Sha Ou. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 167-189). / Abstracts in English and Chinese. / abstract --- p.i / 中文摘要 --- p.iv / acknowledgements --- p.vii / Chapter chapter 1. --- introduction --- p.1 / Chapter 1.1 --- Overview --- p.1 / Chapter 1.2 --- Ribosome-inactivating proteins (RIPs) --- p.1 / Chapter 1.2.1 --- Classification --- p.2 / Chapter 1.2.2 --- Structure --- p.3 / Chapter 1.2.3 --- Enzymatic activities --- p.3 / Chapter 1.3 --- Type II RIPs --- p.5 / Chapter 1.3.1 --- Ricin --- p.5 / Chapter 1.3.2 --- Ricinus communis agglutinin (RCA) --- p.6 / Chapter 1.3.3 --- Intracellular mechanism --- p.7 / Chapter 1.3.4 --- Application of RIPs in neuroscience research: suicide axonal transport --- p.10 / Chapter 1.4 --- Type I RIPs --- p.12 / Chapter 1.4.1 --- Trichosanthin (TCS) --- p.12 / Chapter 1.4.2 --- Ricin A chain (RTA) --- p.15 / Chapter 1.4.3 --- Medical applications: immunolesioning and immunotherapy --- p.16 / Chapter 1.5 --- The types of Cell death --- p.17 / Chapter 1.5.1 --- Necrosis --- p.18 / Chapter 1.5.2 --- Apoptosis --- p.18 / Chapter 1.6 --- Inflammations --- p.21 / Chapter 1.6.1 --- Acute inflammation --- p.21 / Chapter 1.6.2 --- Chronic inflammation --- p.22 / Chapter 1.6.3 --- Retinitis --- p.22 / Chapter 1.7 --- Eye model for neurotoxicity studies in CNS --- p.23 / Chapter 1.8 --- Objective of present study --- p.24 / Chapter CHAPTER 2. --- MATERIALS AND METHODS --- p.25 / Chapter 2.1 --- Plan of this chapter --- p.25 / Chapter 2.2 --- Toxins and methods used --- p.25 / Chapter 2.3 --- Animals --- p.26 / Chapter 2.4 --- Preparation of toxin solutions --- p.27 / Chapter 2.4.1 --- RIP solutions --- p.27 / Chapter 2.4.2 --- Labeling type I RIPs with fluorescence --- p.27 / Chapter 2.4.3 --- Control solutions --- p.29 / Chapter 2.5 --- Administrations of solutions --- p.30 / Chapter 2.5.1 --- Basic procedures of vitreous chamber injection --- p.30 / Chapter 2.5.2. --- Injection of trichosanthin (TCS) --- p.31 / Chapter 2.5.3 --- Injection of ricin A chain (RTA) --- p.31 / Chapter 2.5.4 --- Injection of ricinus communis agglutinin (RCA) --- p.32 / Chapter 2.5.5 --- Administration of FITC-TCS --- p.33 / Chapter 2.5.6 --- Administration of FITC-RTA --- p.33 / Chapter 2.6 --- Retinal tissue processing --- p.33 / Chapter 2.6.1 --- Paraffin method --- p.34 / Chapter 2.6.2 --- Cryostatic method --- p.35 / Chapter 2.6.3 --- Electron microscopic method --- p.35 / Chapter 2.7 --- General effects of RIPs on rat retinas --- p.36 / Chapter 2.7.1 --- Hematoxylin-and-eosin staining --- p.36 / Chapter 2.7.2 --- Retinal thickness --- p.37 / Chapter 2.7.3 --- Pathological changes --- p.38 / Chapter 2.7.4 --- Dosage study on TCS --- p.39 / Chapter 2.7.5 --- Statistics --- p.40 / Chapter 2.8 --- Mechanisms of cell death --- p.40 / Chapter 2.8.1 --- Terminal dUTP nick-end labeling (TUNEL) --- p.40 / Chapter 2.8.2 --- Immunohistochemistry for caspase-3 --- p.42 / Chapter 2.8.3 --- Double staining of cleaved caspase-3 and TUNEL --- p.42 / Chapter 2.8.4 --- Electronic microscope observation --- p.43 / Chapter 2.9 --- Entry of type I RIPs into cells --- p.43 / Chapter 2.9.1 --- Propidium iodide staining --- p.43 / Chapter 2.9.2 --- Immunohistochemical localization of Muller cells --- p.44 / Chapter 2.9.3 --- Double staining of Muller cells and TUNEL --- p.44 / Chapter 2.9.4 --- Confocal microscope --- p.44 / Chapter 2.10 --- Reactions of glial cells --- p.45 / Chapter CHAPTER 3. --- RESULTS --- p.47 / Chapter 3.1 --- Preparation of fluorescein-type I RIP conjugates --- p.47 / Chapter 3.1.1 --- Conjugate of FITC-TCS --- p.47 / Chapter 3.1.2 --- Conjugate of FITC-RTA --- p.47 / Chapter 3.2 --- Effects of TCS on retina --- p.47 / Chapter 3.2.1 --- Retina cell count - a dose-dependence study --- p.48 / Chapter 3.2.2 --- Retinal thickness measurement - a time-course study --- p.49 / Chapter 3.2.3 --- Pathological changes --- p.50 / Chapter 3.3 --- Effects of RTA on retina --- p.51 / Chapter 3.3.1 --- Retinal thickness measurement - a time-course study --- p.51 / Chapter 3.3.2 --- Pathological changes --- p.53 / Chapter 3.4 --- Effects of RCA on retina --- p.54 / Chapter 3.4.1 --- Retinal thickness measurement --- p.54 / Chapter 3.4.2 --- Pathological changes --- p.55 / Chapter 3.5 --- Summary of results: general effects of RIPs --- p.56 / Chapter 3.6 --- Cell death - TUNEL method --- p.56 / Chapter 3.6.1 --- TCS experiment --- p.57 / Chapter 3.6.2 --- RTA experiment --- p.58 / Chapter 3.6.3 --- RCA experiment --- p.58 / Chapter 3.7 --- Cell death 一 cleaved caspase-3 immunohistochemistry --- p.58 / Chapter 3.7.1 --- TCS experiment --- p.59 / Chapter 3.7.2 --- RTA experiment --- p.59 / Chapter 3.8 --- EM observation --- p.59 / Chapter 3.8.1 --- TCS experiment --- p.59 / Chapter 3.8.2 --- RTA experiment --- p.60 / Chapter 3.9 --- Summary of results: mode of cell death --- p.60 / Chapter 3.10 --- Localisation of type I RIPs --- p.61 / Chapter 3.10.1 --- FITC-TCS --- p.62 / Chapter 3.10.2 --- FITC-TCS and Muller cell double staining --- p.63 / Chapter 3.10.3 --- Muller cell and TUNEL double staining --- p.64 / Chapter 3.10.4 --- FITC-RTA --- p.64 / Chapter 3.10.5 --- Summary of results: route of intoxication --- p.65 / Chapter 3.11 --- Glial cell reactions after RIP treatment --- p.65 / Chapter 3.11.1 --- TCS experiment --- p.65 / Chapter 3.11.2 --- RTA experiment --- p.66 / Chapter 3.11.3 --- RCA experiment --- p.67 / Chapter 3.11.4 --- Summary of results: glial reactions --- p.67 / Chapter CHAPTER 4. --- DISCUSSION --- p.69 / Chapter 4.1 --- General effects of RIPs on rat retinas --- p.69 / Chapter 4.1.1 --- Effects of trichosanthin (TCS) --- p.69 / Chapter 4.1.2 --- Effects of ricin A chain (RTA) --- p.71 / Chapter 4.1.3 --- Effects of ricinus communis agglutinin (RCA) --- p.73 / Chapter 4.2 --- The mechanisms of cell death --- p.74 / Chapter 4.2.1 --- Cell death caused by TCS --- p.75 / Chapter 4.2.2 --- Caspase-3 and the retina of RCS rat --- p.77 / Chapter 4.2.3 --- Cell death caused by RTA --- p.78 / Chapter 4.2.4 --- Cell death caused by RCA --- p.80 / Chapter 4.2.5 --- Mechanism of RTA - induced necrosis --- p.81 / Chapter 4.3 --- The mechanisms of type I RIPs entering cells --- p.82 / Chapter 4.3.1 --- Transport of TCS in retinal cells --- p.82 / Chapter 4.3.2 --- The uptake of Pure FITC by rat retina --- p.85 / Chapter 4.4 --- Reactions of glial cells --- p.85 / Chapter 4.4.1 --- Glial cell reactions in TCS experiment --- p.86 / Chapter 4.4.2 --- Glial cell reactions in RTA and RCA experiments --- p.87 / Chapter 4.5 --- Possible applications of RIPs on retinal studies --- p.88 / Chapter 4.5.1 --- Potential applications of TCS --- p.88 / Chapter 4.5.2 --- Possible uses of RTA and RCA --- p.90 / Chapter CHAPTER 5. --- CONCLUSIONS --- p.91 / "FIGURES, TABLES, GRAPHS, AND LEGENDS" --- p.93 / APPENDICES --- p.154 / Appendix A Source of materials --- p.154 / Appendix B Dosages for vitreous chamber injection --- p.156 / Appendix C Protocol of conjugate fluorescein to proteins --- p.157 / Appendix D Electronic Microscope methods --- p.160 / Appendix E Histological methods --- p.162 / Appendix F Protocols of TUNEL --- p.163 / Appendix G Protocols of Immunohistochemistry staining --- p.165 / REFERENCES --- p.167

by Wai-Man Tong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 155-174). / Abstracts in English and Chinese. / ABSTRACT --- p.I-IV / Chapter 1. --- INTRODUCTION / Chapter 1.1. --- General / Chapter 1.1.1. --- Ribosome Inactivating Protein --- p.1 / Chapter 1.1.1.1. --- Ricin --- p.2 / Chapter 1.1.1.2. --- Trichosanthin --- p.5 / Chapter 1.1.2. --- In Vitro Study of RIP --- p.6 / Chapter 1.2. --- Uptake of Ribosome Inactivating Proteins / Chapter 1.2.1. --- Suicide Transport --- p.7 / Chapter 1.2.1.1 . --- Endocytic Uptake of Ricin --- p.8 / Chapter 1.2.1.2. --- Endocytic Uptake of Trichosanthin --- p.11 / Chapter 1.2.2. --- Pervious Studies in This Laboratory --- p.11 / Chapter 1.3. --- Apoptosis And Ribosome Inactivation / Chapter 1.3.1. --- Apoptosis / Chapter 1.3.1.1. --- Morphological Feature of Apoptosis --- p.14 / Chapter 1.3.1.2. --- Molecular Changes of Apoptosis --- p.15 / Chapter 1.3.2. --- Toxicity of Ribosome Inactivating Protein / Chapter 1.3.2.1. --- Toxicity of Ricin --- p.20 / Chapter 1.3.2.2. --- Toxicity of Trichosanthin --- p.21 / Chapter 2. --- MATERIALS AND METHODS / Chapter 2.1. --- GENERAL / Chapter 2.1.1. --- Cell Culture / Chapter 2.1.1.1 . --- Schwann Cell Culture --- p.23 / Chapter 2.1.1.2. --- Dorsal Root Ganglion Neuron Culture --- p.24 / Chapter 2.1.1.3. --- Identification of Schwann Cell and Dorsal Root Ganglion Neuron --- p.25 / Chapter 2.1.2. --- Labeling of Toxins --- p.26 / Chapter 2.1.3. --- Administration of Toxin --- p.27 / Chapter 2.2. --- UPTAKE OF RIBOSOME INACTIVATING PROTEINS / Chapter 2.2.1. --- Real-Time Observation of Toxin Uptake by Neurons --- p.27 / Chapter 2.3. --- APOPTOSIS STUDY OF RIBOSOME INACTIVATING PROTEINS' TOXICITY / Chapter 2.3.1. --- TUNEL Staining --- p.28 / Chapter 2.3.2. --- Annexin V Staining --- p.30 / Chapter 2.4. --- MOLECULAR STUDY OF THE DEATH MECHANISM OF RIBOSOME INACTIVATING PROTEINS / Chapter 2.4.1. --- NIH/3T3 Cell Line Culture --- p.33 / Chapter 2.4.2. --- Differential Display / Chapter 2.4.2.1. --- Differential Display --- p.34 / Chapter 2.4.2.2. --- Cloning and Sequencing --- p.38 / Chapter 2.4.2.3. --- RT-PCR --- p.42 / Chapter 2.4.3. --- Two Dimension Gel Electrophoresis --- p.43 / Chapter 2.4.4. --- Ribosomal RNA Analysis --- p.48 / Chapter 3. --- RESULTS / Chapter 3.1. --- General / Chapter 3.1.1. --- Cell Culture / Chapter 3.1.1.1 . --- Schwann Cell Culture --- p.50 / Chapter 3.1.1.2. --- Dorsal Root Ganglion Neuron Culture --- p.51 / Chapter 3.1.1.3. --- Identification of Schwann Cell and Dorsal Root Ganglion Neuron --- p.51 / Chapter 3.1.2. --- RIPs Labeling --- p.52 / Chapter 3.2. --- Uptake of Ribosome Inactivating Protein / Chapter 3.2.1. --- Real-Time Observation of Toxin Uptake --- p.53 / Chapter 3.3. --- Apoptosis Study of Ribosome Inactivating Proteins' Toxicity / Chapter 3.3.1. --- TUNEL Staining --- p.55 / Chapter 3.3.2. --- Annexin V Assay / Chapter 3.3.2.1. --- Schwann Cell Culture --- p.57 / Chapter 3.3.2.2. --- Dorsal Root Ganglion Neuron Culture --- p.58 / Chapter 3.3.2.3. --- Unique Observable Pattern --- p.60 / Chapter 3.4. --- Molecular Study of the Death Mechanism of Ribosome Inactivating Proteins / Chapter 3.4.1. --- NIH/3T3 Cell Line Culture --- p.60 / Chapter 3.4.1.1. --- TUNEL Staining --- p.61 / Chapter 3.4.1.2. --- Annexin V Staining --- p.61 / Chapter 3.4.2. --- Differential Display / Chapter 3.4.2.1. --- Observation --- p.61 / Chapter 3.4.2.2. --- Primer Combination --- p.62 / Chapter 3.4.2.3. --- Differential Display --- p.62 / Chapter 3.4.3. --- Two-Dimensional Gel Electrophoresis / Chapter 3.4.3.1. --- Observation --- p.63 / Chapter 3.4.3.2. --- Comparison of Gels --- p.63 / Chapter 3.4.4. --- Ribosomal RNA Analysis --- p.63 / Chapter 4. --- DISCUSSION / Chapter 4.1. --- General / Chapter 4.1.1. --- The Selection of In Vitro Model / Chapter 4.1.1.1. --- Schwann Cell Culture --- p.65 / Chapter 4.1.1.2. --- Dorsal Root Ganglion Neuron Culture --- p.66 / Chapter 4.1.2. --- Labeling of Toxins with Fluorochromes --- p.67 / Chapter 4.1.3. --- Dosage Used in In Vitro Study --- p.68 / Chapter 4.2. --- Uptake of Ribosome Inactivating Proteins / Chapter 4.2.1. --- Real-Time Examination of Toxin Uptake --- p.69 / Chapter 4.3. --- Involvement of Apoptosis in Ribosome Inactivating Proteins' Intoxication / Chapter 4.3.1. --- TUNEL Staining --- p.75 / Chapter 4.3.2. --- Annexin V and Propidium Iodide Staining --- p.77 / Chapter 4.3.3. --- Special Pattern of Fluorescence Signal in Neuronal Cell Bodies --- p.82 / Chapter 4.4. --- Molecular Study of Death Mechanism of Ribosome Inactivating Proteins / Chapter 4.4.1. --- NIH/3T3 Cell Line Culture --- p.84 / Chapter 4.4.2. --- Differential Display --- p.84 / Chapter 4.4.3. --- Two Dimensional Polyacrylamide Gel Electrophoresis --- p.86 / Chapter 4.4.4. --- Ribosomal RNA Alternation --- p.88 / Chapter 5. --- CONCLUSIONS --- p.89 / Chapter 6. --- "FIGURES, GRAPHS AND LEGENDS" --- p.91 / Chapter 7. --- REFERENCES --- p.155 / APPENDIX / Appendix A Materials --- p.175 / Appendix B Source of Chemicals and Equipments --- p.184 / ACKNOWLEDGEMENTS --- p.186