Ko Wing Hung. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1992. / Includes bibliographical references (leaves 129-138). / Chapter Chapter 1 --- Introduction --- p.1 / Chapter Chapter 2 --- Methodology --- p.16 / Chapter Chapter 3 --- Development of Radioimmunoassay --- p.30 / Chapter Chapter 4 --- Coupling of Trichosanthin to Dextran T40 --- p.38 / Chapter Chapter 5 --- Pharmacokinetic Study --- p.49 / Chapter Chapter 6 --- Renal tubular reabsorption of Trichosanthin --- p.63 / Chapter Chapter 7 --- Biological activities of Dextran-Trichosanthin --- p.77 / Chapter Chapter 8 --- Immunological activities of Dextran-Trichosanthin --- p.91 / Chapter Chapter 9 --- General Discussion --- p.124 / References

by Chung Ka Kin Kenny. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 116-120). / Acknowledgments --- p.i / Abstract --- p.ii / Contents --- p.iii / Abbreviations --- p.viii / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Chemical and Biological Properties of TCS --- p.1 / Chapter 1.1.1 --- Chemical properties --- p.2 / Chapter 1.1.2 --- Biological properties --- p.5 / Chapter 1.2 --- Structure of Antigens --- p.6 / Chapter 1.3 --- Methods Used for the Mapping of Antigenic Sites on the Protein --- p.8 / Chapter 1.4 --- Objective and Strategy of the Study of the Antigenic Determinants of TCS --- p.13 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Bacterial Strains Used --- p.15 / Chapter 2.2 --- General Techniques --- p.16 / Chapter 2.2.1 --- Extraction of DNA by phenol/chloroform --- p.16 / Chapter 2.2.2 --- Ethanol precipitation --- p.16 / Chapter 2.2.3 --- Minipreparation of plasmid --- p.16 / Chapter 2.2.4 --- Preparation of plasmid DNA using Qiagen pack 100 Cartridge --- p.17 / Chapter 2.2.5 --- Preparation of plasmid DNA using Magic´ёØ Minipreps DNA Purification kit from Promega --- p.18 / Chapter 2.2.6 --- Preparation and transformation of Escherichia coli competent cells --- p.19 / Chapter 2.2.7 --- Agarose gel electrophoresis of DNA --- p.21 / Chapter 2.2.8 --- Restriction enzyme digestion of DNA --- p.21 / Chapter 2.2.9 --- Purification of DNA fragment from agarose gel using Gene Clean® kit --- p.22 / Chapter 2.2.10 --- Ligation of insert into vector --- p.22 / Chapter 2.2.11 --- Rapid screening for the presence of plasmid by direct lysis of the bacterial colony --- p.23 / Chapter 2.2.12 --- SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.24 / Chapter 2.2.13 --- Staining of protein in polyacrylamide gel --- p.25 / Chapter 2.2.14 --- Western blot detection of TCS --- p.25 / Chapter 2.2.15 --- Autoradiography --- p.27 / Chapter 2.3 --- Production of Recombinant Anti-TCS Fab Fragment --- p.27 / Chapter 2.3.1 --- Cloning and screening of the recombinant anti-TCS Fab fragment --- p.27 / Chapter 2.3.2 --- Expression of recombinant anti-TCS Fab fragment --- p.28 / Chapter 2.4 --- Construction of Deletion Library of pET58210 by Transposon Mediated Mutagenesis --- p.29 / Chapter 2.5 --- PCR Insertion Mapping of the Transposon Inserted Clones --- p.31 / Chapter 2.6 --- DNA Sequencing of Positive Clones After the PCR Insertion Mapping --- p.32 / Chapter 2.6.1 --- DNA sequencing reaction --- p.33 / Chapter 2.6.2 --- DNA sequencing gel casting and electrophoresis --- p.34 / Chapter 2.7 --- Computer Analysis of the DNA Sequence --- p.35 / Chapter 2.8 --- Subcloning of the Positive Clone that have been Located by DNA Sequencing --- p.35 / Chapter 2.8.1 --- Subcloning of the mutated TCS gene sequence with the Tn1000 delta end facing the N-tenninal of the TCS gene sequence --- p.36 / Chapter 2.8.2 --- Subcloning of the mutated TCS gene sequence with the Tn1000 gamma end facing the N-terminal of the TCS gene sequence --- p.37 / Chapter 2.9 --- Construction of Deletion Mutants that Exclude the 21 Amino Acids in the N-terminal of TCS --- p.39 / Chapter 2.10 --- Overexpression of Mutated TCS Protein with the T7 RNA Polymerase Driven Expression System --- p.41 / Chapter 2.11 --- Analysis of TCS Deletion mutants by Western Blotting --- p.42 / Chapter 2.12 --- Preparation of Recombinant TCS Protein --- p.42 / Chapter 2.13 --- Proteolytic and Chemical Cleavage of Recombinant TCS --- p.44 / Chapter 2.14 --- Tricine-SDS-16.5% Polyacrylamide Gel Electrophoresis --- p.45 / Chapter 2.15 --- Electroblotting Using Polyvinylidene Difluoride Membrane (PVDF) for N-terminal Determination --- p.48 / Chapter 2.16 --- Reagents and Buffers --- p.49 / Chapter 2.16.1 --- Media for bacterial culture --- p.49 / Chapter 2.16.2 --- Reagents for preparation of plasmid DNA --- p.51 / Chapter 2.16.3 --- DNA electrophoresis buffers --- p.51 / Chapter 2.16.4 --- Reagents for SDS-PAGE --- p.52 / Chapter 2.16.5 --- Reagents for Western blot --- p.53 / Chapter 2.16.6 --- Reagents for DNA sequencing --- p.54 / Chapter 2.16.7 --- Reagents for electroblotting using PVDF --- p.54 / Chapter Chapter 3 --- The Study of Antigenic Determinants of TCS by Transposon Mediated Deletion Mutagenesis / Chapter 3.1 --- Introduction --- p.55 / Chapter 3.2 --- Results --- p.56 / Chapter 3.2.1 --- Construction of deletion library of pET58210 by transposon mediated mutagenesis --- p.56 / Chapter 3.2.2 --- PCR insertion mapping of the transposon inserted clones --- p.60 / Chapter 3.2.3 --- DNA sequencing of positive clones after the PCR insertion mapping --- p.63 / Chapter 3.2.4 --- Computer analysis of the DNA sequence --- p.65 / Chapter 3.2.5 --- Expression of mutated plasmids with Tn 1000 --- p.65 / Chapter 3.2.6 --- Subcloning of the positive clones that have been characterized by DNA sequencing --- p.68 / Chapter 3.2.7 --- Overexpression of mutated TCS protein with the T7 RNA polymerase driven expression system --- p.71 / Chapter 3.2.8 --- Analysis of TCS deletion mutants by Western blotting --- p.71 / Chapter 3.2.9 --- Construction of deletion mutants that exclude the 21 amino acids in the N-terminal of TCS --- p.77 / Chapter 3.3 --- Discussion --- p.80 / Chapter Chapter 4 --- The Study of Antigenic Determinants on TCS by Chemical and Enzymatic Cleavages of TCS Protein / Chapter 4.1 --- Introduction --- p.89 / Chapter 4.2 --- Results --- p.91 / Chapter 4.2.1 --- Preparation of recombinant TCS protein --- p.91 / Chapter 4.2.2 --- Proteolytic and chemical cleavages of recombinant TCS --- p.94 / Chapter 4.2.3 --- Western blotting analysis of TCS protein fragment generated by proteolytic and chemical cleavage --- p.96 / Chapter 4.2.4 --- N-terminal determination of the antigenic TCS fragment produced by chemical cleavage --- p.100 / Chapter 4.3 --- Discussion --- p.103 / Chapter Chapter 5 --- General Discussion and Future Study / Chapter 5.1 --- General Discussion --- p.107 / Chapter 5.2 --- Future Study --- p.115 / References --- p.116

by Chan Wah Lun. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1995. / Includes bibliographical references (leaves 252-260). / Acknowledgments --- p.i / Abstract --- p.ii / Contents --- p.vi / Naming of TCS mutants and modified TCS protein --- p.x / Abbreviations --- p.xi / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Physical and chemical properties of Trichosanthin --- p.1 / Chapter 1.2 --- Biological activities of Trichosanthin --- p.3 / Chapter 1.3 --- Renal tubular reabsorption and nephrotoxicity of Trichosanthin --- p.10 / Chapter 1.4 --- Objective and strategies of study --- p.11 / Chapter Chapter 2 --- Materials and methods --- p.19 / Chapter 2.1 --- General Techniques --- p.19 / Chapter 2.2 --- Site directed mutagenesis of Trichosanthin --- p.21 / Chapter 2.3 --- DNA sequencing --- p.37 / Chapter 2.4 --- Overexpression of modified Trichosanthin in E. coli --- p.42 / Chapter 2.5 --- Purification of modified Trichosanthin --- p.43 / Chapter 2.6 --- Breaking of Disulphide bridge between modified TCS --- p.44 / Chapter 2.7 --- Coupling of DX T40 to modified Trichosanthin --- p.44 / Chapter 2.8 --- Biological activities of modified Trichosanthin and Dextran-modified trichosantin conjugates --- p.46 / Chapter 2.9 --- Immunogenicity of modified Trichosanthin and Dextran-trichosanthin conjugates --- p.50 / Chapter 2.10 --- Nephrotoxicity of Trichosanthin and Dextran-trichosanthin conjugates --- p.53 / Chapter Chapter 3 --- Construction of TCS mutants --- p.61 / Chapter 3.1 --- Introduction --- p.61 / Chapter 3.2 --- Method --- p.61 / Chapter 3.3 --- Results --- p.62 / Chapter 3.3.1 --- Construction of K173C mutant --- p.62 / Chapter 3.3.2 --- Construction of R29C mutant --- p.64 / Chapter 3.3.3 --- Construction of K173C R29C double mutant --- p.65 / Chapter 3.4 --- Discussion --- p.66 / Chapter Chapter 4 --- "Expression,Purification and Ribosome- inactivating activities of Modified Trichosanthin proteins" --- p.87 / Chapter 4.1 --- Introduction --- p.87 / Chapter 4.2 --- Method --- p.87 / Chapter 4.3 --- Results --- p.88 / Chapter 4.3.1 --- "Expression, purification and ribosome-inactivating activity of K173C" --- p.88 / Chapter 4.3.2 --- "Expression ,purification and ribosome-inactivating activity of R29C" --- p.89 / Chapter 4.3.3 --- "Expression, purification and ribosome-inactivating activity of K173C R29C" --- p.90 / Chapter 4.4 --- Discussion --- p.91 / Chapter Chapter 5 --- Coupling of Dextran T40 to modified Trichosanthin --- p.108 / Chapter 5.1 --- Introduction --- p.108 / Chapter 5.2 --- Method --- p.109 / Chapter 5.3 --- Results --- p.109 / Chapter 5.3.1 --- Coupling of R29C --- p.109 / Chapter 5.3.2 --- Coupling of K173C --- p.111 / Chapter 5.3.3 --- Coupling of R29CK173C --- p.111 / Chapter 5.4 --- Discussion --- p.111 / Chapter Chapter 6 --- Biological Activities of modified Trichosanthin and Dextran-modified trichosanthin conjugates --- p.128 / Chapter 6.1 --- Introduction --- p.128 / Chapter 6.2 --- Method --- p.128 / Chapter 6.3 --- Results --- p.130 / Chapter 6.3.1 --- In vivo Biological activity- Mid-term abortifacient activity --- p.130 / Chapter 6.3.2 --- In vitro biological activities / Chapter 6.3.2a --- Ribosome-inactivating activity --- p.131 / Chapter 6.3.2b --- Anti-tumour activity --- p.132 / Chapter 6.4 --- Discussion --- p.133 / Chapter Chapter 7 --- Immunogenicity of Dextran-modified trichosanthin conjugates --- p.156 / Chapter 7.1 --- Introduction --- p.156 / Chapter 7.2 --- Method --- p.157 / Chapter 7.3 --- Results / Chapter 7.3.1 --- Immunogenicity without denaturation of protein --- p.158 / Chapter 7.3.2 --- Immunogenicity with denaturation of protein --- p.161 / Chapter 7.4 --- Discussion --- p.162 / Chapter Chapter 8 --- Nephrotoxicity of Trichosanthin and Dextran-Trichosanthin conjugates --- p.199 / Chapter 8.1 --- Introduction --- p.200 / Chapter 8.2 --- Method --- p.202 / Chapter 8.3 --- Results --- p.202 / Chapter 8.3.1 --- Functional study on nephrotoxicity of Trichosanthin --- p.202 / Chapter 8.3.2 --- Morphological study on the nephrotoxicity of Trichosanthin --- p.203 / Chapter 8.3.3 --- The effect of coupling of Dextran T40 on the nephrotoxicity of Trichosanthin --- p.206 / Chapter 8.4 --- Discussion --- p.207 / Chapter Chapter 9 --- General Discussion --- p.244 / References --- p.252

by Wong Yick Fu. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1990. / Bibliography: leaves 201-224. / Acknowledgments --- p.V / Summary --- p.vi / Publications --- p.ix / Statement of Originality --- p.X / List of Abbreviations --- p.xi / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Preamble --- p.2 / Chapter 1.2 --- History of Trichosanthin --- p.6 / Chapter 1.3 --- Preparation of Trichosanthin --- p.8 / Chapter 1.4 --- Chemistry of Trichosanthin --- p.10 / Chapter 1.4.1 --- Primary structure --- p.10 / Chapter 1.4.2 --- Three dimensional structure --- p.12 / Chapter 1.5 --- Pharmacology of Trichosanthin --- p.14 / Chapter 1.5.1 --- Pharmacologic action --- p.14 / Chapter 1.5.2 --- Pharmacokinetics --- p.18 / Chapter 1.5.3 --- Toxicity --- p.21 / Chapter 1.6 --- Clinical Use of Trichosanthin --- p.24 / Chapter 1.6.1 --- Clinical application --- p.24 / Chapter 1.6.2 --- Mechanism of action --- p.29 / Chapter 1.6.3 --- Adverse reactions --- p.31 / Chapter 1.6.4 --- Contraindications --- p.33 / Chapter 1.7 --- Objectives of Project and Organization of Thesis --- p.35 / Chapter Chapter 2 --- Anti-tumour Activity of Trichosanthin In Vitro and In Vivo --- p.37 / Chapter 2.1 --- Cytotoxic Effects of Trichosanthin on Cultured Tumour Cells --- p.38 / Chapter 2.1.1 --- Introduction --- p.38 / Chapter 2.1.2 --- Materials and methods --- p.40 / Chapter 2.1.3 --- Results --- p.49 / Chapter 2.1.4 --- Discussion --- p.59 / Chapter 2.2 --- Effects of Trichosanthin on Co-cultured Cell Lines In Vitro --- p.64 / Chapter 2.2.1 --- Introduction --- p.64 / Chapter 2.2.2 --- Materials and methods --- p.65 / Chapter 2.2.3 --- Results --- p.66 / Chapter 2.2.4 --- Discussion --- p.77 / Chapter 2.3 --- Combination Effects of Trichosanthin with Adriamycin and Cisplatin on Cultured Tumour Cells --- p.80 / Chapter 2.3.1 --- Introduction --- p.80 / Chapter 2.3.2 --- Materials and methods --- p.81 / Chapter 2.3.3 --- Results --- p.84 / Chapter 2.3.4 --- Discussion --- p.93 / Chapter 2.4 --- Effects of Trichosanthin on Choriocarcinoma Cells In Vivo --- p.96 / Chapter 2.4.1 --- Introduction --- p.96 / Chapter 2.4.2 --- Materials and methods --- p.97 / Chapter 2.4.3 --- Results --- p.101 / Chapter 2.4.4 --- Discussion --- p.107 / Chapter 2.5 --- Effects of Trichosanthin Protein and Polysaccharide on Choriocarcinoma Cells In Vitro --- p.110 / Chapter 2.5.1 --- Introduction --- p.110 / Chapter 2.5.2 --- Materials and methods --- p.112 / Chapter 2.5.3 --- Results --- p.114 / Chapter 2.5.4 --- Discussion --- p.117 / Chapter Chapter 3 --- Mechanism of Action of Trichosanthin on Tumour Cells --- p.119 / Chapter 3.1 --- Morphological Study of Effects of Trichosanthin on Cultured Choriocarcinoma Cells --- p.120 / Chapter 3.1.1 --- Introduction --- p.120 / Chapter 3.1.2 --- Materials and methods --- p.121 / Chapter 3.1.3 --- Results --- p.124 / Chapter 3.1.4 --- Discussion --- p.133 / Chapter 3.2 --- Binding of Radiolabelled Trichosanthin with Tumour Cells In Vitro --- p.137 / Chapter 3.2.1 --- Introduction --- p.137 / Chapter 3.2.2 --- Materials and methods --- p.138 / Chapter 3.2.3 --- Results --- p.146 / Chapter 3.2.4 --- Discussion --- p.154 / Chapter 3.3 --- Effects of Trichosanthin on Macromolecule Synthesis of Choriocarcinoma Cells In vitro --- p.159 / Chapter 3.3.1 --- Introduction --- p.159 / Chapter 3.3.2 --- Materials and methods --- p.160 / Chapter 3.3.3 --- Results --- p.163 / Chapter 3.3.4 --- Discussion --- p.167 / Chapter Chapter 4 --- General Discussion --- p.169 / Chapter 4.1 --- Anti-tumour Activity of Trichosanthin --- p.170 / Chapter 4.2 --- Mechanism of Action of Trichosanthin --- p.180 / Chapter 4.3 --- Prospects of Research on Trichosanthin --- p.195 / References --- p.201 / Appendix 1 --- p.225 / Appendix 2 --- p.242

by Wong Kam Bo. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1993. / Includes bibliographical references (leaves 149-153). / Acknowledgments --- p.i / Abstract --- p.ii / Contents --- p.iii / Abbreviations --- p.vii / Short names for mutants --- p.viii / One letter symbol for amino acids --- p.x / Chapter Chapter 1 --- Introduction / Chapter 1.1. --- Chemical and Physical Properties of Trichosanthin --- p.1 / Chapter 1.2. --- Activities of Trichosanthin at the cellular level --- p.2 / Chapter 1.3. --- Activities of Trichosanthin at the molecular level --- p.3 / Chapter 1.4. --- Objective and Strategy of Protein engineering of Trichosanthin --- p.8 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1. --- General Techniques --- p.15 / Chapter 2.1.1. --- Ethanol Precipitation of DNA and RNA --- p.15 / Chapter 2.1.2. --- Spectrophotometric quantification of DNA and RNA --- p.15 / Chapter 2.1.3. --- Minipreparation of Plasmid DNA --- p.15 / Chapter 2.1.4. --- Preparation of Plasmid DNA using Qiagen-pack 100 Cartridge --- p.16 / Chapter 2.1.5. --- Preparation of Plasmid DNA using Magic´ёØ Minipreps DNA Purification kit from Promega --- p.17 / Chapter 2.1.6. --- Preparation and Transformation of Escherichia coli Competent Cell --- p.18 / Chapter 2.1.7. --- Agarose Gel Electrophoresis of DNA --- p.19 / Chapter 2.1.8. --- Purification of DNA from Agarose Gel using GeneClean® (BIO 101 Inc.) kit --- p.20 / Chapter 2.1.9. --- Polymerase Chain Reaction (PGR) --- p.21 / Chapter 2.1.10. --- Restriction Digestion of DNA --- p.23 / Chapter 2.1.11. --- Ligation of DNA fragments --- p.23 / Chapter 2.1.12. --- Autoradiography --- p.24 / Chapter 2.1.13. --- SDS-Polyacrylamide Gel Electrophoresis (SDS- PAGE) --- p.24 / Chapter 2.1.14. --- Staining of Protein in polyacrylamide gel --- p.25 / Chapter 2.1.15. --- Western Blot detection of TCS --- p.25 / Chapter 2.1.16. --- Liquid Scintillation Counting --- p.27 / Chapter 2.1.17. --- Minimization of Ribonuclease (RNAase) activity in experiments involving RNA --- p.27 / Chapter 2.2. --- Site-Directed Mutagenesis of Trichosanthin --- p.28 / Chapter 2.2.1. --- "Construction of E160D,El60A and SEAAR deletion mutants" --- p.28 / Chapter 2.2.2. --- Construction of E189A mutant and El60A E189A double mutant --- p.31 / Chapter 2.2.3. --- Construction of E189D mutant and El60A E189D double mutant --- p.36 / Chapter 2.2.4. --- Construction of Q156A mutant --- p.38 / Chapter 2.2.5. --- Construction of Q156A El60A mutant (Fig. 2.7) --- p.41 / Chapter 2.2.6. --- Construction of Q156A El89A mutant (Fig. 2.8) --- p.43 / Chapter 2.3. --- DNA sequencing --- p.45 / Chapter 2.3.1. --- DNA Sequencing Reaction --- p.45 / Chapter 2.3.2. --- DNA Sequencing Electrophoresis --- p.46 / Chapter 2.3.3. --- Resolving GC band compression --- p.48 / Chapter 2.4. --- Overexpression of mutated TCS in Escherichia coli --- p.48 / Chapter 2.5. --- Purification of mutated TCS --- p.49 / Chapter 2.6. --- Ribosome inactivating activity Assay using Rabbit Reticulocyte Lysate In Vitro Translation system --- p.50 / Chapter 2.7. --- N-glycosidase activity Assay --- p.51 / Chapter 2.7.1. --- Inactivation of ribosome in rabbit reticulocyte lysate --- p.51 / Chapter 2.7.2. --- RNA extraction --- p.51 / Chapter 2.7.3. --- Aniline Degradation --- p.52 / Chapter 2.7.4. --- Electrophoresis of RNA in Agarose Gel containing Formamide --- p.52 / Chapter 2.8. --- Reagents and buffers --- p.53 / Chapter 2.8.1. --- Nucleic Acid Electrophoresis Buffers --- p.53 / Chapter 2.8.2. --- Reagents for preparation of plasmid DNA --- p.54 / Chapter 2.8.3. --- Media for bacterial culture --- p.54 / Chapter 2.8.4. --- Reagents for SDS-PAGE --- p.55 / Chapter 2.8.5. --- Reagents for western blot --- p.56 / Chapter 2.8.6. --- Reagents for DNA sequencing --- p.57 / Chapter Chapter 3 --- Construction of TCS mutants / Chapter 3.1. --- Introduction --- p.59 / Chapter 3.2. --- Results --- p.59 / Chapter 3.2.1. --- "Construction of E160D,El60A and ASEAAR" --- p.59 / Chapter 3.2.2. --- Construction of E189A and E160AE189A mutants --- p.66 / Chapter 3.2.3. --- Construction of E189D and E160AE189D mutants --- p.80 / Chapter 3.2 --- A Construction of Q156A mutant --- p.82 / Chapter 3.2.5. --- Construction of Q156AE160A and Q156AE189A --- p.86 / Chapter 3.3. --- Discussion --- p.90 / Chapter Chapter 4 --- Expression and Purification of mutated TCS proteins / Chapter 4.1. --- Introduction --- p.94 / Chapter 4.2. --- Results --- p.95 / Chapter 4.2.1. --- Expression and purification of E160D and El60A mutants --- p.95 / Chapter 4.2.2. --- Expression and purifcation of E189D and E160AE189D mutants --- p.99 / Chapter 4.2.3. --- Expression and purifcation of E189A and E160AE189A mutants --- p.104 / Chapter 4.2.4. --- Expression and purifcation of Q156A and Q156AE160A mutants --- p.109 / Chapter 4.2.5. --- Expression and purifcation of Q156AE189A mutant --- p.114 / Chapter 4.2.6. --- Analysis of protein purity by SDS-PAGE and Western immunoblotting --- p.114 / Chapter 4.3. --- Discussion --- p.119 / Chapter Chapter 5 --- Biological Assay of mutated proteins / Chapter 5.1. --- Introduction --- p.125 / Chapter 5.2. --- Results --- p.125 / Chapter 5.2.1. --- Ribosome inactivating activity assay --- p.125 / Chapter 5.2.2. --- N-glycosidase activities of El60AE189A mutant --- p.131 / Chapter 5.3. --- Discussion --- p.133 / Chapter 5.3.1. --- Role of glutamate-160 --- p.133 / Chapter 5.3.2. --- A putative mechanism for N-glycosidase activity of TCS --- p.137 / Chapter 5.3.3. --- Role of glutamate-189 and glutamine-156 --- p.143 / Chapter 5.3.4. --- Prospective and future studies --- p.145 / Chapter 5.4. --- Concluding remarks --- p.147 / Appendix / Chapter A.l --- Size of molecule weight markers --- p.148 / Chapter A.2 --- Reference --- p.149

He Xian-hui. / "November 1998." / Thesis (Ph.D.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (p. 218-230). / 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.

No description available.

Finally, NF-kappaB and another transcriptional regulator p53 are usually tightly related in their control of cell survival. Opposite to NF-kappaB, p53 mediates cell death signals, usually activated under DNA damage and subsequently involved in cell growth control, DNA damage repair or apoptosis. It was found in this study, DNA damage and cell cycle arrest responses tended to participate with the anti-HSV-1 activity. Although in HEp-2 cells, TCS induced more DNA damage ratio and S and G2/M phase arrest proportion than HSV-1 infected cells, but more p53 was expressed and activated by phosphorylating at Ser 15 by TCS in HSV-1 infected HEp-2 cells than uninfected ones. In the same time the activation of BAX, which promotes the apoptotic function of p53, increased during TCS treatment when infected with HSV-1, the p53 therefore regulates apoptosis in HSV-1 infected cell during TCS treatment. / Firstly, we demonstrated that TCS reduced HSV-1 antigen and DNA content, The IC50 (half maximal inhibitory concentration) of TCS on HSV-1 replication was 2.5 +/- 0.23 mug/ml. The anti-HSV-1 effect of TCS was related to interfering with viral replication during 3 to 15 hours after infection which coincide with early to late viral replication period. TCS had no effect on HSV-1 attachment, penetration or immediate early gene expression. However, the expression of early gene, late gene and virion release were diminished. / Fourthly, the role of the nuclear factor-kappaB (NF-kappaB) in the anti-HSV-1 effect of TCS was explored. NF-kappaB initiates cell survival pathways. It is widely involved in viral infection and replication to make sure virus overcomes the host cells immune response. We found HSV-1 enhanced the activity of NF-kappaB in HEp-2 cells by triggering its translocation from cytoplasm to nuclear. However, during the anti-HSV-1 effect of TCS, TCS suppressed HSV-1-aroused NF-kappaB translocation in HEp-2 cells, the inhibition of NF-kappaB activity in HSV-1-infected cells by TCS treatment tend to abolish the anti-apoptosis effect developed by HSV-1, so that the host cells suffered more extracellular stress and showed more apoptosis ratio than uninfected ones. / Taken together, this study demonstrated TCS interfered with HSV-1 early to late infection period. TCS selectively induced more HSV-1 infected HEp-2 cells to apoptosis than uninfected ones, the selectivity of TCS was due to apoptotic signaling pathway switching from CD95 (Fas/Apo-1)-mediated type I to type II apoptotic pathway. Furthermore, during TCS induced-apoptosis in HSV-1 infected cells, TCS suppressed NF-kappaB activation that triggered by HSV-1 infection. At the meanwhile, p53 participated in the TCS-induced apoptosis regulation in infected cell. / TCS is toxic to cell because its RIP activity, killing of the viral host cells certainly inhibits virus expansion, only when it kills more infected cells, the material could be considered as an anti-viral agent. It was found TCS induced losing of cell viability and enhancing in apoptosis in HEp-2 cells and HSV-1 infected HEp-2 cells. The decrease of cell viability and increase of apoptosis ratio were enhanced when HEp-2 cells were infected with HSV-1 compared with uninfected ones. The 50% of effect concentration (EC50 ) in cytotoxicity and apoptosis were decreased from 24.64 +/- 1.17 mug/ml and 37.57 +/- 1.47 mug/ml in uninfected HEp-2 cells to 3.01 +/- 1.30 mug/ml and 3.89 +/- 1.31 mug/ml in HSV-1 infected HEp-2 cells respectively. / The reason of type I to type II apoptosis pathway transition might due to the activity change of death receptor on HEp-2 cells. The type I apoptotic pathway induced by TCS was related to CD95 (Fas/Apo-1) system activation and signaling pathway. When HEp-2 cells were infected with HSV-1, the CD95 (Fas/Apo-1) was suppressed by HSV-1 infection. As a result, TCS triggered a less CD95 (Fas/Apo-1) dependent type II apoptotic pathway in the infected cells. / Thirdly, TCS activated different apoptotic pathways, namely type I and type II apoptotic pathways, between uninfected and infected cells. The type I apoptotic pathway bypasses the dependence on the mitochondrial but quickly activates a large amount of caspase-8 at the CD95 (Fas/Apo-1) formed death inducing signaling complex (DISC), which amplifies the signal. By contrast, the formation of the DISC in the type II apoptotic pathway is strongly reduced. It depends on loss of the mitochondrial transmembrane potential (DeltaPsi m) and release of cytochrome c and capase-9 activation to mediate apoptosis signal transduction. We found in HSV-1 infected an uninfected HEp-2 cells, TCS induced the loss of DeltaPsim, this DeltaPsim losing was increased when HEp-2 cells were infected with HSV-1. Furthermore, when there were no HSV-1 infection, TCS induced caspase-dependent type I apoptosis pathway that quickly activated large amount of caspase-8 after TCS treatment. However, when infected with HSV-1, this pathway turned into mitochondrial dependent type II pathway involving caspase-9 response, whose apoptosis ratio was diminished by over expressed Bcl-2, which is a hallmark defining type I or type II apoptosis. / Trichosanthin (TCS) is a type I ribosome inactivating protein (RIP), it was found to inhibit human simplex virus type 1 (HSV-I) but the anti-HSV-I mechanism is unclear. HSV-1 is a widely distributed DNA virus, it causes large range of human diseases. During the lytic life cycle of HSV-1, highly regulated cascade of genes are expressed to interfere with host cell metabolism and immune response. In this context the anti-HSV-1 mechanism of TCS in human epithelial carcinoma HEp-2 cells was studied. / He, Dongxu. / Advisers: Wing Ho Yung; Siu Cheung Tam. / Source: Dissertation Abstracts International, Volume: 73-06, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 124-138). / 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.

Chan Siu Hong. / "December 1999." / Thesis (Ph.D.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (p. 172-192). / 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.

by Lam Ying Hoo. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 145-149). / Acknowledgements --- p.i / Abstract --- p.ii / Contents --- p.iv / Abbreviations --- p.x / Chapter Chapter 1 --- Introduction / Chapter 1.1. --- Trichosanthin --- p.1 / Chapter 1.2. --- Anti-plant viral and fungal properties of RIPs --- p.3 / Chapter 1.3. --- Agrobacterium-mediated transformation --- p.5 / Chapter 1.3.1. --- Ti (tumor inducing) plasmid --- p.6 / Chapter 1.3.2. --- Role of vir proteins in T-DNA transfer --- p.6 / Chapter 1.3.3. --- Integration of T-DNA into plant genome --- p.11 / Chapter 1.3.4. --- Use of Agrobacterium plasmid as transformation vectors --- p.13 / Chapter 1.4. --- Objective and strategy of producing transgenic plants that express TCS --- p.15 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1. --- Bacterial Strains used --- p.19 / Chapter 2.2. --- General Techniques --- p.20 / Chapter 2.2.1. --- Growth of bacterial strains --- p.20 / Chapter 2.2.2. --- Restriction Enzyme Digestion of DNA --- p.21 / Chapter 2.2.3. --- Agarose Gel Electrophoresis of DNA --- p.21 / Chapter 2.2.4. --- Purification of DNA fragments from Agarose Gel using GeneClean II® ( BIO 101 Inc.) kit --- p.22 / Chapter 2.2.5. --- Purification of DNA fragments by Phenol/Chloroform Extraction --- p.23 / Chapter 2.2.6. --- Ligation of DNA fragments --- p.24 / Chapter 2.2.7. --- Preparation and Transformation of Escherichia coli Competent Cells --- p.24 / Chapter 2.2.8. --- Minipreparation of Plasmid DNA --- p.26 / Chapter 2.2.9. --- Preparation of Plasmid DNA using Magic´ёØ Minipreps DNA Purification kit from Promega --- p.27 / Chapter 2.2.10. --- Preparation of Plasmid DNA using Qiagen-pack 100 Cartridge --- p.29 / Chapter 2.2.11. --- SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.30 / Chapter 2.2.12. --- Western Blot detection of TCS --- p.33 / Chapter 2.2.13. --- Polymerase Chain Reaction (PCR) --- p.34 / Chapter 2.3. --- Construction of Plant Transformation Vectors --- p.36 / Chapter 2.3.1. --- Construction of pSLJ58210 --- p.36 / Chapter 2.3.2. --- Construction of pSLJ TCS1 and pSLJ TCS2 --- p.38 / Chapter 2.3.3. --- Conjugation of pSLJ TCS1 and pSLJ TCS2 into A. tumefaciens by Triparental Mating --- p.41 / Chapter 2.4. --- Transformation of Tobacco Leaf Explants by Agrobacterium tumefaciens --- p.43 / Chapter 2.4.1. --- Growth of A. tumefaciens LBA4404 (pSLJ TCS1) --- p.43 / Chapter 2.4.2. --- Surface Sterilization of tobacco leaves --- p.43 / Chapter 2.4.3. --- Inoculation of tobacco leaf explants with A. tumefaciens LBA4404 (pSLJ TCS1) --- p.44 / Chapter 2.4.4. --- Regeneration of shoots from Transformed explants --- p.45 / Chapter 2.4.5. --- Rooting of Transformed shoots --- p.45 / Chapter 2.4.6. --- Re-establishment of cultured Plantlets in soil --- p.45 / Chapter 2.5. --- Analysis of the Regenerated Transgenic Tobacco --- p.46 / Chapter 2.5.1. --- Isolation of plant leaf protein --- p.46 / Chapter 2.5.2. --- SDS-PAGE and Western blot detection of TCS --- p.48 / Chapter 2.5.3. --- Anti-viral assay of Transgenic tobacco against TuMV --- p.48 / Chapter 2.6. --- Bioassay of Inhibitory activity of TCS protein against TuMV --- p.49 / Chapter 2.6.1. --- Preparation of biologically active TCS protein --- p.49 / Chapter 2.6.2. --- Purification of TuMV from infected plant leaves --- p.51 / Chapter 2.6.3. --- Mechanical Inoculation of virus onto host plant --- p.53 / Chapter 2.6.4. --- Anti-viral assay on Local Lesion host --- p.54 / Chapter 2.6.5. --- Anti-viral assay on Systemic host --- p.55 / Chapter 2.7 --- Establishment of the plant culture medium for efficient Regeneration from tissue explants of Brassica parachinensis --- p.56 / Chapter 2.7.1. --- Preparation and Sterilization of culture medium --- p.57 / Chapter 2.7.2. --- Preparation of Sterile seedlings of B. parachinensis --- p.57 / Chapter 2.7.3. --- Regeneration from Cotyledon petiole and Hypocotyl segment explants --- p.58 / Chapter 2.7.4. --- Regeneration from Internode stem segment explants of shoot culture --- p.60 / Chapter 2.8. --- Reagents and Buffers --- p.61 / Chapter 2.8.1. --- Media for Bacterial culture --- p.61 / Chapter 2.8.2. --- Media for Plant tissue culture --- p.64 / Chapter 2.8.3. --- Restriction Enzymes --- p.66 / Chapter 2.8.4. --- Buffers for Agarose Gel Electrophoresis --- p.66 / Chapter 2.8.5. --- DNA ligation Buffer --- p.67 / Chapter 2.8.6. --- Reagents for preparation of E.coli competent cells --- p.67 / Chapter 2.8.7. --- Reagents for preparation of Plasmid DNA --- p.68 / Chapter 2.8.8. --- Reagents for Qiagen-pack 100 Cartridge --- p.69 / Chapter 2.8.9. --- Reagents for SDS-PAGE --- p.70 / Chapter 2.8.10. --- Reagents for Western Blotting --- p.71 / Chapter Chapter 3 --- Construction of Plant Transformation Vectors / Chapter 3.1. --- Introduction --- p.73 / Chapter 3.2. --- Results --- p.74 / Chapter 3.2.1. --- Construction of pSLJ58210 --- p.74 / Chapter 3.2.2. --- Construction of the recombinant binary vectors pSLJ TCSl and pSLJ TCS --- p.78 / Chapter 3.2.3. --- Conjugation ofpSLJ TCS 1 and pSLJ TCS 2 into Agrobacterium tumefaciens via Triparental Mating --- p.82 / Chapter 3.3. --- Discussion --- p.90 / Chapter Chapter 4 --- Transformation of Tobacco Leaf Explants by Agrobacterium tumefaciens / Chapter 4.1. --- Introduction --- p.94 / Chapter 4.2. --- Results --- p.95 / Chapter 4.2.1. --- Regeneration of leaf explants after transformation --- p.95 / Chapter 4.2.2. --- The level of expression of TCS in transgenic tobacco leaf --- p.100 / Chapter 4.3. --- Discussion --- p.104 / Chapter 4.3.1. --- Regeneration of transgenic tobacco plants --- p.104 / Chapter 4.3.2. --- Expression of TCS in transgenic tobacco plants --- p.108 / Chapter Chapter 5 --- Two approaches to study the Inhibitory effect of TCS on TuMV / Chapter 5.1. --- Introduction --- p.112 / Chapter 5.2. --- Results --- p.113 / Chapter 5.2.1. --- Expression and purification of recombinant TCS --- p.113 / Chapter 5.2.2. --- Purification of TuMV --- p.119 / Chapter 5.2.3. --- Anti-viral assay on local lesion host --- p.119 / Chapter 5.2.4. --- Anti-viral assay on Systemic host --- p.124 / Chapter 5.2.5. --- Anti-viral assay of Transgenic tobacco against TuMV --- p.126 / Chapter 5.3. --- Discussion --- p.129 / Chapter Chapter 6 --- Establishment of plant culture conditions for efficient shoot regeneration from tissue explants of B.parachinensis / Chapter 6.1. --- Introduction --- p.133 / Chapter 6.2. --- Results --- p.133 / Chapter 6.3. --- Discussion --- p.137 / Chapter Chapter 7 --- Conclusion / Appendix / Chapter A.1. --- Size of molecular weight markers --- p.143 / Chapter A.2. --- References --- p.145