注射EPO可促进骨形成。但是EPO/EPOR在骨骼发育和再生中的作用机制尚不明确。 / 实验发现EPO/EPOR在生长板的前肥大软骨和肥大软骨区富集，且可促进软骨细胞增殖。但利用siRNA技术将软骨细胞EPOR沉默后，软骨细胞增殖会受抑制。随后，我们用阿利新蓝对软骨细胞外基质中的的蛋白聚糖进行染色，发现EPO可促进软骨细胞分化。此外软骨细胞标志基因包括SOX9、SOX5、SOX6、2型胶原蛋白和蛋白聚糖表达上调。实验还发现EPO可促进MSCs增殖。同时EPO可促进上述软骨细胞标志基因的表达增加。当基因沉默EPOR或使用EPO封闭肽后，软骨细胞标志基因的表达降低。以上数据表明， EPO促进软骨细胞增殖和分化的功能至少部分通过其同源受体EPOR介导。 / 我们还探讨了在低氧环境下，EPO对软骨细胞的作用。在低氧环境中， EPO/EPOR和HIF-1α mRNA和蛋白质表达水平均上调，且EPO促进软骨祖细胞集落形成。此结果提示EPO在低氧条件下介导软骨细胞增殖。同时在软骨细胞中标，EPO可激活JAK2和STAT3磷酸化，该结果表明JAK/STAT信号介导软骨细胞的生物学功能。 / 体外内皮细胞出芽实验发现EPO明显促进跖骨表面内皮细胞出芽。在小鼠骨折处局部注射EPO 14天后μCT血管成像发现，EPO可促进骨折处血管生成。体外与体内试验结果同时证实EPO可促进血管生成。 / 在骨折术后第7天和14天，藏红O染色发现EPO促进软骨骨痂形成。在术后第28天，X光和μCT扫描发现，三维重建和定量分析显示EPO促进骨形成，且伴随着骨量和骨面积的增加，以及骨生物力学特性的改善。以上结果表明， EPO可有效促进骨折修复。 / 综上所述， EPO/ EPOR信号调控软骨细胞和MSCs增殖及其软骨细胞分化。EPO还调控软骨细胞在低氧环境下的生物学特性。EPO/ EPOR信号分子有助于骨愈合过程中血管生成和骨形成。因此，EPO/EPOR可作为一个新的治疗靶点促进骨骼修复与再生。 / Erythropoietin (EPO) and EPO receptor (EPOR) are essential cytokine signals regulating erythropoiesis. Growing evidences suggest that EPO/EPOR signaling involves in the development and regeneration of non-hematopoietic organs including heart, brain and bone, et al. Several recent studies indicate that administration of EPO locally or systemically promotes bone formation. However, the underlying mechanisms of EPO/EPOR in skeletal development and regeneration remain unknown. / Our results show that EPO and EPOR are abundantly expressed in the pre-hypertrophic and hypertrophic zone of the growth plates. The proliferation rate of chondro-progenitors is increased following EPO treatment Alcian blue staining for extracellular matrix proteoglycan indicates that EPO promotes the differentiation of chondrocytes. This is accompanied by up-regulated chondrogenic marker genes including SOX9, SOX5, SOX6, type 2 collagen and aggrecan. In a parallel study, the proliferation rate of MSCs is increased following EPO treatment. The mRNA expression of above chondrogenic marker genes is also up-regulated. These effects are eliminated following knockdown of EPOR in chondrocytes by siRNA or treatment with EPO block peptide. These findings indicate that EPO promotes the proliferation and differentiation of primary chondrocytes at least partially mediated by its cognate receptor EPOR. / We next examined the role of EPO in chondrocytes under hypoxia. The mRNA and protein levels of EPO/EPOR and HIF-1α are up-regulated under hypoxia. EPO also enhances the colony forming efficiency of chondro-progenitors under hypoxia. This result suggests that EPO may serve as a mediator to regulate proliferation of chondro-progenitors under hypoxic condition. In addition, we show that EPO up-regulates the phosphorylation states of Janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3) in chondrocytes, suggesting that the function of EPO in chondrocytes is mediated through JAK/STAT signaling. / To address the function of EPO in angiogenesis, we performed metatarsal endothelial sprouting assay. The endothelial sprouting is significantly enhanced in metatarsals treated with EPO. This coincide with our in vivo data that local delivery of EPO increases vascularity of the healing bone at day 14 post-fracture in mice as indicated by micro-CT angiography analysis. / Interestingly, in the mouse fracture model, EPO promotes cartilaginous callus formation at days 7 and 14 post-surgery. This results in accelerated osteogenesis at day 28 post-surgery indexed by the radiographical scoring and micro-CT analysis characterized by increased bone volume and bone surface. This is accompanied by improved biomechanical properties of the healing bone. These results indicate that administration of EPO may serve as an efficient therapy to facilitate bone regenerartion. / In conclusion, EPO/EPOR signal regulates of the proliferation and differentiation of chondrocytes and MSCs to promote chondrogenesis. EPO may also function as a positive mediator in chondrocytes in response to low oxygen tension during chondrogenesis. EPO/EPOR signaling also contributes to angiogenesis and osteogenesis during bone healing. Therefore, EPO/EPOR may serve as a novel therapeutic target to promote skeletal regeneration. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Wan, Lin. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 130-152). / Abstracts also in Chinese. / Wan, Lin.

Xing Hongtao. / "February 2003." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (p. 149-176). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Chan, Chung Kit. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 98-110). / Abstracts in English and Chinese. / Abstract --- p.i / Abstract in Chinese --- p.iv / Acknowledgements --- p.v / Table of Abbreviations --- p.vi / Table of Contents --- p.vii / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter Chapter 2 --- Functions of hyaluronan in the development of retinofugal pathway / Introduction --- p.18 / Materials and Methods --- p.19 / Results --- p.23 / Discussion --- p.26 / Figures --- p.32 / Chapter Chapter 3 --- Characterization of Nogo and its receptor in retinofugal pathway using Western blot analysis / Introduction --- p.40 / Materials and Methods --- p.42 / Results --- p.50 / Discussion --- p.52 / Figures --- p.57 / Chapter Chapter 4 --- Expression patterns and functions of Sonic hedgehogin retinofugal pathway / Introduction --- p.62 / Materials and Methods --- p.64 / Results --- p.69 / Discussion --- p.76 / Figures --- p.81 / Chapter Chapter 5 --- General Discussion --- p.91 / Figures --- p.95 / References --- p.98

Islet plasticity has proven to be an important platform for the engineering of alternative islet tissue for transplantation. In vitro studies have shown the ability of islets to transdifferentiate into duct-like epithelial structures (DLS) thought to possess progenitor cells capable of replenishing damaged tissue within the pancreas. The aim of this study was to investigate the natural history of human derived duct-like epithelial structures transplanted into nude mice. / Human islet derived duct-like structures from three cadaver pancreases were subcutaneously transplanted into 6-8 week old male HSD athymic nude-Foxn1 mice. Six mice were sacrificed at day 3, 7, 14 and 21 from each time period. DLS were also placed in matrigel for in-vitro control samples. DLS were processed for immunohistochemistry for endocrine markers, epithelial markers, cell death and proliferation markers, islet maturation markers and angiogenic factors. / Our results show that as DLS are transplanted, there is an increase in cell death and proliferation. This increase in cell death and proliferation causes an increase in PDX-1 expression as well as VEGF, an angiogenic factor. But over time, transplanted DLS do not show an increase in cell death and show a small decrease in cell proliferation from pre-transplanted DLS. At day 3 of engraftment, DLS show a significant expression of PDX-1. We see a small increase in endocrine tissue after 3 days of transplantation, then an increase in endocrine cell death, which returns the percentage of endocrine cells back to pre-transplantation levels at day 21. DLS were shown to express VEGF, and once transplanted into an initial hypoxic environment there is a substantial increase in expression, followed by a recruitment of microvessels. Although there is a dynamic change in expression of cell markers throughout engraftment, there is no significant change in DLS size, nuclei per DLS or cell morphology over time. / DLS have been shown to survive subcutaneous transplantation and possess an initial increase in cell proliferation leading to increases in PDX-1 and VEGF expression. Transplanted DLS have shown to possess significant angiogenic properties with the recruitment of microvessels into subcutaneous DLS grafts. Subcutaneous DLS transplantation could be used in combination with islet transplantation to alleviate current problems with islet transplantation such as islet cell death and insufficient blood supply.

The proprotein convertase subtilisin/kexin type 9, PCSK9, belongs to the proprotein convertase (PC) family. Human mutations in the gene encoding PCSK9 lead to either familial hyper- or hypocholesterolemia, resulting from a gain or loss of function, respectively. Mice lacking PCSK9 are viable and show a 42% decrease in plasma cholesterol levels. The enzyme triggers the degradation of the low density lipoprotein receptor (LDLR) through a partially unknown mechanism. / PCSK9 is very abundant in the liver and intestine during development and adulthood. Hepatocytes have a capacity to reproduce themselves and, upon injury, can repopulate the liver. For a better understanding of the role of PCSK9 in the liver, partial hepatectomy was performed on Pcsk9 +/+, Pcsk9+/- and Pcsk9-/- mice. The absence of PCSK9 resulted in defective liver regeneration, while wild type (WT) and heterozygous mice had no phenotype. Regeneration defects could be prevented by a high cholesterol diet. PCSK9 deficiency, by contributing to maintaining low circulating cholesterol levels may thus hamper liver regeneration. This knowledge is critical for the analysis of future PCSK9 inhibitors expected to be developed in the near future. / Key words. Proprotein convertase subtilisin/kexin 9 (PCSK9), a familial hyper- or hypocholesterolemia, low density lipoprotein receptor, knockout mouse model, partial hepatectomy.

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Regrowth in the lesioned central nervous system is impeded by inhibitory molecules including myelin-associated inhibitors (MAIs) and chondroitin sulfate proteoglycans (CSPGs). Inhibitory molecules engage neuronal cell surface receptors and activate the small GTPase RhoA in injured neurons to mediate neurite outgrowth inhibition through targeted modifications to the cytoskeleton. Inhibition of RhoA with the ribosyltransferase C3 attenuates neurite outgrowth inhibition in vitro and in vivo but the ubiquitous expression and multifunctionality of RhoA may limit the specificity of therapeutic RhoA antagonists. The hypothesis of the thesis is that molecules that functionally interact with RhoA to mediate myelin-dependent inhibition may represent more specific targets for therapeutic intervention. We have explored the contribution of two RhoA interacting proteins to the neurite outgrowth inhibitory effects of MAIs. In Chapter 2 we describe the contribution of the rho effector, Rho kinase (ROCK) to MAI responses in neurons. In Chapter 3 we identify the cytosolic phosphoprotein CRMP4b (Collapsin Response Mediator Protein 4b) as a novel RhoA binding partner that mediates neuronal responses to CNS inhibitors. By structure function analysis we have developed a molecular antagonist of CRMP4b-RhoA binding that promotes neurite outgrowth on inhibitory substrates in vitro and has the potential to be a potent and specific molecular therapeutic for spinal cord injury. In Chapter 4 we identify glycogen sythase kinase 3b (GSK3b) as an important kinase in the MAI pathway that regulates protein interactions with RhoA. This thesis provides insights into the signal transduction machinery that is engaged in response to CNS inhibitors and suggests several novel therapeutic targets to promote axon regeneration following CNS injury.

Indiana University-Purdue University Indianapolis (IUPUI) / The aim of this current study was to determine if the exposure of human dental pulp stem cells (HDPSC) to the DynaMatrix membrane will result in an increased production of angiogenic cytokines that are critical for pulp/root regeneration. Angiogenesis cytokine arrays have been established as a viable method for assessing expression of cytokines.20 HDPSC were chosen as they are expected to be found in the apical papilla and the infected immature root canal system of teeth that current regenerative endodontic techniques are designed to treat.