Cells experiencing a low oxygen tension show relative resistance to the lethal effects of radiation. It is believed that the effectiveness of the treatment of certain human tumors is hindered by the existence of such radioresistant cells within the tumor. The purpose of this work was to study the drug Ro-07-0582 both for its toxic effects and its ability to preferentially sensitize hypoxic cells to the lethal effects of radiation (radiosensitize). These properties were examined in vitro in two Chinese hamster cell lines, CHO and CH2B2, and also in the mouse tumour cell line EMT6. Ro-07-0582 is shown to have a chemotherapeutic potential in that it demonstrates a very selective toxicity for hypoxic cells after a few hours exposure. It is much less toxic to aerobic cells. These toxic properties were studied extensively, both in hypoxic and aerobic cell suspensions. The measured endpoint was the ability of a cell to multiply and form a colony of 50 or more cells within an allotted incubation time. Hypoxic toxicity was greater at 37°C than at 22°C and was affected by small changes (~30ppm) in 0₂ concentration in the cell suspension. The toxic effects were similar in the three cell lines. The radiosensitizing capability of Ro-07-0582 was determined by measuring the Dose Modifying Factors (DMF's) for various drug concentrations with each cell line. DMF's were calculated by comparison of survival curves for cell suspensions irradiated under hypoxia in the presence of drug with the survival curve for cell suspensions irradiated under hypoxia in the absence of drug. The DMF for the irradiation of aerobic cells in the absence of drug is called the Oxygen Enhancement Ratio (OER) and was approximately 3.0 in all three cell lines. Ro-07-0582 was found to selectively radiosensitize hypoxic cells in suspension with high efficiency. For each cell line, sensitization was observed with drug concentrations as low as 0.1mM, while concentrations of 10mM or greater yielded DMF's within the measured range of OER values. The presence of 1mM Ro-07-0582 during irradiation of hypoxic cells yields a DMF of 1.8. Introduction of the drug before or after irradiation, instead of during irradiation, had little if any effect. Radiosensitization measurements were also carried out at high cell concentrations (cell pellets), where many sensitizers are ineffective. Results showed that the 0582 radiosensitization attained in cell pellets is quite comparable with that attained in dilute suspension. The attributes of Ro-07-0582 as a potential radiosensi-tizer were considered. The sensitization achieved by Ro-07-0582 is very good, and surpasses that of metronidazole, a chemical under study for clinical use. For drug doses necessary to achieve high levels of sensitization the toxicity of Ro-07-0582 to aerobic cells is quite acceptable. The toxicity to hypoxic cells, however, is much increased over the toxicity to aerobic cells, and this may prove to be a useful adjunct to the drug's sensitizing properties in destroying hypoxic tumour cells. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

Wang Xiansong. / "February 2004." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (p. 247-285). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Lee, Hoi Ying. / "September 2010." / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 171-189). / Abstracts in English and Chinese. / Abstract (English) --- p.i / Abstract (Chinese) --- p.iii / Acknowledgement --- p.v / Publications --- p.vi / Abbreviations --- p.vii / Table of Content --- p.x / Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Human mast cells and its activation --- p.1 / Chapter 1.2 --- Role of mast cells in inflammation --- p.2 / Chapter 1.3 --- Mast cell heterogeneity --- p.5 / Chapter 1.4 --- Interaction of bone and immune system --- p.1 / Chapter 1.5 --- Introduction of bone system --- p.8 / Chapter 1.6 --- Bone remodeling --- p.9 / Chapter 1.7 --- Regulation of bone remodeling --- p.10 / Chapter 1.8 --- Introduction of Osteoporosis --- p.12 / Chapter 1.9 --- Pathophysiology of osteoporosis --- p.13 / Chapter 1.10 --- Pharmacological interventions in osteoporosis --- p.14 / Chapter 1.11 --- Involvement of mast cells in bone metabolism --- p.18 / Chapter 1.12 --- Aim of study --- p.20 / Chapter 2 --- Materials and Methods --- p.27 / Chapter 2.1 --- Materials --- p.27 / Chapter 2.2 --- Methods --- p.34 / Chapter 2.2.1 --- Human mast cells culture --- p.34 / Chapter 2.2.2 --- Human mast cells characterization --- p.35 / Chapter 2.2.3 --- Histamine release assay --- p.36 / Chapter 2.2.4 --- Immunofluorescence staining of estrogen receptors --- p.37 / Chapter 2.2.5 --- Reverse Transcriptase Polymerase Chain Reaction --- p.37 / Chapter 2.2.6 --- TNF measurement --- p.38 / Chapter 2.2.7 --- Calcium mobilization studies of mast cells --- p.38 / Chapter 2.2.8 --- Statistical analysis --- p.39 / Chapter 3 --- Effects of estrogen and selective estrogen receptor modulators (SERMs) on mediators release from human mast cells --- p.41 / Chapter 3.1 --- Introduction --- p.41 / Chapter 3.2 --- Materials and methods --- p.50 / Chapter 3.3 --- Results --- p.51 / Chapter 3.3.1 --- Characterization of human mast cells --- p.51 / Chapter 3.3.2 --- Effect of estrogen on mediator release from human mast cells --- p.52 / Chapter 3.3.2.1 --- Basal histamine release after treatment of estrogen --- p.52 / Chapter 3.3.2.2 --- Histamine release induced by immunological stimulus --- p.52 / Chapter 3.3.2.3 --- Histamine release induced by chemical secretagogues --- p.54 / Chapter 3.3.3 --- Effect of selective estrogen receptor modulators (SERMs) on mast cell activity --- p.54 / Chapter 3.3.3.1 --- Basal histamine release after SERMs treatment --- p.54 / Chapter 3.3.3.2 --- Histamine release induced by immunological stimulus --- p.55 / Chapter 3.3.3.3 --- Histamine release induced by chemical secretagogues --- p.57 / Chapter 3.3.4 --- Effect of estradiol on TNF-α release from human mast cells --- p.57 / Chapter 3.3.5 --- Effect of SERMs on TNE-α release from human mast cells --- p.58 / Chapter 3.3.6 --- Expression of estrogen receptors on human mast cells --- p.59 / Chapter 3.3.6.1 --- Expression of estrogen receptor after treatment of estradiol --- p.59 / Chapter 3.3.7 --- Expression of various bone remodeling molecules on human mast cells --- p.60 / Chapter 3.3.7.1 --- Expression of bone remodeling molecule after treatment of estradiol --- p.61 / Chapter 3.4 --- Discussion --- p.63 / Chapter 4 --- Effects of anti-osteoporosis Chinese herbal medicines on activity of human mast cells --- p.98 / Chapter 4.1 --- Introduction --- p.98 / Chapter 4.2 --- Materials and methods --- p.103 / Chapter 4.3 --- Results --- p.104 / Chapter 4.3.1 --- Effect of the anti-osteoporosis Chinese herbal formulation ELP on histamine release from human mast cells --- p.104 / Chapter 4.3.1.1 --- Histamine release induced by immunological stimulus --- p.104 / Chapter 4.3.1.2 --- Histamine release induced by chemical secretagogues --- p.105 / Chapter 4.3.2 --- Effect of Herba Epimedii (HEP) on histamine release from human mast cells --- p.105 / Chapter 4.3.2.1 --- Histamine release induced by immunological stimulus --- p.106 / Chapter 4.3.2.2 --- Histamine release induced by chemical secretagogues --- p.106 / Chapter 4.3.3 --- Effect of Fructus Ligustri Lucidi (FLL) on histamine release from human mast cells --- p.107 / Chapter 4.3.3.1 --- Histamine release induced by immunological stimulus --- p.107 / Chapter 4.3.3.2 --- Histamine release induced by chemical secretagogues --- p.107 / Chapter 4.3.4 --- Effect of Fructus Psoraleae (FP) on histamine release from human mast cells --- p.108 / Chapter 4.3.4.1 --- Histamine release induced by immunological stimulus --- p.108 / Chapter 4.3.4.2 --- Histamine release induced by chemical secretagogues --- p.109 / Chapter 4.3.5 --- Effect of various partitions from solvent extraction of HEP on histamine release from human mast cells --- p.109 / Chapter 4.3.5.1 --- Histamine release induced by immunological stimulus --- p.110 / Chapter 4.3.5.2 --- Histamine release induced by chemical secretagogue --- p.111 / Chapter 4.3.6 --- Effect of various partitions from solvent extraction of FLL on histamine release from human mast cells --- p.112 / Chapter 4.3.6.1 --- Histamine release induced by immunological stimulus --- p.113 / Chapter 4.3.6.2 --- Histamine release induced by chemical secretagogue --- p.114 / Chapter 4.3.7 --- Effect of ELP and its herbal constituents on the production of cytokine from human mast cells --- p.115 / Chapter 4.3.8 --- Modulation in calcium mobilization in activated human mast cell by ELP and its herbal constituents --- p.117 / Chapter 4.4 --- Discussion --- p.119 / Chapter 5 --- General discussion --- p.163 / Reference --- p.171

The place of estrogen in women's health remains controversial. Premenopausal women have a lower prevalence of cardiovascular disease (CVD) than men and in observational studies hormone replacement therapy (HRT) decreases CVD in postmenopausal women. However, prospective randomized trials of secondary and primary prevention have failed to substantiate an overall protective effect from HRT and have even shown some harm. To explain this paradox it is necessary to better understand the effects of estrogen on the vascular wall. Estrogen rapidly mediates the activation of eNOS and increases the production of nitric oxide (NO), an important factor for endothelial health. In ovariectomized rats estrogen reduces production of superoxide (O2-) by NAD(P)H oxidase. The decreased function is associated with a decrease in the p47phox component of NAD(P)H oxidase and its interaction with the multicomponent enzyme. In these rats estrogen did not alter eNOS expression and bioavailability of NO, which is in contrast to its acute effects. This highlights the difference between chronic and acute studies. The decrease in O2-production suggests the intracellular signaling. / Estrogen has antiapoptotic effects. Oxidized low-density lipoprotein (oxLDL) and the inflammatory cytokine TNFalpha increased apoptosis which is associated with atherosclerosis. In human umbilical vein endothelial cells (HUVEC), estrogen decreased the extent of TNFalpha and oxLDL induced apoptosis as indicated by the expression of cleaved caspase-3 and FACS assay. Estrogen also preserves the antiapoptotic mitochondrial Bcl-2 and Bcl-xL proteins. / Estrogen has angiogenic properties that can help a healthy endothelium respond to injury. However, estrogen increases the angiogenesis caused by TNFalpha and this could lead to revascularization in the plaques of women with advanced disease. / Overall the balance between the positive and negative aspects of the effects of estrogen on the vascular wall could explain the paradoxical response in older women.

No description available.

Kam Man Fai Afia. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves [195]-224). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgement --- p.v / Publications --- p.vi / Abbreviations --- p.vii / Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- The Mast Cell --- p.2 / Chapter 1.1.1 --- Historical Perspective --- p.2 / Chapter 1.1.2 --- Mast Cell Origin and Development --- p.3 / Chapter 1.1.3 --- Mast Cell Heterogeneity --- p.5 / Chapter 1.1.3.1 --- Rodent Mast Cell Heterogeneity --- p.5 / Chapter 1.1.3.2 --- Human Mast Cell Heterogeneity --- p.7 / Chapter 1.1.4 --- Mast Cell Mediators --- p.10 / Chapter 1.1.4.1 --- Preformed Mediators --- p.11 / Chapter 1.1.4.2 --- Newly Synthesized Lipid Mediators --- p.14 / Chapter 1.1.4.3 --- Cytokines --- p.16 / Chapter 1.1.5 --- Mast Cell Activation --- p.17 / Chapter 1.1.5.1 --- Immunological Activation --- p.19 / Chapter 1.1.5.1.1 --- FcεIR Activation and Protein Tyrosine Phosphorylation --- p.19 / Chapter 1.1.5.1.2 --- Activation of Phospholipases --- p.20 / Chapter 1.1.5.1.3 --- The Role of Calcium --- p.22 / Chapter 1.1.5.1.3.1 --- Intracellular Calcium Mobilization --- p.23 / Chapter 1.1.5.1.3.2 --- Calcium Influx --- p.24 / Chapter 1.1.5.1.3.3 --- Mechanisms of Action of Calcium in Mast Cells --- p.28 / Chapter 1.1.5.1.4 --- The Role of G-proteins --- p.30 / Chapter 1.1.5.1.5. --- The Role of Cylic AMP --- p.33 / Chapter 1.1.5.1.2.1 --- Mechanisms of Action of Cyclic AMP in Mast Cells --- p.36 / Chapter 1.1.5.1.2.2 --- Implications for the Inhibitory Role of Cyclic AMP in Mast Cell Activation --- p.37 / Chapter 1.2 --- The Cyclic Nucleotide Phosphodiesterases --- p.39 / Chapter 1.2.1 --- Introduction --- p.39 / Chapter 1.2.2 --- Classification and Structure --- p.41 / Chapter 1.2.3 --- Distribution and Physiological Functions of the Different PDE Families --- p.45 / Chapter 1.2.4 --- Phosphodiesterase Inhibitors --- p.49 / Chapter 1.2.4.1 --- Non-selective PDE Inhibitors --- p.50 / Chapter 1.2.4.2 --- Selective PDE Inhibitors --- p.52 / Chapter 1.2.4.2.1 --- PDE1 and PDE2 Inhibitors --- p.52 / Chapter 1.2.4.2.2 --- PDE3 Inhibitors --- p.53 / Chapter 1.2.4.2.3 --- PDE4 Inhibitors --- p.54 / Chapter 1.2.4.2.4.1 --- PDE5 Inhibitors --- p.56 / Chapter 2. --- Materials and Methods --- p.59 / Chapter 2.1 --- Materials --- p.60 / Chapter 2.1.1 --- Drugs --- p.60 / Chapter 2.1.1.1 --- Phosphodiesterase Inhibitors --- p.60 / Chapter 2.1.1.2 --- Mast Cell Secretagogues --- p.61 / Chapter 2.1.2 --- Materials for Rat Peritoneal Mast Cell Experiments --- p.61 / Chapter 2.1.2.1 --- Materials for Rat Sensitization --- p.61 / Chapter 2.1.2.2 --- Materials for Buffers --- p.62 / Chapter 2.1.2.3 --- Materials for Histamine Assay --- p.62 / Chapter 2.1.2.4 --- Miscellaneous --- p.63 / Chapter 2.1.3 --- Materials for RBL-2H3 Cell Line Experiments --- p.63 / Chapter 2.1.3.1 --- Materials for Cell Culture --- p.63 / Chapter 2.1.3.2 --- Materials for Cell Sensitization and Enzyme Release --- p.64 / Chapter 2.1.3.3 --- Materials for β-Hexosaminidase Assay --- p.64 / Chapter 2.1.3.4 --- Miscellaneous --- p.64 / Chapter 2.2 --- Rat Peritoneal Mast Cell Experiments --- p.65 / Chapter 2.2.1 --- Preparation of Buffers --- p.65 / Chapter 2.2.2 --- Preparation of Stock Solutions --- p.66 / Chapter 2.2.2.1 --- Mast Cell Secretagogue Stock Solutions --- p.66 / Chapter 2.2.2.2 --- Phosphodiesterase Inhibitor Stock Solutions --- p.66 / Chapter 2.2.3 --- Animals and Cell Isolation --- p.71 / Chapter 2.2.3.1 --- Animals --- p.71 / Chapter 2.2.3.2 --- Sensitization of Animals --- p.71 / Chapter 2.2.3.3 --- Cell Isolation --- p.71 / Chapter 2.2.3.4 --- Cell Purification --- p.72 / Chapter 2.2.3.5 --- Determination of Cell Number and Viability --- p.73 / Chapter 2.2.4 --- General Protocol for Histamine Release and Histamine Measurement --- p.75 / Chapter 2.2.4.1 --- Histamine Release --- p.75 / Chapter 2.2.4.2 --- Spectrofluorometric Determination of Histamine Content --- p.76 / Chapter 2.2.4.2.1 --- Manual Histamine Assay --- p.76 / Chapter 2.2.4.2.2 --- Automated Histamine Assay --- p.78 / Chapter 2.2.4.3 --- Calculation of Histamine Levels --- p.78 / Chapter 2.2.4.4 --- Presentation and Statistics --- p.79 / Chapter 2.3 --- RBL-2H3 Cell Line Experiments --- p.80 / Chapter 2.3.1 --- Preparation of Stock Solutions --- p.80 / Chapter 2.3.2 --- Preparation of Materials for Enzyme Release and Assay --- p.81 / Chapter 2.3.2.1 --- Cell Culture --- p.81 / Chapter 2.3.2.2 --- Preparation of Cells for β-Hexosaminidase Release Experiments --- p.82 / Chapter 2.3.2.3 --- β-Hexosaminidase Release --- p.82 / Chapter 2.3.2.4 --- β-Hexosaminidase Assay --- p.83 / Chapter 3. --- Effects of Phosphodiesterase Inhibitors on Mediator Release from Rat Mast Cells --- p.84 / Chapter 3.1 --- Introduction --- p.85 / Chapter 3.2 --- Materials and Methods --- p.87 / Chapter 3.2.1 --- Rat Peritoneal Mast Cells --- p.87 / Chapter 3.2.1.1 --- Experiments Employing Immunological Stimulus in RPMCs --- p.87 / Chapter 3.2.1.2 --- Experiments Employing Non-Immunological Stimuli in RPMCs --- p.88 / Chapter 3.2.2 --- Rat Basophilic Leukemia Cells --- p.88 / Chapter 3.3 --- Results --- p.89 / Chapter 3.3.1 --- Rat Peritoneal Mast Cells --- p.89 / Chapter 3.3.1.1 --- Immunologically Activated Rat Peritoneal Mast Cells --- p.89 / Chapter 3.3.1.1.1 --- Effects of Non-Selective PDE Inhibitors on Anti-IgE-Mediated Histamine Release from RPMCs --- p.89 / Chapter 3.3.1.1.2 --- Effects of Selective PDE1 and PDE2 Inhibitors on Anti-IgE- Mediated Histamine Release from RPMCs --- p.90 / Chapter 3.3.1.1.3 --- Effects of Selective PDE3 Inhibitors on Anti-IgE-Mediated Histamine Release from RPMCs --- p.90 / Chapter 3.3.1.1.4 --- Effects of Selective PDE4 Inhibitors on Anti-IgE-Mediated Histamine Release from RPMCs --- p.91 / Chapter 3.3.1.1.5 --- Effects of Selective PDE5 Inhibitors on Anti-IgE-Mediated Histamine Release from RPMCs --- p.91 / Chapter 3.3.1.2 --- Non-Immunologically Activated Rat Peritoneal Mast Cells --- p.92 / Chapter 3.3.1.2.1 --- Effects of Selective PDE Inhibitors on Compound 48/80- Mediated Histamine Release from RPMCs --- p.92 / Chapter 3.3.1.2.2 --- Effects of Selective PDE Inhibitors on Histamine Release from RPMCs Stimulated by Calcium Ionophores --- p.93 / Chapter 3.3.2 --- Rat Basophilic Leukemia Cells --- p.93 / Chapter 3.3.2.1 --- Effects of Non-Selective PDE Inhibitors on Antigen-Mediated β-Hexosaminidase Release from RBL-2H3 Cells --- p.93 / Chapter 3.3.2.2 --- Effects of Selective PDE Inhibitors on Antigen-Mediated β-Hexosaminidase Release from RBL-2H3 Cells --- p.94 / Chapter 3.4 --- Discussion --- p.95 / Chapter 3.4.1 --- Rat Peritoneal Mast Cells --- p.95 / Chapter 3.4.1.1 --- Immunologically Activated RPMCs --- p.95 / Chapter 3.4.1.2 --- Non-Immunologically Activated RPMCs --- p.99 / Chapter 3.4.2 --- Rat Basophilic Leukemia Cells --- p.103 / Chapter 4. --- Combined Effects of Selective Phosphodiesterase Inhibitors on Immunologically Induced Histamine from Rat Mast Cells --- p.143 / Chapter 4.1 --- Introduction --- p.144 / Chapter 4.2 --- Materials and Methods --- p.144 / Chapter 4.2.1 --- Simultaneous Addition of PDE3 and PDE4 Inhibitors --- p.145 / Chapter 4.2.2 --- Sequential Addition of PDE3 and PDE4 Inhibitors --- p.145 / Chapter 4.3 --- Results --- p.146 / Chapter 4.3.1 --- Effects of the Selective Inhibitors for PDE3 and PDE4 Alone: Calculation of the Expected Inhibition Curve --- p.146 / Chapter 4.3.2 --- Effects of the Simultaneous Addition of PDE3 and PDE4 Inhibitors on Anti-IgE-Mediated Histamine Release from RPMCs --- p.148 / Chapter 4.3.2.1 --- Rolipram and Siguazodan --- p.148 / Chapter 4.3.2.2 --- Ro 20-1724 and Siguazodan --- p.149 / Chapter 4.3.2.3 --- Rolipram and Quazinone --- p.149 / Chapter 4.3.2.4 --- Ro 20-1724 and Quazinone --- p.150 / Chapter 4.3.3 --- Effects of the Sequential Addition of PDE3 and PDE4 Inhibitors on Anti-IgE-Mediated Histamine Release from RPMCs --- p.150 / Chapter 4.3.3.1 --- Rolipram and Siguazodan --- p.150 / Chapter 4.3.3.2 --- Ro 20-1724 and Siguazodan --- p.151 / Chapter 4.3.3.3 --- Rolipram and Quazinone --- p.151 / Chapter 4.3.3.4 --- Ro 20-1724 and Quazinone --- p.152 / Chapter 4.4 --- Discussion --- p.153 / Chapter 5. --- Future Directions --- p.191 / Chapter 5.1 --- Future Directions --- p.192 / References --- p.195

Ho Yi-Fong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 120-131). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.iv / Table of Contents --- p.v / List of Figures --- p.ix / List of Tables --- p.xi / Abbreviations --- p.xii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Corticosteroids in ophthalmology --- p.1 / Chapter 1.1.1 --- History of the clinical use of corticosteroids --- p.1 / Chapter 1.1.2 --- Administration --- p.1 / Chapter 1.1.3 --- General biological effects of corticosteroids --- p.4 / Chapter 1.1.4 --- Application of corticosteroids in ocular diseases --- p.5 / Chapter 1.1.5 --- Side effects of ocular corticosteroid treatment --- p.6 / Chapter 1.1.6 --- General introduction to commonly used corticosteroids in ophthalmology --- p.6 / Chapter 1.1.6.1 --- Hydrocortisone --- p.6 / Chapter 1.1.6.2 --- Dexamethasone --- p.7 / Chapter 1.1.6.3 --- Triamcinolone --- p.7 / Chapter 1.1.6.4. --- Chemical structures and relative anti-inflammatory potencies --- p.8 / Chapter 1.1.7 --- Cytotoxicity of triamcinolone --- p.12 / Chapter 1.2 --- Retinal ganglion cells --- p.13 / Chapter 1.2.1 --- Basic structures of the eye --- p.13 / Chapter 1.2.2 --- Anatomical structure of retina --- p.13 / Chapter 1.2.3 --- Functions of retinal ganglion cells --- p.18 / Chapter 1.2.4 --- Culture models to study RGCs --- p.20 / Chapter 1.3 --- Aim of study --- p.25 / Chapter Chapter 2 --- Methodology --- p.26 / Chapter 2.1 --- Intravitreal injection of TA (IVTA) --- p.26 / Chapter 2.1.1 --- Materials --- p.26 / Chapter 2.1.1.1 --- Animals --- p.26 / Chapter 2.1.1.2 --- Chemicals --- p.26 / Chapter 2.1.1.3 --- Instruments --- p.26 / Chapter 2.1.2 --- Procedures --- p.26 / Chapter 2.2 --- Peripheral Nerve - Optic Nerve Grafting (PN-ON) Procedure --- p.27 / Chapter 2.3 --- Retrograde Labeling of regenerating RGCs --- p.27 / Chapter 2.3.1 --- Materials --- p.27 / Chapter 2.3.2 --- Procedures --- p.27 / Chapter 2.3.3 --- Statistical analysis --- p.28 / Chapter 2.4 --- Immunohistochemistry --- p.28 / Chapter 2.4.1 --- Materials --- p.28 / Chapter 2.4.2 --- Procedures --- p.29 / Chapter 2.4.3 --- Statistical analysis --- p.29 / Chapter 2.5 --- Histology --- p.29 / Chapter 2.5.1 --- Materials --- p.29 / Chapter 2.5.2 --- Procedures --- p.29 / Chapter 2.6 --- Primary rat retinal ganglion cell culture --- p.30 / Chapter 2.6.1 --- Materials --- p.30 / Chapter 2.6.1.1 --- Animals --- p.30 / Chapter 2.6.1.2 --- Chemicals --- p.30 / Chapter 2.6.1.3 --- Solutions and buffers --- p.30 / Chapter 2.6.1.4 --- Instruments --- p.31 / Chapter 2.6.2 --- Preparations --- p.31 / Chapter 2.6.2.1 --- Working media --- p.31 / Chapter 2.6.2.2 --- Plate coating --- p.32 / Chapter 2.6.3 --- Cell culture process --- p.32 / Chapter 2.6.3.1 --- Dissection of retinal tissues --- p.32 / Chapter 2.6.3.2 --- Purification of RGCs --- p.33 / Chapter 2.6.3.3 --- Culture condition and cell seeding --- p.34 / Chapter 2.7 --- Corticosteroid treatment --- p.34 / Chapter 2.7.1 --- Materials --- p.34 / Chapter 2.7.2 --- Preparations --- p.34 / Chapter 2.7.3 --- Treatment --- p.35 / Chapter 2.8 --- Cell viability assay and morphometric study --- p.36 / Chapter 2.8.1 --- Materials --- p.36 / Chapter 2.8.2 --- Calcein-AM staining --- p.36 / Chapter 2.8.3 --- Cell viability --- p.37 / Chapter 2.8.4 --- Morphometry study --- p.37 / Chapter 2.9 --- TUNEL Assay --- p.38 / Chapter 2.9.1 --- Materials --- p.38 / Chapter 2.9.2 --- Procedure --- p.38 / Chapter 2.9.3 --- Statistical analysis --- p.39 / Chapter 2.10 --- Quantitative Reverse transcription - Polymerase Chain Reaction (qRT-PCR) --- p.39 / Chapter 2.10.1 --- Materials --- p.39 / Chapter 2.10.1.1 --- "Chemicals, reagents, and kits" --- p.39 / Chapter 2.10.1.2 --- Primers --- p.40 / Chapter 2.10.1.3 --- Equipment --- p.41 / Chapter 2.10.1.4 --- Software --- p.41 / Chapter 2.10.2 --- Procedures --- p.41 / Chapter 2.10.2.1 --- Cell collection and RNA isolation --- p.41 / Chapter 2.10.2.2 --- Reverse Transcription --- p.42 / Chapter 2.10.2.3 --- Real-time PCR --- p.43 / Chapter 2.10.3 --- Statistical analysis --- p.43 / Chapter 2.11 --- Western blotting --- p.44 / Chapter 2.11.1 --- Sample preparation --- p.44 / Chapter 2.11.1.1 --- Materials --- p.44 / Chapter 2.11.1.1.1 --- Chemicals and materials --- p.44 / Chapter 2.11.1.1.2 --- Equipment --- p.44 / Chapter 2.11.1.2 --- Procedures --- p.44 / Chapter 2.11.2 --- Protein assay --- p.45 / Chapter 2.11.2.1 --- Materials --- p.45 / Chapter 2.11.2.1.1 --- Chemicals and materials --- p.45 / Chapter 2.11.2.1.2 --- Equipment and software --- p.46 / Chapter 2.11.2.2 --- Procedures --- p.46 / Chapter 2.11.3 --- SDS-polyacrylamide gel electrophoresis (SDS-PAGE) --- p.46 / Chapter 2.11.3.1 --- Materials --- p.46 / Chapter 2.11.3.1.1 --- Chemicals and reagents --- p.46 / Chapter 2.11.3.1.2 --- Equipment --- p.46 / Chapter 2.11.3.1.3 --- Solutions and buffers --- p.47 / Chapter 2.11.3.2 --- Gel preparation --- p.48 / Chapter 2.11.3.3 --- Electrophoresis --- p.49 / Chapter 2.11.3.4 --- Transblotting (semi-dry transfer) --- p.49 / Chapter 2.11.3.5 --- Band visualization --- p.49 / Chapter 2.11.4 --- Immunostaining --- p.50 / Chapter 2.11.4.1 --- Materials --- p.50 / Chapter 2.11.4.1.1 --- Antibodies --- p.50 / Chapter 2.11.4.1.2 --- Chemicals and reagents --- p.50 / Chapter 2.11.4.1.3 --- Equipment --- p.50 / Chapter 2.11.4.2 --- Procedures --- p.50 / Chapter 2.12 --- Gas-chromatography-electron-capture negative-ion mass spectrometry (GC-NCI-MS) --- p.51 / Chapter 2.12.1 --- Standard and reagents --- p.51 / Chapter 2.12.2 --- Chromatography and mass spectrometry --- p.52 / Chapter 2.12.3 --- Sample collection --- p.52 / Chapter 2.12.3 --- Standard and sample preparation --- p.53 / Chapter 2.12.4 --- Validation --- p.54 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Effect of triamcinolone on RGCs in vivo --- p.55 / Chapter 3.2 --- Cell viability of RGCs after IVTA plus PN-ON grafting --- p.55 / Chapter 3.3 --- Abnormal retinal morphology under different IVTA conditions --- p.59 / Chapter 3.4 --- Cell viability assay --- p.66 / Chapter 3.4.1 --- Effects of triamcinolone on RGC viability --- p.66 / Chapter 3.4.2 --- Effects of dexamethasone on RGC viability --- p.68 / Chapter 3.4.3 --- Effects of hydrocortisone on RGC viability --- p.70 / Chapter 3.4.4 --- Effects of filtered fraction of triamcinolone on RGC viability --- p.72 / Chapter 3.5 --- Morphometric study analysis of RGCs --- p.74 / Chapter 3.5.1 --- Percentage of RGCs showing neurite outgrowth --- p.74 / Chapter 3.5.2 --- Average neurite length --- p.77 / Chapter 3.5.3 --- Neurite spanning area --- p.77 / Chapter 3.5.4 --- Neurite count --- p.80 / Chapter 3.5.5 --- Neurite branching --- p.83 / Chapter 3.6 --- Determination of concentration of TA in culture media by GC-NCI-MS --- p.85 / Chapter 3.7 --- TUNEL assay --- p.90 / Chapter 3.8 --- Real-time quantitative Reverse transcription - Polymerase Chain Reaction --- p.93 / Chapter 3.9 --- Western blot --- p.99 / Chapter Chapter 4 --- Discussion --- p.102 / Chapter Chapter 5 --- References --- p.120

Diss. (sammanfattning) Stockholm : Karol. inst., 2001. / Härtill 6 uppsatser.

Hoi, Chu Peng. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 81-103). / Abstracts in English and Chinese. / Acknowledgements --- p.II / Abstract --- p.III / Abstract (in Chinese) --- p.V / List of Abbreviations --- p.VI / List of Figures --- p.VIII / List of Tables --- p.X / Table of Contents --- p.XI / Chapter Chapter One --- General introduction --- p.1 / Chapter 1.1 --- Alzheimer's disease --- p.1 / Chapter 1.1.1 --- Epidemiology and risk factors --- p.2 / Chapter 1.1.2 --- Clinical manifestation and course --- p.4 / Chapter 1.1.3 --- Clinical diagnosis --- p.5 / Chapter 1.1.4 --- Neuropathology and pathogenesis of AD --- p.8 / Chapter 1.1.5 --- Drug therapy of AD --- p.11 / Chapter 1.1.5.1 --- Drugs for symptomatic treatment --- p.11 / Chapter 1.1.5.2 --- Drugs based on epidemiology --- p.12 / Chapter 1.1.5.3 --- Drugs with potential disease-modifying effects --- p.14 / Chapter 1.1.5.4 --- Herbal supplements --- p.15 / Chapter 1.2 --- Models for drug discovery in Alzheimer Disease --- p.15 / Chapter 1.2.1 --- In vivo (animal) models --- p.16 / Chapter 1.2.2 --- In vitro (cellular) models --- p.18 / Chapter 1.3 --- Chinese herbs for the treatment of AD --- p.20 / Chapter 1.3.1 --- Ginkgo biloba L --- p.21 / Chapter 1.3.2 --- Magnolia officinalis --- p.24 / Chapter 1.3.3 --- Acori graminei Rhizoma (AGR) --- p.26 / Chapter 1.3.4 --- Gastrodia elata (G. elata) --- p.27 / Chapter 1.3.5 --- Rhodiola rosea L.( R. rosea) --- p.29 / Chapter 1.3.6 --- Scutellariae baicalensis --- p.30 / Chapter 1.3.7 --- Curcuma longa L.(Zingiberaceae) --- p.31 / Chapter 1.4 --- Aims of the study --- p.33 / Chapter Chapter Two --- Materials and Methods --- p.34 / Chapter 2.1 --- Materials --- p.34 / Chapter 2.1.1 --- Chemicals and reagents --- p.34 / Chapter 2.1.2 --- Materials for cell culture --- p.35 / Chapter 2.1.3 --- Instruments --- p.35 / Chapter 2.2 --- Methods --- p.36 / Chapter 2.2.1 --- Cell culture --- p.36 / Chapter 2.2.2 --- MTT cell viability assay --- p.38 / Chapter 2.2.3 --- Characterization of the cytotoxicity of Aβ peptide in NGF-differentiated PC 12 cells --- p.38 / Chapter 2.2.4 --- Screening of the neuroprotective effect of major principles from selected herbs on PC 12 cells against Aβ-induced cytotoxicity --- p.39 / Chapter 2.2.5 --- Measurement of reactive oxygen species (ROS) --- p.40 / Chapter 2.2.6 --- Measurement of intracellular calcium levels --- p.41 / Chapter 2.2.7 --- Measurement of caspase-3 activity --- p.42 / Chapter 2.2.8 --- Propidium iodide (PI) staining to evaluate apoptosis and necrosis --- p.43 / Chapter 2.3 --- Statistics --- p.45 / Chapter Chapter Three --- Results --- p.46 / Chapter 3.1 --- NGF-differentiated PC 12 cells --- p.46 / Chapter 3.1.1 --- Determination of an appropriate cell density for the screening experiments --- p.46 / Chapter 3.1.2 --- Characterization of Aβ-induced cytotoxicity in NGF-differentiated PC 12 cells --- p.47 / Chapter 3.1.2.1 --- Cytotoxicity of Aβ-related fragments in NGF-differentiated PC 12 cells --- p.48 / Chapter 3.1.2.2 --- Dose-dependent cytotoxic effect of Aβ on PC 12 cells --- p.48 / Chapter 3.1.2.3 --- Time-dependent effect of Aβ-induced toxicity on PC12 cells --- p.50 / Chapter 3.1.3 --- Protective effect of selected active principles against Aβ1-4-induced toxicity in PC 12 cells --- p.51 / Chapter 3.2 --- Measurement of reactive oxygen species (ROS) --- p.54 / Chapter 3.2.1 --- Measurement of ROS induced by H202 --- p.54 / Chapter 3.2.2 --- Measurement of ROS induced by Aβ --- p.56 / Chapter 3.3 --- Measurement of Intracellular calcium levels --- p.57 / Chapter 3.4 --- Measurement of caspase-3 activity --- p.58 / Chapter 3.4.1 --- AMC reference standard curve --- p.59 / Chapter 3.4.2 --- Measurement of caspase-3 activity --- p.59 / Chapter 3.5 --- PI staining for evaluate apoptosis and necrosis --- p.60 / Chapter Chapter Four --- Discussion --- p.64 / Chapter 4.1 --- Aβ-induced cytotoxicity in NGF-differentiated PC 12 cells as an in vitro model of Alzheimer's disease --- p.64 / Chapter 4.1.1 --- Cell line selection --- p.65 / Chapter 4.1.2 --- Characterization of Aβ-induced cytotoxicity in NGF-differentiated PC 12 cells --- p.66 / Chapter 4.2 --- Screening of the neuroprotective effects of selected active principles against Aβ-induced cytotoxicity in NGF-differentiated PC 12 cells --- p.67 / Chapter 4.3 --- Neuroprotection via inhibition of the ROS generation --- p.71 / Chapter 4.4 --- Neuroprotection via suppression of calcium homeostasis --- p.73 / Chapter 4.5 --- Neuroprotective via inhibition of Aβ-induced apoptosis --- p.75 / Chapter 4.5.1 --- Inhibition of caspase-3 activation --- p.75 / Chapter 4.5.2 --- PI staining for evaluation of apoptosis and necrosis --- p.76 / Chapter Chapter Five --- Conclusion and future work --- p.79 / Chapter 5.1 --- Conclusion --- p.79 / Chapter 5.2 --- Future work --- p.80 / References --- p.81

Os doentes transplantados renais têm alto risco para desenvolver queratoses actínicas e câncer de pele. Para verificar o efeito quimioprofilático da acitretina estudamos a evolução de 13 doentes transplantados renais com queratoses actínicas múltiplas e história de carcinomas cutâneos submetidos a tratamento por 12 meses (20mg/dia). Fez-se a avaliação clínica e laboratorial regularmente em todo o período do estudo. Realizou-se exame histopatológico, demonstração imuno-histoquímica de sub-populações de linfócitos T (CD4, CD8), células natural killer e células de Langerhans, sua quantificação e comparação em biopsias de pele, sem lesão, de área exposta e protegida do sol antes, após seis e 12 meses de tratamento. Observou-se melhora das lesões cutâneas e ausência de aparecimento de novos tumores em 12 dos 13 pacientes. Não ocorreram alterações laboratoriais relacionadas a função renal, hepatotoxicicidade e hiperlipidemia. Não houve diferenças significativas histopatológicas e da população de linfócitos T e células natural killer da pele exposta e protegida do sol com o tratamento. Verificou-se aumento numérico de células de Langerhans epidérmicas aos 12 meses quando comparado aos da pele antes e após seis meses de tratamento (p = 0,002 e p = 0,003). Em nossa casuística o uso de acitretina em doses baixas foi útil para melhorar o aspecto cutâneo e prevenir lesões cutâneas pré-cancerosas e carcinomas. O aumento das células de Langerhans epidérmicas estaria relacionado ao efeito imunomodular da acitretina. / Renal transplant recipients have an increased incidence of actinic keratosis and skin cancer. In order to examine the chemoprophylatic effects of low-dose acitretin on skin cancer development we submitted 13 renal transplanted patients to acitretin therapy (20 mg/day) for 12 month. The patients were assessed at monthly intervals during the first 6 months and every two months until the 12th month for new skin lesions and for acitretin toxicity. Normal skin biopsies of sun exposed and sun protected area were taken for histopathological exam and submitted to immunohistochemistry technique to demonstrate CD4+ and CD8+ T lymphocytes, natural killer cells and Langerhans cells wich were counted and compared in the beginning, after 6th month and 12th month of the treatment. There was an improvement of actinic keratosis and all patients but one did not develop new skin cancer. Side-effects were well-tolerated and no significant biochemical effects were observed. Although there were no differences in the microscopic aspects of the skin and in the number of CD4+ and CD8+ T lymphocytes and natural killer cells, there was a significant increase in the number of epidermal Langerhans cells after 12 months of acitretin therapy. The data obtained permit us to conclude that low dose acitretin therapy is safe, well-tolerated and partially effective in chemoprophylaxis of skin cancer in renal transplant recipients. The increase in epidermal Langerhans cells observed may be an expression of the immunomodulatory effect of acitretin.