Conclusion. The present results indicate that, baicalein with optimal dosage of 30 muM suppressed collagen deposition in AngII stimulated SHR CF cultures. In animal model of hypertension, high dose of baicalein feeding for 12 week showed optimal antifibrotic effect in hypertensive hearts. (Abstract shortened by UMI.) / For in-vivo study, comparing to control group, HW/BW (x1000) of SHR was significantly reduced in 12 weeks-high dose baicalein and (-0.78+/-0.23, p=0.014) 12 weeks-Valsartan group (-0.71+/-0.22, p=0.021), however, no significant change was observed in the LW/BW ratio. / In Blood pressure control, no effects on attenuation of SBP were observed after 4 weeks and 12 weeks daily administration of baicalein, only 12 weeks feeding of Valsartan significantly down-regulated the systolic blood pressure by -19.25+/-10.09 mmHg, p=0.049. / In the in-vivo study, SHR was used as a model of genetic hypertension. The objectives were: firstly, to determine the efficacy of baicalein in the prevention of myocardial fibrosis (interstitial fibrosis) in SHR, & compared with WKY rats as normal controls. Secondly, to determine if over-expression of pro-collagen I (and III, if any) gene in the ventricles could be normalized by baicalein. Thirdly, to determine if left ventricular hypertrophy in SHR is improved by baicalein. Furthermore, to determine if blood pressure and blood biochemistry parameters (plasma level of brain natriuretic peptides (BNP), and serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) level could be alternated by baicalein. Besides, to determine the body weight (BW), heart weight to body ratio (HW/BW), liver weight to body weight ratio (LW/BW), serum AST and ALT level could be alternated by baicalein. Finally to evaluate by echocardiography if there are changes of ivss and ivsd in SHR after administration of baicalein. / Keywords. baicalein, wogonin, collagen, cardiac fibrosis, hypertension / Objectives. In the in-vitro study, cardiac fibroblast culture was prepared from neonatal SHR and WKY rats. The objectives were multi-fold: firstly, to determine over-expression of pro-collagen I mRNA (and III, if any) in cardiac fibroblasts cultures could be normalized by baicalein and wogonin after AngII activation. Secondly, to evaluate the efficacy of baicalein and wogonin on the suppression of total collagen protein production in cardiac fibroblasts cultures after AngII activation. Thirdly, to evaluate the mechanism (in protein level) of baicalein and wogonin on regulating collagen deposition in cardiac fibroblasts after AngII activation. Furthermore, to determine if there were any effects on cytotoxicity and membrane integrity of baicalein and wogonin towards cardiac fibroblasts cultures. Finally, to determine the optimal concentration of baicalein and wogonin for the above actions in-vitro. / Results. For in-vitro study, incubation of AngII resulted in significant up-regulation of COL-I and COL-III mRNA and total collagen protein production. Addition of either baicalein or wogonin significantly suppressed the mRNA synthesis and total collagen protein in CF with an optimal dosage of 30 muM. No effects on viability and membrane integrity were observed on baicalein and wogonin towards cardiac fibroblasts cultures. / Kong, Kam Chuen Ebenezer. / Advisers: Cheuk-Man Yu; Gabriel W. K. Yip. / Source: Dissertation Abstracts International, Volume: 71-01, Section: B, page: 0242. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 176-204). / 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. / Abstracts in English and Chinese.

As exogenously administered RA suppressed the expression of the RA synthesizing enzymes, further investigation on whether this would lead to deficiency in endogenous RA concentrations was conducted. Results showed that exogenously administered RA significantly reduced the endogenous RA level in the head region with C57 embryos showing a greater reduction than ICR embryos. / In addition, detailed morphological and histological studies were conducted to determine if RA treatment caused early embryonic changes with strain difference. When compared with ICR embryos, C57 embryos exhibited more pronounced responses to RA, including developmental retardation, underdevelopment of the anterior neural plate and absence of or smaller optic pit/optic vesicle formation. However, RA treatment did not cause abnormal apoptosis in the early stages in both strains. / Since the teratogenic effect of RA is highly developmental stage-dependent, it is possible that there is a difference in the developmental stage between these 2 mouse strains at the time of RA injection. Indeed, it was found that the developmental stage of ICR embryos was approximately 6 hours ahead of C57 embryos. However, the role that this factor plays in the differential strain susceptibility to RA can be excluded since C57 fetuses were still 3 times more susceptible to developing anophthalmia/microphthalmia than ICR fetuses that were subject to RA treatment at equivalent developmental stages. Comparison of susceptibility to RA-induced anophthalmia/microphthalmia was also made among heterozygous fetuses obtained from reciprocal matings between C57 and ICR male and female mice, and those in homozygous ICR and C57 fetuses. Results showed that the C57 strain has conferred both genetic predisposition and maternal effects in increasing the embryo's susceptibility to RA-induced ocular defects. / Since the type of RA-induced ocular defects mimic those that developed in Raldh2 null mutant embryos, the effect of RA treatment on the expression of RA synthesizing enzymes, Raldh2 and Raldh3, and the RA-inducible gene Cyp26a1, as well as some early eye development genes were examined. Exogenously administered RA reduced the mRNA expression levels of Raldh2, Raldh3 and Cyp26a1 in the head region, with C57 embryos showing a greater reduction than ICR embryos. / Taken together, results of this thesis suggest that there is a strain difference in susceptibility to RA-induced ocular defects in which exogenously applied RA suppresses the expression of RA synthesizing enzymes and leads to endogenous RA deficiency. This finding may shed light on understanding why both excess and deficiency of RA can lead to similar types of ocular defects. / To determine if there are strain differences in the susceptibility to RA-induced ocular defects, two mouse strains were used. They are C57BL/6J (C57), mice that spontaneously develop ocular defects and ICR mice, which are not prone to developing ocular defects. Detailed time and dose response studies were conducted and eye defects were examined in near-term fetuses. C57 fetuses were found to be significantly more susceptible to RA-induced anophthalmia/microphthalmia than ICR fetuses. / Vitamin A (retinol) and its most active metabolite, all- trans retinoic acid (RA) is essential for vision in the adult and for eye development in the embryo. It is well documented that in humans, excess intake or deficiency of vitamin A or RA is associated with congenital ocular defects such as microphthalmia. However, the underlying mechanism remains unclear. The aim of this study is to examine the pathogenic mechanism of RA-induced developmental ocular defects. / Lau, Wing Sze Josephine. / Source: Dissertation Abstracts International, Volume: 71-01, Section: B, page: 0240. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 186-211). / 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. / Abstracts in English and Chinese.

A confocal scanning laser ophthalmoscope (CSLO) was used to image the axonal and dendritic aborizations of RGCs in the Thy-1 YFP mice. With quantitative analysis of cell body area, axon diameter, dendritic field, number of terminal branches, total dendritic branch length, branching complexity, symmetry and distance from the optic disc, the morphologies of RGCs and the patterns of axonal and dendritic degeneration were analyzed. After optic nerve crush, RGC damage was observed prospectively to begin with progressive dendritic shrinkage, followed by loss of the axon and the cell body. Similar pattern of RGC degeneration was observed after 90 minutes of retinal ischemia although no morphological changes were detected when the duration of ischemia was shortened to 30 minutes. The rate of dendritic shrinkage was variable and estimated on average 2.0% per day and 11.7% per day with linear mixed modeling, after optic nerve crush and retinal ischemic injury, respectively. RGCs with a larger dendritic field had a slower rate of dendritic shrinkage. / In summary, we demonstrated that dendritic shrinkage could be evident even before axonal degeneration after optic nerve crush and retinal ischemic injury. We have established a methodology for in vivo and direct visualization of RGCs and retinal microglia, which could provide reliable and early markers for neuronal damage. Measuring the rate of dendritic shrinkage and tracking the longitudinal activation of microglia would provide new paradigms to study the mechanism of neurodegenerative diseases and offer new insights in testing novel therapies for neuroprotection. / Progressive neuronal cell death and microglial activation are the key pathological features in most neurodegenerative diseases. While investigating the longitudinal profiles of neuronal degeneration and microglial activation is pertinent to understanding disease mechanism and developing treatment, analyzing progressive changes has been obfuscated by the lack of a non-invasive approach that allows long term, serial monitoring of individual neuronal and microglial cells. Because of the clear optical media in the eye, direct visualization of the retinal ganglion cells (RGCs) and microglia is possible with high resolution in vivo imaging technique. In this study, we developed experimental models to visualize and characterize the cellular morphology of RGCs and retinal microglia in vivo in the Thy-1 YFP and the CX3CR1 +/GFP transgenic mice, described the patterns of axonal and dendritic shrinkage of RGCs, discerned the dynamic profile of microglial activation and investigated the relationship between RGC survival and microglial activation after optic nerve crush and retinal ischemic injury induced by acute elevation of intraocular pressure. / The longitudinal profile of microglial activation was investigated by imaging the CX3CR1GFP/+ transgenic mice with the CSLO. Activation of retinal microglia was characterized with an increase in cell number reaching a peak at a week after optic nerve crush and retinal ischemic injury, which was followed by a gradual decline falling near to the baseline at the 4 th week. The activation of retinal microglia was proportional to the severity of injury. The number of RGCs survival at 4 weeks post-injury was significantly associated with the number of activated retinal microglia. / Li, Zhiwei. / Adviser: Leung Kai Shun. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 50-66). / 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.

Baicalin, which is a flavonoid, was previously shown to exert neuroprotective effects against ischemic injury and oxidative insults. In this study, baicalin was found to induce neuronal differentiation on both C17.2 NSC and primary mouse NSC originated from hippocampuses of E14.5 mouse embryos. The baicalin-mediated differentiation of C17.2 NSC was noted in dose- and time-dependent manners. Baicalin-treated NSC displayed long processes of neurites. The gene expression of neuronal markers, NF-L, TUBB3 and MAP2 was also significantly increased after treated with 20 to 50 muM baicalin on C17.2 NSC. Treating C17.2 NSC with baicalin significantly increased the number of TUBB3 positive cells by 300%. A significant increase in the gene expression of TUBB3 was also observed on primary NSC upon baicalin treatment at 5 to 10 muM. The number of TUBB3 positive cells was increased by 100% after treating with 10 muM baicalin. C17.2 NSC treated with baicalin also increased the gene expression of GABAergic and serotonergic neuronal subtype specific enzymes GAD1 and TPH1. / Nature provides a vast pool of natural compounds with neuroprotection and neurotrophism. A few of these compounds can induce the differentiation of neural stem cells (NSC). There are ample opportunities to discover more natural compounds with differentiation inducing effect on NSC. One of the objectives of this project is to look for novel natural compounds showing neurogenic effect on NSC. This project has established a platform for screening medicinal materials and natural compounds with neural differentiation promoting effect on C17.2 mouse neural stem cell line. Screening results identified total Sanqi saponins, total Renshen saponins, Huangqin extracts and baicalin as potent candidates for inducing this differentiation of NSC. / This project also aims at characterizing the mechanisms involved in the neuronal differentiation effect of baicalin on NSC. Annotation from microarray analysis indicated that baicalin treatment on C17.2 NSC is related to development of tissue and nervous system. qPCR study attested the increased gene expression of nerve growth factor-beta, neurotrophin-3, pro-neural transcriptional factors Ngn1, Ngn2 and NeuroD2. Western blotting showed that baicalin activated ERK1/2 MAP kinase but not JNK and p38 MAP kinases. / This project demonstrated the neurogenic potential of natural resources on NSC. A novel neuronal induction effect of baicalin on NSC was also demonstrated with its mechanisms characterized. This project also revealed that baicalin can be used for promoting functional recovery of post-ischemia animals. / This study showed for the first time that baicalin exerts neuronal differentiation inducing effect on NSC. Another objective of this project is to study whether baicalin can promote functional recovery of animals with ischemia brain injury. Mice having undergone transient occlusion of the bilateral common carotid arteries with blood-reperfusion to induce global cerebral ischemia were treated with baicalin and/or EGFP-NSC. Ischemia animals received implantation of EGFP-NSC into the caudate putamen and/or intravenous injection of baicalin on alternate days for two-week on day seven post-ischemia displayed significant improvement of the cognitive function in terms of the incident of error and escape time in the water T-maze task compared to the control arm of ischemia mice. Data of the study suggested that the therapeutic effect of baicalin would be comparable to that of neural stem cell transplant in improving the cognitive function in a mouse ischemic stroke model. / Li, Ming. / Adviser: P. C. Shaw. / Source: Dissertation Abstracts International, Volume: 73-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 199-232). / 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.

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.

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

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