In this study, the brain of senescence accelerated mouse prone 8 (SAMP8) mice model was adopted to investigate PTMs state (especially methylation patterns) of core histones (H2A, H2B, H3 and H4). Seven methylated sites (H3K24, H3K27, H3K36, H3K79, H3R128, H4K20 and H2A R89) were detected by tandem matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) analysis. The methylation of H3K27 and H3K36 demonstrated a modulating relationship and methylated H3K27 might contribute to the hypermethylation state and gene repression in aged brain. Western blotting results showed that mono-methylated H4K20 decreased during SAMP8 mice aging and di-methylated H3K79 decreased in the brain of 12-month-old SAMP8 mice compared with age-matched senescence accelerated-resistant mouse (SAMR1) control. Di-methylated H3K79 could express in neuron cells of cerebral cortex and hippocampus. Whereas, the number of H3K79 methylation negative cells was higher in the cortex of 12-month old SAMP8 mice than that of age-matched control SAMR1 mice. Chromatin immunoprecipitation (ChIP) result indicated homeodomain transcription factor Pbx1 isoform 1 (Pbx1), transcription factors and transcriptional regulator proteins, such as T-box isoform 20, TetR family precursor BAZ2B and ribosomal protein, were recruited to methylated H3K79 site. Therefore, a model of methylated H3K79 on gene transcriptional regulation was proposed. Furthermore, the consequences of decreased H3K79 methylation in Neuro-2a (N2a) cells were investigated via transfection with Dot1 (disruptor of telomeric silencing) siRNA. After transfection, N2a cells displayed shorter neurite and less dendrite. Proteomic change in the N2a cells provided convincing evidence for the multi-function of decreased H3K79 methylation on transcriptional regulation, protein translation and folding, stress response and DNA breaks repair, which would contribute to brain dysfunction during neurodegenerative disease or aging. / Nowadays, many countries including China are experiencing aging populations. Aging has become the major risk factor for many diseases, such as neurodegenerative disease. The studies on the role of epigenetics in the aging process have grown tremendously in recent years. However, no systematic investigations have provided the information on histone post-translational modifications (PTMs) in aged brain and the roles of histone PTMs in brain aging are still unknown. / This study gave a new insight into the link between histone PTMs and brain aging. It could provide the experimental evidence for future studies and help us to better understand aging or neurodegenerative disease at epigenetic level. Furthermore, it could benefit for setting up the strategies for epigenetic therapy to neurodegenerative disease. / Wang, Chunmei. / Adviser: Ngai Saiming. / Source: Dissertation Abstracts International, Volume: 73-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaf 136). / 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, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Chiasm cells, which include glia and neurons, are generated early before any retinal axon arrives at the midline of the mouse ventral diencephalon. These cells have been shown to affect retinal axon growth and patterning in the optic chiasm. In this study, we used EdU (5-ethyny1-2'-deoxyuridine) for birthdating these chiasm cells, aiming to find out when these cells are generated; then we tried to trace their fates at later stages of development. EdU injection at embryonic day (E) 9.5 to El 1 labeled a number of chiasmatic neurons and radial glial cells at E13, which were immunoreactive for SSEA-1 and RC2, respectively. After colocalization studies, we found that most of these neurons were born as early as E9.5, while a large number of radial glial cells were born as from El 1. Both E9.5-born chiasmatic neurons and Ell-born radial glia decreased by E14-E16; the radial glia even disappeared finally from the midline. Furthermore, we found that some chiasmatic neurons underwent apoptotic cell death as from El 4, and that the radial glia likely differentiated into other cell types after finishing their retinal axon guidance mission at the midline. So it is reasonable that some of the earliest born chiasm cells disappear during development. / During development, retinal ganglion cell axons grow from the eye to the ventral diencephalon, where axons from the two eyes converge and segregate into crossed and uncrossed projections, forming the optic chiasm. This pattern is critical for binocular vision. Although significant progress has been obtained over the past decades, how retinal axon growth and guidance are regulated at the chiasm is largely unknown. Our research will focus on those problems. / In the last part of this thesis, we investigated the retinal axon pathway in the ventral diencephalon of the Sox10Dom mutant embryos and gamma-crystallin mutant embryos. Our findings indicate that Sox10 may not contribute to axon guidance in the developing optic pathway whereas gammaA-crystallin may only play a role in the later uncrossed axons. / N-methyl-D-aspartate (NMDA) receptor is one of the ionotropic glutamate receptors, which are important in synaptic plasticity, apart from implications in dendritic spine remodeling, neurite outgrowth, elongation and branching and glutamate neurotoxicity. There are several subtypes of NMDA receptor channel subunits, NR1, NR2A-D, NR3A&B. The functional diversity of NMDA receptor resides in the different assembly of subunits. In this study, we used RT-PCR to analyze the mRNA expression of all the NMDA receptor subunits in mouse embryos. After that we chose the NR1, NR2B and NR3A antibodies to investigate NMDA receptor subunit expression in the optic pathway during mouse optic pathway development. Using immunohistochemistry, we found that NR1, NR2B and NR3A were expressed in the mouse retina and optic pathway as from E13 when the optic chiasm is forming. Expression of the NMDA receptor subunits were found in the inner cell layers and along retinal axons. Colocalization studies showed that NR1, NR2B and NR3A were localized on the ganglion cells and their axons. In the ventral diencephalon, these subunits were expressed extensively, but NR1 and NR3A were particularly strong along the optic nerve and optic tract. Furthermore, to identify the function of NMDA receptor during optic chiasm development, we cultured E14 retinal explants on laminin and poly-D-ornithine in the presence of the NMDA receptor antagonists MK-801 or Dextrorphan-D-tartrate. These two antagonists can significantly inhibit the retinal axon outgrowth, suggesting that the NMDA receptor promotes retinal axon outgrowth in the retinofugal pathway during optic chiasm development. / Li, Jia. / Adviser: Chan Sun On. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 145-158). / 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, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Wong Hei Lui. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 192-226). / Abstracts in English and Chinese. / Publications Based On The Work In This Thesis --- p.i / Abstract --- p.ii / Acknowledgements --- p.vii / Abbreviations --- p.viii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Normal joint --- p.1 / Chapter 1.11 --- Biology of joint --- p.1 / Chapter 1.12 --- Structure of synovial joint --- p.1 / Chapter 1.13 --- Components of the mature synovial joint --- p.3 / Chapter 1.131 --- Articular cartilage --- p.3 / Chapter 1.1311 --- Water --- p.4 / Chapter 1.1312 --- Cartilage matrix --- p.4 / Chapter 1.1313 --- Chondrocyte --- p.5 / Chapter 1.132 --- Synovium --- p.5 / Chapter 1.1321 --- Synovium vasculature --- p.6 / Chapter 1.1322 --- Synovial blood flow --- p.7 / Chapter 1.133 --- Synovial fluid --- p.8 / Chapter 1.134 --- Bone --- p.9 / Chapter 1.2 --- Pathological processes of arthritis --- p.11 / Chapter 1.21 --- Activation of immune cells in arthritis --- p.11 / Chapter 1.22 --- Synovial proliferation --- p.13 / Chapter 1.221 --- Synovial lining cell activation --- p.13 / Chapter 1.222 --- Pannus invasion --- p.14 / Chapter 1.23 --- Cartilage and bone degradation --- p.14 / Chapter 1.231 --- Depletion of proteoglycan (GAG) --- p.15 / Chapter 1.232 --- Collagen denature --- p.15 / Chapter 1.3 --- Tachykinins (TKs) --- p.17 / Chapter 1.31 --- History --- p.17 / Chapter 1.32 --- "Synthesis, storage and release of TKs" --- p.17 / Chapter 1.33 --- Tachykinin receptors --- p.18 / Chapter 1.331 --- Characterization of NK1 receptor --- p.19 / Chapter 1.332 --- Characterization of NK2 receptor --- p.19 / Chapter 1.333 --- Characterization of NK3 receptor --- p.20 / Chapter 1.34 --- Effector systems of TKs --- p.21 / Chapter 1.35 --- Termination of TK signals --- p.21 / Chapter 1.351 --- Enzymatic breakdown --- p.21 / Chapter 1.352 --- Receptor desensitization --- p.22 / Chapter 1.353 --- Receptor endocytosis --- p.22 / Chapter 1.36 --- TK receptor antagonists --- p.23 / Chapter 1.361 --- Selective NK1 receptor antagonists --- p.23 / Chapter 1.362 --- Selective NK2 receptor antagonists --- p.24 / Chapter 1.363 --- Selective NK3 receptor antagonists --- p.25 / Chapter 1.4 --- Roles of tachykinins in arthritis --- p.28 / Chapter 1.41 --- Correlation between tachykinins and joint inflammation --- p.28 / Chapter 1.42 --- Roles of tachykinins in immune cell activation --- p.30 / Chapter 1.43 --- Roles of tachykinins in synovial proliferation --- p.31 / Chapter 1.44 --- Roles of tachykinins in cartilage degradation --- p.32 / Chapter 1.5 --- Animal model of arthritis --- p.33 / Chapter 1.51 --- Instability model --- p.33 / Chapter 1.52 --- Immobilization model --- p.34 / Chapter 1.53 --- Noxious agent-induced model --- p.34 / Chapter 1.531 --- Collagen-induced erosive arthritis --- p.34 / Chapter 1.532 --- Cartilage oligometric matrix protein-induced arthritis --- p.35 / Chapter 1.533 --- Oil-induced arthritis --- p.35 / Chapter 1.534 --- Streptococcal cell wall-induced arthritis --- p.35 / Chapter 1.535 --- Adjuvant-induced arthritis --- p.36 / Chapter 1.536 --- Pristane-induced arthritis --- p.36 / Chapter 1.6 --- Current anti-arthritic therapies --- p.39 / Chapter 1.61 --- Non steroid anti-inflammatory drugs --- p.39 / Chapter 1.62 --- Glucocorticoid --- p.44 / Chapter 1.63 --- Second-line treatment --- p.46 / Chapter 1.631 --- Sulfasalazine --- p.46 / Chapter 1.632 --- Gold salts --- p.47 / Chapter 1 633 --- D-penicillamine --- p.48 / Chapter 1.634 --- Antimalarial --- p.49 / Chapter 1 .635 --- Methotrexate --- p.51 / Chapter 1.64 --- New trends for treatment of arthritis --- p.53 / Chapter 1.641 --- Anti-cytokine therapy --- p.53 / Chapter 1.642 --- Anti-angiogenesis therapy --- p.54 / Chapter 1.7 --- Aims of study --- p.57 / Chapter Chapter 2 --- Material and drugs --- p.62 / Chapter Chapter 3 --- Methodology --- p.62 / Chapter 3.1 --- Animals used and anaesthetization --- p.62 / Chapter 3.2 --- Measurement of plasma protein extravasation --- p.63 / Chapter 3.3 --- Measurement of knee joint sizes --- p.64 / Chapter 3.4 --- Measurement of knee joint blood flow --- p.65 / Chapter 3.5 --- Measurement of histological changes --- p.65 / Chapter 3.51 --- Dissection and fixation --- p.65 / Chapter 3.52 --- Decalcification --- p.66 / Chapter 3.53 --- Processing --- p.66 / Chapter 3.54 --- Embedding --- p.67 / Chapter 3.55 --- Sectioning --- p.67 / Chapter 3.56 --- Staining --- p.69 / Chapter 3.6 --- Data analysis --- p.69 / Chapter 3.61 --- Scoring systems --- p.72 / Chapter Chapter 4 --- A model of monoarthritis in rats --- p.72 / Chapter 4.1 --- Introduction --- p.72 / Chapter 4.2 --- Method --- p.73 / Chapter 4.3 --- Results --- p.73 / Chapter 4.31 --- Lewis rats --- p.73 / Chapter 4.32 --- Sprague-Dawley (SD) rats --- p.74 / Chapter 4.33 --- Comparison of FCA-induced changes in Lewis and SD rats --- p.74 / Chapter 4.34 --- Histological studies on arthritic SD rats --- p.75 / Chapter 4.4 --- Discussion --- p.93 / Chapter 4.5 --- Conclusions --- p.95 / Chapter Chapter 5 --- Effect of Substance P on adjuvant-induced arthritis --- p.96 / Chapter 5.1 --- Introduction --- p.96 / Chapter 5.2 --- Method --- p.98 / Chapter 5.3 --- Results --- p.99 / Chapter 5.31 --- Evans blue extravasation --- p.99 / Chapter 5.32 --- Joint size --- p.100 / Chapter 5.33 --- Knee joint blood flow --- p.101 / Chapter 5.34 --- Histology results --- p.102 / Chapter 5.341 --- Infiltration of immune cells in synovial tissue --- p.102 / Chapter 5.342 --- Synovial tissue proliferation --- p.102 / Chapter 5.343 --- Cartilage degradation --- p.103 / Chapter 5.344 --- Bone degradation --- p.103 / Chapter 5.4 --- Discussion --- p.120 / Chapter 5.5 --- Conclusions --- p.125 / Chapter Chapter 6 --- Effects of tachykinin receptor antagonists on FCA-induced arthritis / Chapter 6.1 --- Introduction --- p.126 / Chapter 6.2 --- Method --- p.128 / Chapter 6. 21 --- Intravenous NK1 receptor antagonists on FCA-induced arthritis --- p.128 / Chapter 6. 22 --- Intraperitoneal TK receptor antagonists on FCA-induced arthritis --- p.128 / Chapter 6.3 --- Results --- p.129 / Chapter 6.31 --- Intravenous NK1 227}0اreceptor antagonists on FCA-induced arthritis Evans blue extravasation and joint swelling --- p.129 / Chapter 6.32 --- Intraperitoneal tachykinin receptor antagonists on FCA- induced arthritis Evans blue extravasation and joint swelling --- p.129 / Chapter 6.33 --- Intraperitoneal tachykinin receptor antagonists on FCA- induced immune cell accumulation --- p.130 / Chapter 6.34 --- Intraperitoneal tachykinin receptor antagonists on FCA- induced synovial tissue proliferation --- p.131 / Chapter 6.35 --- Intraperitoneal tachykinin receptor antagonists on FCA- induced cartilage degration and bone erosion --- p.131 / Chapter 6.4 --- Discussion --- p.159 / Chapter 6.5 --- Conclusions --- p.162 / Chapter Chapter 7 --- Individual and combined effects of dexamethasone and TK receptor antagonists on FCA-induced arthritis --- p.163 / Chapter 7.1 --- Introduction --- p.163 / Chapter 7.2 --- Method --- p.166 / Chapter 7.3 --- Results --- p.167 / Chapter 7.31 --- Evans blue extravasation --- p.167 / Chapter 7.32 --- Knee joint size --- p.167 / Chapter 7.33 --- Body weight --- p.168 / Chapter 7.34 --- Cellular infiltration --- p.168 / Chapter 7.35 --- Synovial tissue proliferation --- p.168 / Chapter 7.36 --- Cartilage degradation --- p.169 / Chapter 7.4 --- Discussion --- p.184 / Chapter 7.5 --- Conclusions --- p.187 / Chapter Chapter 8 --- General discussions and conclusions --- p.188 / References --- p.192

Chan Hoi-Huen. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 104-123). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.v / Publications --- p.vi / Table of contents --- p.viii / List of Figures --- p.xii / List of Tables --- p.xiv / Abbreviations --- p.xv / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Blood vessels and blood pressure --- p.1 / Chapter 1.2 --- Smooth muscle --- p.2 / Chapter 1.3 --- Endothelium --- p.3 / Chapter 1.4 --- Vasodilation and Vasoconstriction --- p.5 / Chapter 1.5 --- Calcitonin gene-related peptide (CGRP) --- p.6 / Chapter 1.5.1 --- Discovery of CGRP --- p.6 / Chapter 1.5.2 --- Localization and distribution of CGRP --- p.7 / Chapter 1.5.3 --- Structure profile of CGRP --- p.8 / Chapter 1.5.4 --- CGRP and the vascular system --- p.10 / Chapter 1.6 --- Nitric oxide --- p.11 / Chapter 1.6.1 --- Production of NO by nitric oxide synthase (NOS) --- p.12 / Chapter 1.6.2 --- Actions of NO in smooth muscle --- p.14 / Chapter 1.6.3 --- Synergism with CGRP --- p.14 / Chapter 1.7 --- Other research of CGRP in the laboratory of Professor Ronald R. Fiscus --- p.15 / Chapter 1.8 --- "Aging, diabetes mellitus, sex hormones and cardiovascular system" --- p.16 / Chapter 1.9 --- Aim of study --- p.18 / Chapter Chapter 2 --- Methods and materials --- p.19 / Chapter 2.1 --- General experimental methods --- p.19 / Chapter 2.1.1 --- Measurement of blood pressure in anaesthetized rats --- p.19 / Chapter 2.1.2 --- Tissue bath experiments --- p.20 / Chapter 2.1.2.1 --- Preparation of isolated rat aortic rings --- p.20 / Chapter 2.1.2.2 --- Measurement of contractile and relaxant responses in the rat aortic rings --- p.21 / Chapter 2.1.3 --- Culture of aortic rat vascular smooth muscle cells --- p.22 / Chapter 2.1.4 --- Immunostaining for smooth muscle α-actin in cultured smooth muscle cells --- p.23 / Chapter 2.1.5 --- Determination of nitrite levels in smooth muscle cell culture media --- p.24 / Chapter 2.1.6 --- Measurement of protein contents --- p.25 / Chapter 2.1.7 --- Reversed Transcription- Polymerase Chain Reaction (RT-PCR) --- p.26 / Chapter 2.1.7.1 --- mRNA isolation --- p.26 / Chapter 2.1.7.2 --- Reverse transcription (RT) --- p.27 / Chapter 2.1.7.3 --- Polymerase chain reaction (PCR) --- p.27 / Chapter 2.1.7.4 --- Agarose slab gel electrophoresis --- p.29 / Chapter 2.1.7.5 --- Capillary electrophoresis --- p.29 / Chapter 2.2 --- Reagents --- p.30 / Chapter Chapter 3 --- Impairment of hypotension to calcitonin gene-related peptide in female rats with streptozotocin-induced diabetes mellitus or ovariectomy --- p.40 / Chapter 3.1 --- Introduction --- p.40 / Chapter 3.2 --- Methods --- p.45 / Chapter 3.2.1 --- Animal Preparation --- p.45 / Chapter 3.2.2 --- Statistical analysis --- p.46 / Chapter 3.3 --- Results --- p.47 / Chapter 3.3.1 --- "Body weight, blood glucose and initial blood pressure" --- p.47 / Chapter 3.3.2 --- Hypotensive responses to CGRP in ovariectomized rats --- p.48 / Chapter 3.3.3 --- Hypotensive responses to CGRP in diabetic rats --- p.49 / Chapter 3.3.4 --- Hypotensive responses to CGRP in rats with diabetes and ovariectomy --- p.50 / Chapter 3.4 --- Discussion --- p.50 / Chapter 3.5 --- Conclusions --- p.56 / Chapter Chapter 4 --- Severe impairment of CGRP-induced hypotension in vivo and vasorelaxation in vitro in elderly rats --- p.61 / Chapter 4.1 --- Introduction --- p.61 / Chapter 4.2 --- Methods --- p.64 / Chapter 4.2.1 --- Tissue preparation for vascular rings --- p.64 / Chapter 4.2.2 --- Vasorelaxation studies in vitro --- p.65 / Chapter 4.2.3 --- Animal preparation for in vivo studies --- p.65 / Chapter 4.2.4 --- Measurement of hypotensive responses to CGRP --- p.66 / Chapter 4.2.5 --- Statistical analysis --- p.66 / Chapter 4.3 --- Results --- p.67 / Chapter 4.3.1 --- Effect of age on CGRP-induced vasorelaxations in rings of thoracic aorta and caudal arteries --- p.67 / Chapter 4.3.2 --- Effect of age on acetylcholine-induced responses in aortic rings --- p.68 / Chapter 4.3.3 --- CGRP-induced hypotension in young female and male rats --- p.68 / Chapter 4.3.4 --- CGRP-induced hypotension in elderly female and male rats --- p.68 / Chapter 4.3.5 --- CGRP-induced hypotension in elderly female rats with ovariectomy --- p.69 / Chapter 4.4 --- Discussion --- p.69 / Chapter 4.5 --- Conclusions --- p.73 / Chapter Chapter 5 --- "Effects of CGRP on interleukin-Iβ-, lipopolysaccharides- and ginseng extract-induced production of nitrite oxide in vascular smooth muscle cells of elderly rats" --- p.82 / Chapter 5.1 --- Introduction --- p.82 / Chapter 5.2 --- Methods --- p.83 / Chapter 5.2.1. --- Animal model --- p.83 / Chapter 5.2.2. --- Culture of vascular smooth muscle cells --- p.84 / Chapter 5.2.3 --- Extraction of total RNA --- p.84 / Chapter 5.2.4 --- Reverse transcription and polymerase chain reaction (RT-PCR) --- p.35 / Chapter 5.2.5 --- Capillary electrophoresis with laser-induced fluorescence detector (CE-LIF) --- p.85 / Chapter 5.2.6 --- Determination of nitrite levels in smooth muscle cell culture media --- p.85 / Chapter 5.2.7 --- Measurement of protein contents --- p.86 / Chapter 5.3 --- Results --- p.86 / Chapter 5.3.1 --- "Effects of IL-Iβ, alone and in combination with CGRP, on NO production in young and elderly VSMCs" --- p.86 / Chapter 5.3.2 --- "Effects of LPS, alone and in combination with CGRP, on NO production in young and elderly VSMCs" --- p.89 / Chapter 5.3.3 --- "Effects of ginseng extract, alone and in combination with CGRP, on NO production in VSMCs" --- p.89 / Chapter 5.4 --- Discussion --- p.90 / Chapter 5.5 --- Conclusions --- p.93 / Chapter Chapter 6 --- General discussion and Conclusions --- p.100 / References --- p.104

Leung, Jackie. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 117-131). / Abstract also in Chinese. / Abstract --- p.i / 論文摘要 --- p.iii / Acknowledgement --- p.iv / List of publications --- p.vi / List of Tables --- p.vi / List of Figures --- p.vi / Table of Contents --- p.ix / Chapter Chapter 1: --- Introduction --- p.1 / Chapter 1.1. --- Liver cancer --- p.1 / Chapter 1.1.1. --- Hepatocellular Carcinoma (HCC) --- p.2 / Chapter 1.2. --- Lung Cancer --- p.5 / Chapter 1.3. --- Pteris semipinnata L --- p.8 / Chapter 1.4. --- Extract of PsL: 5F --- p.10 / Chapter 1.5. --- Animal models in chemotherapy researches --- p.13 / Chapter 1.5.1. --- Model of HCC --- p.13 / Chapter 1.5.2. --- Model of lung cancer --- p.15 / Chapter 1.6. --- Apoptosis: Significance of programmed cell death --- p.17 / Chapter 1.6.1. --- The extrinsic pathway --- p.18 / Chapter 1.6.2. --- The intrinsic pathway --- p.19 / Chapter 1.7. --- Apoptotic molecules related to this study --- p.22 / Chapter 1.7.1. --- Bcl-2 family --- p.22 / Chapter 1.7.1.1. --- Bax --- p.22 / Chapter 1.7.1.2. --- Bcl-2 --- p.23 / Chapter 1.7.2. --- Nuclear factor kappa B --- p.25 / Chapter 1.7.3. --- Inducible nitric oxide synthase --- p.27 / Chapter 1.8. --- Side-effects of chemotherapy --- p.29 / Chapter 1.8.1. --- Chemotherapy and liver dysfunction --- p.30 / Chapter 1.8.2. --- Nephrotoxicity of chemotherapeutic agents --- p.31 / Chapter 1.9. --- Aim of study --- p.33 / Chapter Chapter 2: --- Materials and Methodology --- p.34 / Chapter 2.1. --- Animals --- p.34 / Chapter 2.1.1. --- HCC model --- p.34 / Chapter 2.1.2. --- Lung cancer model --- p.35 / Chapter 2.2. --- Tumors induction --- p.36 / Chapter 2.2.1. --- HCC induction in C3H/HeJ mice --- p.36 / Chapter 2.2.2. --- Lung cancer induction in A/J mice --- p.37 / Chapter 2.3. --- 5F preparation --- p.38 / Chapter 2.4. --- 5F treatment --- p.39 / Chapter 2.5. --- Harvest of samples and tissues --- p.41 / Chapter 2.6. --- Tumor assessment --- p.43 / Chapter 2.7. --- Investigation of apoptosis and cell proliferation --- p.44 / Chapter 2.8. --- Immunohistochemistry --- p.47 / Chapter 2.9. --- Biochemical test --- p.51 / Chapter 2.9.1. --- Liver Function Tests (LFT) --- p.52 / Chapter 2.9.1.1. --- Aspartate aminotransferase (AST) & Alanine aminotransferase (ALT) --- p.52 / Chapter 2.9.2. --- Renal Function Test (RFT) --- p.53 / Chapter 2.9.2.1. --- Serum creatinine level (CRE) --- p.53 / Chapter 2.9.2.2. --- Blood Urea Nitrogen index (BUN) --- p.54 / Chapter 2.10. --- Statistical analysis --- p.55 / Chapter Chapter 3: --- Results --- p.56 / Chapter 3.1. --- Anti-tumor effect of 5F is dose- dependent --- p.56 / Chapter 3.2. --- 5F reduces cell proliferation and induces apoptosis in-vivo --- p.60 / Chapter 3.3. --- Effects of 5F on apoptotic signaling molecules --- p.68 / Chapter 3.3.1. --- 5F up-regulates pro-apoptotic Bax and Bak --- p.68 / Chapter 3.3.2. --- 5F down-regulates anti-apoptotic NF-kappa B and Bcl-2 --- p.76 / Chapter 3.3.3. --- 5F up-regulated iNOS in HCC but not in lung cancer --- p.88 / Chapter 3.3.4. --- Regulation on Erk1/2 was associated with treatment of 5F --- p.93 / Chapter 3.4. --- Side-effect studies of 5F --- p.97 / Chapter Chapter 4: --- Discussion --- p.105 / Chapter Chapter 5: --- Conclusion --- p.116 / Bibliography --- p.117

Ng, Sze Ying. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 110-124). / Abstract also in Chinese. / Thesis Committee --- p.i / Acknowledgements --- p.ii / Contents --- p.iii / Abstract --- p.vii / 論文摘要 --- p.x / Abbreviations --- p.xi / List of Figures --- p.xiii / List of Tables --- p.xvii / Chapter CHAPTER ONE --- INTRODUCTION --- p.1 / Chapter 1.1 --- Embryonic Stem Cells (ESCs) --- p.1 / Chapter 1.1.1 --- What are ESCs and the characteristics of ESCs --- p.1 / Chapter 1.1.1.1 --- Pluripotent markers --- p.2 / Chapter 1.1.1.2 --- Germ layers' markers --- p.3 / Chapter 1.1.2 --- Mouse ESCs (mESCs) --- p.4 / Chapter 1.1.2.1 --- mESCs co-culture with mitotically inactivated mouse embryonic fibroblast (MEF) feeder layers --- p.4 / Chapter 1.1.2.2 --- Feeder free mESCs --- p.4 / Chapter 1.1.3 --- Promising uses of ESCs and their shortcomings --- p.5 / Chapter 1.1.4 --- Characteristics of ESC-derived cardiomyocytes (ESC-CMs) --- p.6 / Chapter 1.2 --- Cardiovascular diseases (CVD) --- p.7 / Chapter 1.2.1 --- Background --- p.7 / Chapter 1.2.2 --- Current treatments --- p.8 / Chapter 1.2.3 --- Potential uses of ESC-CMs for basic science research and therapeutic purposes --- p.9 / Chapter 1.2.4 --- Current hurdles in application of ESC-CMs for clinical uses --- p.10 / Chapter 1.3 --- Cardiac gene markers --- p.13 / Chapter 1.3.1 --- Atrial-specific --- p.13 / Chapter 1.3.2 --- Ventricular-specific --- p.19 / Chapter 1.4 --- Lentiviral vector-mediated gene transfer --- p.27 / Chapter 1.5 --- Cell cycle in ESCs --- p.29 / Chapter 1.5.1 --- Cell cycle --- p.29 / Chapter 1.5.2 --- Characteristics of cell cycle in ESCs --- p.30 / Chapter 1.6 --- Potassium (K+) channels --- p.31 / Chapter 1.6.1 --- Voltage gated potassium (Kv) channels --- p.32 / Chapter 1.6.2 --- Role of Kv channels in maintenance of membrane potential --- p.32 / Chapter 1.7 --- Objectives and significances --- p.33 / Chapter CHAPTER TWO --- MATERIALS AND METHODS --- p.35 / Chapter 2.1 --- Mouse embryonic fibroblast (MEF) culture --- p.35 / Chapter 2.1.1 --- Derivation of MEF --- p.3 5 / Chapter 2.1.2 --- MEF culture --- p.37 / Chapter 2.1.3 --- Irradiation of MEF --- p.37 / Chapter 2.2 --- mESC culture and their differentiation --- p.38 / Chapter 2.2.1 --- mESC culture --- p.38 / Chapter 2.2.2 --- Differentiation of mESCs --- p.39 / Chapter 2.3 --- Subcloning --- p.40 / Chapter 2.3.1 --- Amplification of Irx4 --- p.40 / Chapter 2.3.2 --- Purification of DNA products --- p.41 / Chapter 2.3.3 --- Restriction enzyme digestion --- p.42 / Chapter 2.3.4 --- Ligation of Irx4 with iDuet101A vector --- p.43 / Chapter 2.3.5 --- Transformation of ligation product into competent cells --- p.43 / Chapter 2.3.6 --- Small scale preparation of bacterial plasmid DNA --- p.44 / Chapter 2.3.7 --- Confirmation of positive clones by restriction enzyme digestion --- p.45 / Chapter 2.3.8 --- DNA sequencing of the cloned plasmid DNA --- p.45 / Chapter 2.3.9 --- Large scale preparation of target recombinant expression vector --- p.45 / Chapter 2.4 --- Lentiviral vector-mediated gene transfer to mESCs --- p.47 / Chapter 2.4.1 --- Lentivirus packaging --- p.47 / Chapter 2.4.2 --- Lentivirus titering --- p.48 / Chapter 2.4.3 --- Multiple transduction to mESCs --- p.48 / Chapter 2.4.4 --- Hygromycin selection on mESCs --- p.49 / Chapter 2.5 --- Selection of stable clone --- p.49 / Chapter 2.5.1 --- Monoclonal establishment and clone selection --- p.49 / Chapter 2.6 --- Differentiation of cell lines after selection --- p.50 / Chapter 2.7 --- Gene expression study on control and Irx4-overexpressed mESC lines --- p.50 / Chapter 2.8 --- Analysis of mESCs at different phases of the cell cycle --- p.55 / Chapter 2.8.1 --- Go/Gi and S phase synchronization --- p.55 / Chapter 2.8.2 --- Cell cycle analysis by propidium iodide (PI) staining followed by flow cytometric analysis --- p.55 / Chapter 2.8.3 --- Gene expression study by qPCR of Kv channel isoforms --- p.56 / Chapter 2.8.4 --- Membrane potential measurement by membrane potential-sensitive dye followed by flow cytometry --- p.57 / Chapter 2.9 --- Apoptotic study --- p.58 / Chapter 2.10 --- Determination of pluripotent characteristic of mESCs --- p.59 / Chapter 2.10.1 --- Expression of germ layers' markers by qPCR --- p.59 / Chapter 2.10.2 --- Differentiation by hanging drop method and suspension method --- p.61 / Chapter CHAPTER THREE --- RESULTS --- p.62 / Chapter 3.1 --- mESC culture --- p.62 / Chapter 3.1.1 --- Cell colony morphology of feeder free mESCs --- p.62 / Chapter 3.2 --- Subcloning --- p.63 / Chapter 3.2.1 --- PCR cloning of Irx4 --- p.63 / Chapter 3.2.2 --- Restriction digestion on iDuet101A --- p.64 / Chapter 3.2.3 --- Ligation of Irx4 to iDuet101A backbone --- p.66 / Chapter 3.2.4 --- Confirmation of successful ligation --- p.67 / Chapter 3.3 --- Lentivirus packaging --- p.68 / Chapter 3.3.1 --- Transfection --- p.68 / Chapter 3.4 --- Multiple transduction of mESCs and hygromycin selection of positively-transduced cells --- p.69 / Chapter 3.5 --- FACS --- p.70 / Chapter 3.6 --- Irx4 and iduet clone selection --- p.71 / Chapter 3.7 --- Characte rization of mESCs after clone selection --- p.74 / Chapter 3.7.1 --- Immunostaining of pluripotent and differentiation markers --- p.74 / Chapter 3.8 --- Differentiation of cell lines after selection --- p.77 / Chapter 3.8.1 --- Size of EBs of the cell lines during differentiation --- p.77 / Chapter 3.9 --- Gene expression study by qPCR --- p.79 / Chapter 3.10 --- Kv channel expression and membrane potential of mESCs at Go/Gi phase and S phases --- p.84 / Chapter 3.10.1 --- Expression of Kv channels subunits at G0/Gi phase and S phase --- p.86 / Chapter 3.10.2 --- Membrane potential at Go/Gi phase and S phase --- p.87 / Chapter 3.11 --- Effects of TEA+ on feeder free mESCs --- p.89 / Chapter 3.11.1 --- Apoptotic study --- p.89 / Chapter 3.11.2 --- Expression of germ layers´ة markers --- p.91 / Chapter 3.11.3 --- Embryo id bodies (EBs) measurement after differentiation --- p.92 / Chapter CHAPTER FOUR --- DISCUSSION --- p.95 / Chapter 4.1 --- Effect of overexpression of Irx4 on the cardiogenic potential of mESCs --- p.95 / Chapter 4.2 --- Role of Kv channels in maintaining the chacteristics of mESCs --- p.99 / Chapter 4.2.1 --- Inhibition of Kv channels led to a redistribution of the proportion of cells in different phases of the cell cycle: importance of Kv channels in cell cycle progression in native ESCs --- p.99 / Chapter 4.2.2 --- Inhibition of Kv channels led to a loss of pluripotency at molecular and functional levels: importance of Kv channels in the fate determination of mESCs --- p.102 / Chapter 4.3 --- Insights from the present investigation on the future uses of ESCs --- p.105 / Conclusions --- p.108 / References --- p.110

Further studies of Stk31 in spermatogenesis in vivo would allow the identification of the asymmetry machinery of GSCs and the signaling mechanism underlying cell fate determination. Further studies of STK31 in cancer stem cells would allow the development of new diagnostic and therapeutic approaches. / In the first part of the experiment, the expression and cellular localization of STK31 were investigated. RT-PCR results showed that STK31 was reactivated in 47 -- 86% of multiple cancers. Immunofluorescent study and GFP tagging experiment showed that STK31 was localized in the cytoplasm and formed aggregated granules that divide asymmetrically during mitosis. Further study by co-staining with E-cadherin demonstrated that the mouse homolog, Stk31, was expressed in the transition state between undifferentiated and differentiated spermatogonia. These data suggest the possible involvement of STK31 in mouse spermatogonia and cancer development. / In the second part of the experiment, the function of Stk31 in mouse spermatogonia was investigated- A GSC culture on an STO feeder layer was established. Studies on growing properties, expression of molecular markers and germ cell transplantation showed that GSC culture maintained spermatogonial stem cell activity. Retinoic acid was then used to induce differentiation of GSC. The differentiation status was confirmed by monitoring the expression of molecular markers. RT-PCR and immunofluorescent study showed that the expression of Stk31 was induced in RA-induced differentiation and Stk31 proteins were asymmetrically distributed during GSC division. Overexpression of Stk31 in GSCs using retroviral transduction induced the differentiation phenotypes. These data indicate the involvement of Stk31 in mouse spermatogonia cell fate determination. / In the third part of the experiment, the function of STK31 in human colon cancer was investigated. A stable STK31 knock-down Caco2 cells were established by stably transfecting two miR RNAi designs with different efficiency into Caco2 cells. Flow cytometry analysis showed that knock-down of STK31 resulted in G1 phase arrest. Cell counts and MTS assays suggested that knock-down of STK31 decreased cell proliferation in confluent cultures. Knock-down of STK31 also enhanced cell attachment to several ECM proteins and decreases cell migration as suggested by attachment assays and migration assays. Moreover, knock-down of STK31 enhanced enterocytic differentiation and inhibited tumorigenicity both in vitro and in vivo as indicated by colony formation assays and xenograft assays. Date obtained from whole genome microarray studies indicate that STK31 regulates these "stemness" properties through altering the expression of key players in various pathways including KIT, SMAD1 and Cyclin D2. These results suggest the involvement of STK31 in colon cancer as a regulator of "sternness". / Spermatogenesis is a complicated process involving mitosis, meiosis and post-meiotic differentiation. Due to the lack of in vitro models, genes that are involved in mammalian spermatogenesis are largely unknown. Spermatogenesis and tumorigenesis share important biological similarities. This co-relation can be signified by a special group of genes called cancer/testis (CT) antigens, which are only expressed in the testes and cancer. Although cancer biology has been extensively studied for decades, promising therapeutic methods are not available for every type of cancer. Recent discovery of cancer stem cells and functional genomics studies have shed light on the development of new diagnostic and therapeutic approaches. This thesis describes the expression, cellular localization and function of a novel CT gene, STK31, in spermatogonia and cancer development. / Fok, Kin Lam Ellis. / "December 2009." / Adviser: H.C. Chan. / Source: Dissertation Abstracts International, Volume: 72-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 143-169). / 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. / Abstract also in Chinese.

Attention-deficit hyperactivity disorder (ADHD) is a common neuropsychiatric disorder with onset at preschool age Approximately 5-10% of school-aged children worldwide have ADHD. Psychostimulants are the most common treatments for ADHD, although the precise etiology and pathological mechanisms underlying ADHD are poorly understood. Animal models could help to elucidate and further the understanding of this disorder. Among the major rodent models of ADHD of the genetic and neurotoxin-exposed animal models, the spontaneously hypertensive rats (SHR) are more extensively studied. Nevertheless, the mechanism of ADHD is complex and the evidence of SHR model for ADHD has been conflicting. Objective. In this work, we combined behavioral, neurochemical, neuroimaging, pharmacological and molecular studies to examine SHR as an animal model of ADHD. At the same time, the results of our studies could help us to explore the potential mechanism of ADHD. Material and methods. We compared the locomotor activity, attention, inhibition, learning and memory of juvenile male SHR with those of age- and gender-matched genetic control Wistar-Kyoto rats (WKY) by using the open field test, Morris water maze and prepulse inhibition test. We employed magnetic resonance imaging (MRI) to measure potential morphological differences between different brain areas of SHR and WKY, and the functional MRI (fMRI) for functional differences in these brain areas. We also measured dopamine concentration and dopamine related genes expression in the different dopamine pathways by using enzyme-linked immunosorbent assay (ELISA) to measure the dopamine concentration and by using real time PCR to assay genes expression. We examined SHR responses to D-amphetamine (D-AMP), which is psychostimulant. These included locomotor activity and inhibition ability during D-AMP treatment, expression of dopamine related genes after D-AMP treatment measured by real time PCR and c-fos protein after repeated treatment of D-AMP by the Western Blotting. Results . Hyperactivity, impulsivity and attention deficit were observed in SHR. Decreased brain volume in caudate-putamen and vermis cerebelli in SHR were demarcated using MRI. Functional MRI (fMRI) and altered c-fos expression indicated plasticity changes of the prefrontal cortex (PFC) in SHR. Dopamine content was found to decrease in mesocortical and mesolimbic dopamine pathways, but increased in the striatum. Dopamine D4 receptors gene and protein expression were decreased in the PFC in SHR. We also found that the expression of the synaptosomal-associated protein 25 (SNAP-25) gene was initially lower in the PFC but higher in the striatum in SHR. However, this disparity of SNAP-25 in the PFC vanished after repeated treatment of D-AMP between SHR and WKY. Conclusions. In the present study, we demonstrated that SHR could be established as an ADHD model by completing complex assessments of face validity, construct validity and prediction validity. We suggested that the "synaptogenesis hypotheses" might contribute to the abnormal release of dopamine and dysfunction of PFC and the striatum in SEER. In conclusion, our results have provided further new information relevant to the understanding of ADHD in human via the analysis of the SHR model. / Li, Qi. / Adviser: David Yen. / Source: Dissertation Abstracts International, Volume: 69-02, Section: B, page: 1375. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 108-125). / 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. / Abstract also in Chinese. / School code: 1307.

Law, Sau Kwan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 111-117). / Abstract also in Chinese. / Thesis Committee --- p.i / Acknowledgements --- p.ii / Contents --- p.iii / Abstract --- p.vii / 論文摘要 --- p.x / Abbreviations --- p.xi / List of Figures --- p.xiii / List of Tables --- p.xxiii / Chapter CHAPTER ONE --- INTRODUCTION / Chapter 1.1 --- Embryonic Stem (ES) Cells / Chapter 1.1.1 --- Characteristics of ES Cells l / Chapter 1.1.2 --- Therapeutic Potential of ES Cells --- p.3 / Chapter 1.1.3 --- Myocardial Infarction and ES cells-derived Cardiomyocytes --- p.4 / Chapter 1.1.4 --- Current Hurdles of Using ES cells-derived Cardiomyocytes for Research and Therapeutic Purposes --- p.6 / Chapter 1.2 --- Transcription Factors for Cardiac Development / Chapter 1.2.1 --- GATA-binding Protein 4 (GATA-4) --- p.8 / Chapter 1.2.2 --- Myocyte Enhancer Factor 2C (MEF2C) --- p.10 / Chapter 1.2.3 --- "NK2 Transcription Factor Related, Locus 5 (Nkx2.5)" --- p.11 / Chapter 1.2.4 --- Heart and Neural Crest Derivatives Expressed 1 /2 (HANDI/2) --- p.11 / Chapter 1.2.5 --- T-box Protein 5 (Tbx5) --- p.13 / Chapter 1.2.6 --- Serum Response Factor (SRF) --- p.14 / Chapter 1.2.7 --- Specificity Protein 1 (Spl) --- p.15 / Chapter 1.2.8 --- Activator Protein 1 (AP-1) --- p.16 / Chapter 1.3 --- Reactive Oxygen Species (ROS) / Chapter 1.3.1 --- Cellular Production of ROS --- p.18 / Chapter 1.3.2 --- Maintenance of Redox balance --- p.18 / Chapter 1.3.3 --- Redox Signaling --- p.19 / Chapter 1.4 --- Nitric Oxide (NO) and NO Signaling --- p.20 / Chapter 1.5 --- Aims of the Study --- p.22 / Chapter CHAPTER TWO --- MATERIALS AND METHODS / Chapter 2.1 --- Mouse Embryonic Fibroblast (MEF) Culture / Chapter 2.1.1 --- Derivation of MEF --- p.23 / Chapter 2.1.2 --- Maintenance of MEF Culture --- p.24 / Chapter 2.1.3 --- Irradiation of MEF --- p.25 / Chapter 2.2 --- Mouse ES Cell Culture / Chapter 2.2.1 --- Maintenance of Undifferentiated Mouse ES Cell Culture --- p.26 / Chapter 2.2.2 --- Differentiation of Mouse ES Cells --- p.26 / Chapter 2.2.3 --- Exogenous addition of hydrogen peroxide (H2O2) and NO --- p.27 / Chapter 2.3 --- ROS Localization Study / Chapter 2.3.1 --- Frozen Sectioning --- p.28 / Chapter 2.3.2 --- Confocal microscopy for ROS detection --- p.28 / Chapter 2.4 --- Intracellular ROS Measurement / Chapter 2.4.1 --- "Chemistry of 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA)" --- p.29 / Chapter 2.4.2 --- Flow Cytometry for ROS Measurement --- p.29 / Chapter 2.5 --- Gene Expression Study / Chapter 2.5.1 --- Primer Design --- p.30 / Chapter 2.5.2 --- RNA Extraction --- p.31 / Chapter 2.5.3 --- DNase Treatment --- p.32 / Chapter 2.5.4 --- Reverse Transcription --- p.32 / Chapter 2.5.5 --- Quantitative Real Time PCR --- p.33 / Chapter 2.5.6 --- Quantification of mRNA Expression --- p.34 / Chapter 2.6 --- Protein Expression Study / Chapter 2.6.1 --- Total Protein Extraction --- p.34 / Chapter 2.6.2 --- Nuclear and Cytosolic Protein Extraction --- p.35 / Chapter 2.6.3 --- Measurement of Protein Concentration --- p.36 / Chapter 2.6.4 --- De-sumoylation Assay --- p.36 / Chapter 2.6.5 --- De-phosphorylation Assay --- p.37 / Chapter 2.6.6 --- De-glycosylation Assay --- p.38 / Chapter 2.6.7 --- Western Blot --- p.39 / Chapter 2.7 --- Statistical Analysis --- p.41 / Chapter CHAPTER THREE --- RESULTS / Chapter 3.1 --- Study of Endogenous ROS / Chapter 3.1.1 --- Level and Distribution of Endogenous ROS --- p.47 / Chapter 3.1.2 --- Quantification of intracellular ROS --- p.48 / Chapter 3.2 --- Effect of Exogenous Addition of Nitric Oxide (NO) on Cardiac Differentiation / Chapter 3.2.1 --- Beating Profile of NO-treated Embryoid Bodies (EBs) --- p.50 / Chapter 3.3 --- Effect of Exogenous Addition of H2O2 on Cardiac Differentiation / Chapter 3.3.1 --- Beating Profile of H2O2-treated EBs --- p.51 / Chapter 3.3.2 --- mRNA Expression of Cardiac Structural Genes --- p.52 / Chapter 3.3.3 --- Protein Expression of Cardiac Structural Genes --- p.54 / Chapter 3.3.4 --- mRNA Expression of Cardiac Transcription Factors --- p.58 / Chapter 3.3.5 --- Protein Expression of Cardiac Transcription Factors --- p.67 / Chapter 3.3.6 --- Post-translational Modifications of Cardiac Transcription Factors --- p.74 / Chapter 3.3.7 --- Translocation of Cardiac Transcription Factors --- p.89 / Chapter CHAPTER FOUR --- DISCUSSION / Chapter 4.1 --- Changes in the Level of Endogenous ROS During Cardiac Differentiation of Mouse ES Cells --- p.96 / Chapter 4.2 --- H2O2 and NO Have Opposite Effects Towards Cardiac Differentiation --- p.97 / Chapter 4.3 --- Exogenous Addition of H2O2 Advances Differentiation of Mouse ES Cells into Cardiac Lineage --- p.99 / Chapter 4.4 --- Possible Role of H2O2 in Mediating Cardiac Differentiation of Mouse ES Cells --- p.103 / Chapter 4.5 --- Future Directions --- p.108 / Conclusions --- p.110 / References --- p.111

Serotonin (5-hydroxytryptamine, 5-HT) is a neurotransmitter involved in the embryonic neural development and adult neurogenesis. But the effects of 5-HT on stem cells are not fully known. In this study, the effects and underlying signal pathways of 5- HT on proliferation and neural differentiation of mouse embryonic stem (ES) cells, neural progenitor (NP) cell line C 17.2 and embryonic neural stem (NS) cells were explored. Molecular analysis, immunostaining and western blotting revealed that NP/NB cells expressed the rate-limiting enzyme tryptophan hydroxylase (TPH) and produced endogenous 5-HT. While mouse ES cells showed no expression of TPH. Quantitative PCR demonstrated that ES cells and NPINS cells expressed majority of 5-HT receptor sUbtypes. In serum free propagation culture, WST1, BrdU incorporation and neural colony forming cell assay demonstrated that 5-HT enhanced proliferation of ES cells and NPINS cells in a dose-dependent manner. Tryptophan hydroxylase (TPH) inhibitor para-chlorophenylalanine (PCPA) which can inhibit biosynthesis of endogenous 5-HT decreased viability of mouse NP/NS cells. Mouse ES cells derived embryoid bodies (EB) and NS/NP cells were subjected to neural induction in serum-free medium with and without 5-HT or PCPA. On day 8 of EB cultures, immunofluorescence staining displayed a less percentage of SSEA-1+ cells derived from cultures supplemented with 5-HT. Nestin positivity are comparable. Quantitative PCR analysis suggested that supplement of 5-HT in EB culture inhibit neural differentiation of ES cells and induce mesodermal commitment. On day 21 of ES cells neural induction, compared to cultures without 5-HT treatment, a significantly less number of ß-tubulin III+ neurons, GEAP+ astrocytes and GaIC+ oligodendrocytes were noted in 5-HT -supplemented cultures. For NS/NP cells, the inhibitory effects of 5-HT on neuronal and oligodendrocytic commitment were also observed. And the application of PCPA exerted a promoting effect on neural differentiation of NS cells. Manipulating 5-HT level can affect the expression level of key genes which involved in 5-HT metabolism. ES and NS/NP cells treated with 5-HT showed decreased production of endogenous reactive oxygen species (ROS). 5-HT demonstrated a significant anti-apoptotic effect on NP cells and this antiapoptotic effect may be mediated by up-regulated expression of anti-apoptotic gene Bel- 2. Whole genome cDNA microarray analysis and quantitative RT-PCR revealed that notch signal pathway was involved in mediating the biological effects of 5-HT. Western blotting further confirmed that 5-HT treatment up-regulated the protein level of NICD and notch downstream effectors Hes-l and Hes-5. Finally, the therapeutic effects of ES cell-derived neural cells were testified in a mouse model of global ischemia. Two weeks post-transplantation, BrdU labeled ES cell-derived neural cells survived and migrated throughout brain parenchyma. A majority of transplanted cells remained nestin positive. The cognitive functions of cell transplanted groups showed significant recovery compared with untransplanted arms, but no significant difference was observed between transplanted groups treated with and without 5-HT. Taken together, data of this study indicated 5-HT play an important role in neural development and ES cell-derived neural cells might be applicable in the treatment of stroke. / Li, Jin. / "November 2011." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 195-241). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstracts in English. / ACKNOWLEDGEMENTS --- p.i / LIST OF PUBLICATIONS --- p.ii / ABSTRACT --- p.iii / ABSTRACT [in Chinese] --- p.v / TABLE OF CONTENT --- p.vi / LISTS OF FLOWCHARTS --- p.xii / LISTS OF FIGURES --- p.xiii / LIST OF TABLES --- p.xvi / LIST OF EQUIPMENTS --- p.xvii / LIST OF ABBREVATIONS --- p.xvii / Chapter Chapter1 --- Introduction --- p.1 / Chapter 1.1 --- Central nervous system disorder --- p.1 / Chapter 1.1.1 --- Stroke --- p.1 / Chapter 1.1.2 --- Spinal cord injuries --- p.4 / Chapter 1.1.3 --- Parkinson's disease --- p.6 / Chapter 1.1.4 --- Amyotrophic Lateral Sclerosis --- p.8 / Chapter 1.2 --- Stem cell therapy --- p.10 / Chapter 1.2.1 --- General considerations in stem cell therapy --- p.11 / Chapter 1.2.2 --- Stem cell therapy for stroke --- p.11 / Chapter 1.2.3 --- Stem cell therapy for spinal cord injury --- p.15 / Chapter 1.2.4 --- Stem cell therapy for Parkinson's disease --- p.16 / Chapter 1.2.5 --- Stem cell therapy for ALS --- p.18 / Chapter 1.3 --- Stem cells --- p.20 / Chapter 1.3.1 --- Embryonic stem cells --- p.21 / Chapter 1.3.1.1 --- Derivation and characterization --- p.21 / Chapter 1.3.1.2 --- Biology of ES cells --- p.21 / Chapter 1.3.1.2.1 --- Pluripotency of ES cells --- p.21 / Chapter 1.3.1.2.2 --- Differentiation of ES cells to multiple lineages --- p.24 / Chapter 1.3.1.2.2.1 --- Ectodermal differentiation --- p.25 / Chapter 1.3.1.2.2.2 --- Mesodermal differentiation --- p.27 / Chapter 1.3.1.2.2.3 --- Endodermal differentiation --- p.28 / Chapter 1.3.2 --- Neural stem cells --- p.30 / Chapter 1.3.2.1 --- Derivation and characterization --- p.30 / Chapter 1.3.2.2 --- Biology of NS cells --- p.32 / Chapter 1.3.3 --- Induced pluripotent stem cells --- p.34 / Chapter 1.3.4 --- Mesenchymal stem cells --- p.35 / Chapter 1.4 --- Serotonin (5-HT) --- p.36 / Chapter 1.4.1 --- Distribution --- p.37 / Chapter 1.4.2 --- Metabolism --- p.37 / Chapter 1.4.3 --- Biological effects of 5-HT --- p.38 / Chapter 1.4.4 --- Serotonin receptor subtypes and receptor signal transduction pathways --- p.40 / Chapter Chapter2 --- Aim --- p.43 / Chapter 2.1 --- Hypothesis and study objectives --- p.43 / Chapter Chapter3 --- Materials and Methods --- p.49 / Chapter 3.1 --- Chemicals and Reagents --- p.49 / Chapter 3.1.1 --- Cell culture --- p.49 / Chapter 3.1.2 --- Serotonin, serotonin receptor subtypes specific agonists/antagonists and drugs that regulate serotonin metabolism --- p.51 / Chapter 3.1.3 --- Cell proliferation assay --- p.52 / Chapter 3.1.4 --- Cell apoptosis assay --- p.52 / Chapter 3.1.5 --- Immunohistochemistry and staining --- p.52 / Chapter 3.1.6 --- Western blotting --- p.55 / Chapter 3.1.7 --- Molecular biology --- p.56 / Chapter 3.1.8 --- Whole genome cDNA micro array --- p.58 / Chapter 3.1.9 --- MAO activity assay --- p.58 / Chapter 3.1.10 --- Endogenous ROS production assay --- p.58 / Chapter 3.2 --- Consumable --- p.58 / Chapter 3.3 --- Cells --- p.60 / Chapter 3.3.1 --- Feeder cell --- p.60 / Chapter 3.3.1.1 --- Mouse embryonic fibroblasts --- p.60 / Chapter 3.3.2 --- ES cells --- p.61 / Chapter 3.3.2.1 --- ES cell D3 --- p.61 / Chapter 3.3.2.2 --- ES cell-E14TG2a --- p.61 / Chapter 3.3.3 --- NS cells --- p.61 / Chapter 3.3.3.1 --- Neural progenitor cells line C172 --- p.61 / Chapter 3.3.3.2 --- Mouse embryonic neural stem cells --- p.61 / Chapter 3.4 --- In-house prepared solutions --- p.62 / Chapter 3.4.1 --- Stock solution ofInsulin, Transferrin, Selentine (ITS) Supplement --- p.63 / Chapter 3.4.2 --- Gelatin solution 01% --- p.62 / Chapter 3.4.3 --- Paraformaldehyde solution 4% (PFA) --- p.62 / Chapter 3.4.4 --- Tritox X-lOO solution 03% --- p.63 / Chapter 3.4.5 --- Popidium iodide solution 1 ug/ml (PI) --- p.63 / Chapter 3.4.6 --- Poly-L-ornithine solution --- p.63 / Chapter 3.4.7 --- Laminin solution --- p.64 / Chapter 3.4.7 --- MEF Maintenance medium --- p.64 / Chapter 3.4.9 --- Cryopreservation Media for MEF and C172 (2X) --- p.64 / Chapter 3.4.10 --- Cryopreservation Media for mouse ES cell (2X) --- p.65 / Chapter 3.4.11 --- Cryopreservation Media for mouse NS cell (2X) --- p.65 / Chapter 3.4.12 --- Serum based maintenance medium for C172 --- p.65 / Chapter 3.4.13 --- Serum free maintenance medium for C172 --- p.66 / Chapter 3.4.14 --- Serum-based propagation medium for ES cells --- p.66 / Chapter 3.4.15 --- Serum-free propagation medium forES cells --- p.67 / Chapter 3.4.16 --- Serum-free induction medium for ES cells --- p.67 / Chapter 3.4.16.1 --- Serum-free induction medium I --- p.67 / Chapter 3.4.16.2 --- Serum-free induction medium II --- p.68 / Chapter 3.4.16.3 --- Serum-free induction medium III --- p.68 / Chapter 3.4.17 --- Tris-HCl (1 M), pH 74 --- p.68 / Chapter 3.4.18 --- Tris-HCl (1 M), pH 87 --- p.69 / Chapter 3.4.19 --- Tris-HCI (1 M), pH 69 --- p.69 / Chapter 3.4.20 --- APS 10% (wt/vol) --- p.69 / Chapter 3.4.21 --- Protease inhibitor (10X) --- p.70 / Chapter 3.4.22 --- RIPA --- p.70 / Chapter 3.4.23 --- Resolving buffer (8X) --- p.70 / Chapter 3.4.24 --- Stacking buffer (4X) --- p.71 / Chapter 3.4.25 --- Protein running buffer (lOX) --- p.71 / Chapter 3.4.26 --- Transfer buffer (10X) --- p.72 / Chapter 3.4.27 --- Transfer buffer (IX) --- p.72 / Chapter 3.4.28 --- Blocking buffer (lOX) --- p.72 / Chapter 3.4.29 --- TBS (10X) --- p.73 / Chapter 3.4.30 --- TBS-T (IX) --- p.73 / Chapter 3.4.31 --- Stacking gel --- p.73 / Chapter 3.4.32 --- Resolving gel --- p.74 / Chapter 3.5 --- Methods --- p.75 / Chapter 3.5.1 --- Cell culture --- p.75 / Chapter 3.5.1.1 --- Preparation of acid washed cover slips --- p.75 / Chapter 3.5.1.2 --- Preparation of gelatinized culture wares --- p.75 / Chapter 3.5.1.3 --- Poly-L-omithine and laminin coating --- p.76 / Chapter 3.5.1.4 --- Thawing cryopreserved cells --- p.76 / Chapter 3.5.1.5 --- Passage of culture --- p.77 / Chapter 3.5.1.5 --- 6 Cell count --- p.78 / Chapter 3.5.1.7 --- Cytospin --- p.78 / Chapter 3.5.1.8 --- Trypan blue dye exclusion test --- p.78 / Chapter 3.5.1.9 --- Cryopreservation --- p.79 / Chapter 3.5.1.10 --- Derivation and culture of mouse embryonic fibroblasts (MEF) --- p.79 / Chapter 3.5.1.11 --- Propagation of ES cells in serum-based/free medium --- p.81 / Chapter 3.5.1.12 --- Neural differentiation ofES cells --- p.83 / Chapter 3.5.1.13 --- Propagation ofNP cell C172 in serum-based or serum-free medium --- p.84 / Chapter 3.5.1.14 --- Neural differentiation ofC172 --- p.85 / Chapter 3.5.1.15 --- Derivation and propagation of embryonic NS cells --- p.85 / Chapter 3.5.1.13 --- Neural differentiation of embryonic NS cells --- p.86 / Chapter 3.5.1.17 --- BrdU labeling of the ES cells derived products --- p.87 / Chapter 3.5.2 --- Cell proliferation assay --- p.87 / Chapter 3.5.2.1 --- Cell morphology --- p.87 / Chapter 3.5.2.2 --- WST-1 assay --- p.88 / Chapter 3.5.2.3 --- BrdU incorporation assay --- p.88 / Chapter 3.5.2.4 --- NCFC assay --- p.89 / Chapter 3.5.3 --- Conventional and quantitative RT-PCR --- p.89 / Chapter 3.5.3.1 --- RNA extraction --- p.89 / Chapter 3.5.3.2 --- RNA quantitation --- p.90 / Chapter 3.5.3.3 --- Reverse Transcription ofthe First Strand complementary DNA --- p.90 / Chapter 3.5.3.4 --- Polymerase chain reaction --- p.91 / Chapter 3.5.3.5 --- RNA Integrity Check --- p.91 / Chapter 3.5.3.6 --- Electrophoresis and visualization of gene products --- p.91 / Chapter 3.5.3.7 --- Real-time quantitative PCR --- p.92 / Chapter 3.5.4 --- Microarray --- p.94 / Chapter 3.5.5 --- Immunofluoresent staining --- p.94 / Chapter 3.5.6 --- Western blot --- p.95 / Chapter 3.5.6.1 --- Harvesting samples --- p.95 / Chapter 3.5.6.2 --- Protein extraction --- p.96 / Chapter 3.5.6.3 --- Protein quantification --- p.96 / Chapter 3.5.6.4 --- SDS-PAGE --- p.97 / Chapter 3.5.6.5 --- Wet transfer of protein to PVDF membrane --- p.97 / Chapter 3.5.6.6 --- Blocking the membrane --- p.97 / Chapter 3.5.6.7 --- Immunoblotting --- p.97 / Chapter 3.5.6.8 --- Signal detection --- p.98 / Chapter 3.5.7 --- Cell apoptosis assay --- p.98 / Chapter 3.5.7.1 --- ANNEXINV-FITC apoptosis detection --- p.98 / Chapter 3.5.7.2 --- TUNEL --- p.99 / Chapter 3.5.8 --- Endogenous ROS assay --- p.100 / Chapter 3.5.9 --- In vivo studies --- p.101 / Chapter 3.5.9.1 --- Induction of cerebral ischemia in mice --- p.101 / Chapter 3.5.9.2 --- Transplantation --- p.101 / Chapter 3.5.9.3 --- Assessment of learning ability and memory --- p.102 / Chapter 3.5.10 --- Histological analysis --- p.103 / Chapter 3.5.10.1 --- Animal sacrifice for brain harvest --- p.103 / Chapter 3.5.10.2 --- Cryosectioning --- p.103 / Chapter 3.5.10.3 --- Haematoxylin and eosin staining --- p.104 / Chapter 3.6 --- Data analysis --- p.104 / Chapter Chapter4 --- Results --- p.113 / Chapter 4.1 --- Expression profile of 5-HT receptors and metablism of endogenous 5-HT --- p.113 / Chapter 4.1.1 --- Expression profiles of 5-HT receptors in stem cells --- p.113 / Chapter 4.1.2 --- Biosynthesis of endogenous 5-HT --- p.115 / Chapter 4.2 --- Effects of 5-HT on proliferation of mouse ES cells and NS cells --- p.115 / Chapter 4.2.1 --- Effects of 5-HT on proliferation ofES cells --- p.115 / Chapter 4.2.2 --- Effects of 5-HT on proliferation ofNP and NS cells --- p.117 / Chapter 4.3 --- Effects of 5-HT on differentiation of mouse ES cells and NS cells --- p.119 / Chapter 4.3.1 --- Neural differentiation ofES cells --- p.119 / Chapter 4.3.2 --- Effects of 5-HT on differentiation ofES cells --- p.119 / Chapter 4.3.3 --- Neural differentiation ofNP and NS cells --- p.120 / Chapter 4.3.4 --- Effects of 5-HT on differentiation ofNP and NS cells --- p.121 / Chapter 4.4 --- 5-HT metabolism in mouse ES cells and NS cells --- p.122 / Chapter 4.4.1 --- Expression of key 5-HT metablic genes in stem cells --- p.122 / Chapter 4.4.2 --- Detection ofROS generation in mouse NS cells --- p.123 / Chapter 4.4.3 --- Effects of 5-HT on expression level of MAO-A, MAO-B and SERT --- p.123 / Chapter 4.5 --- Anti-apoptotic effect of 5-HT on NP and NS cells in neural induction --- p.127 / Chapter 4.6 --- Potential signaling pathways mediated by 5-HT --- p.130 / Chapter 4.7 --- Therapeutic effects of 5-HT treated mouse ES cell-derived cells in a stoke model --- p.130 / Chapter 4.7.1 --- Induction of global ischemia by transient BCCAO --- p.130 / Chapter 4.7.1.1 --- HE staining of post ischemic brain --- p.131 / Chapter 4.7.1.2 --- TUNEL analysis of cell apoptosis at post ischemia day 3 --- p.132 / Chapter 4.7.2 --- Cell labelling --- p.132 / Chapter 4.7.3 --- Cognition monitoring post transplantation --- p.133 / Chapter 4.7.4 --- Survival, migration and differentiation of transplanted neural cells --- p.135 / Chapter Chapter5 --- Discussion --- p.180 / Chapter Chapter6 --- Conclusions --- p.192 / References --- p.195