Thesis (Ph. D.)--University of Queensland, 2002. / Includes bibliographical references.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv. / Härtill 4 uppsatser.

Li Chi Ho. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 137-162). / Abstracts in English and Chinese. / Statement --- p.iii / Acknowledgments --- p.iv / Abstract --- p.vi / Abstract (Chinese version) --- p.viii / Abbreviations --- p.x / Table of contents --- p.xii / List of Figure --- p.xviii / List of Table --- p.xx / Chapter Chapter One --- Literature Review / Chapter 1.1 --- The Genus Schistosoma --- p.1 / Chapter 1.2 --- Biology of Schistosoma japonicum --- p.3 / Chapter 1.2.1 --- The History of discovery of Schitosoma japonicum --- p.3 / Chapter 1.2.2 --- Life cycle of Schistosoma japonicum --- p.3 / Chapter 1.2.2.1 --- Egg --- p.6 / Chapter 1.2.2.2 --- Miracidia --- p.6 / Chapter 1.2.2.3 --- Sporocysts --- p.7 / Chapter 1.2.2.4 --- Cercaria --- p.9 / Chapter 1.2.2.5 --- Schislosomula --- p.10 / Chapter 1.2.2.6 --- Adult worms --- p.10 / Chapter 1.2.3 --- Genetics of Schistosoma japonicum --- p.11 / Chapter 1.2.3.1 --- Genome analysis --- p.12 / Chapter 1.2.3.2 --- Schistosome genome --- p.13 / Chapter 1.2.4 --- Tegumental membrane of Schistosomes --- p.14 / Chapter 1.3 --- Pathology of Schistosomiasis --- p.15 / Chapter 1.3.1 --- Acute Schistosomiasis --- p.15 / Chapter 1.3.2 --- Intestinal Disease --- p.16 / Chapter 1.3.3 --- Hepatosplenic Disease --- p.16 / Chapter 1.3.4 --- Cerebral Schistosomiasis --- p.16 / Chapter 1.4 --- Treatment of Schistosomiasis --- p.17 / Chapter 1.4.1 --- Chemotherapy --- p.17 / Chapter 1.4.2 --- Schistosoma Vaccine --- p.17 / Chapter 1.5 --- GPI anchor --- p.19 / Chapter 1.5.1 --- Function of GPI anchored proteins --- p.19 / Chapter 1.5.2 --- Synthesis of GPI anchor --- p.21 / Chapter 1.5.3 --- "Phosphatidylinositol Glycan, Class N (PIG-N)" --- p.26 / Chapter 1.6 --- The role of GPI anchor proteins in Schistosome --- p.26 / Chapter 1.7 --- Aim of study --- p.29 / Chapter Chapter two --- Materials and Methods / Chapter 2.1 --- Materials --- p.31 / Chapter 2.1.1 --- Cell lines and Bacterial Strains --- p.31 / Chapter 2.1.2 --- Chemicals --- p.32 / Chapter 2.1.3 --- Kits and Reagents --- p.34 / Chapter 2.1.4 --- Nucleic acids --- p.34 / Chapter 2.1.5 --- Reagents for Cell culture --- p.34 / Chapter 2.1.6 --- Solutions --- p.35 / Chapter 2.1.7 --- Enzymes --- p.37 / Chapter 2.1.8 --- Primer List --- p.37 / Chapter 2.1.9 --- Antibodies --- p.39 / Chapter 2.2 --- Methods / Chapter 2.2.1 --- Screening of the S. japonicum cercaria stage cDNA library --- p.40 / Chapter 2.2.1.1 --- λ phage plating --- p.40 / Chapter 2.2.1.2 --- Single plaque isolation --- p.40 / Chapter 2.2.1.3 --- Conversion of Lambda TriplEx to pTriplEx --- p.41 / Chapter 2.2.1.4 --- preparation of plasmid DNA --- p.41 / Chapter 2.2.1.5 --- cycle DNA sequencing --- p.42 / Chapter 2.2.2. --- RT-PCR --- p.44 / Chapter 2.2.2.1 --- Isolation of Total RNA by Guandidium Thiocyanate - Cesium Chloride ultracentrifugation --- p.44 / Chapter 2.2.2.2 --- Synthesis of Fist Strand cDNA by reverse transcriptation reaction --- p.45 / Chapter 2.2.2.3 --- PGR amplification of RT product --- p.46 / Chapter 2.2.3 --- Rapid Amplification of cDNA Ends (RACE) --- p.46 / Chapter 2.2.3.1 --- Synthesis of first strand cDNA for RACE reaction --- p.46 / Chapter 2.2.3.2 --- 5 ´ةRACE for Sj-PIG-N gene --- p.47 / Chapter 2.2.3.3 --- 3´ة RACE for Sj-PIG-N gene --- p.48 / Chapter 2.2.3.4 --- Purification of DNA fragment from agarose gel --- p.48 / Chapter 2.2.3.5 --- Ligation of purified PCR fragments and pBluescriptII KS(+) T-vector --- p.49 / Chapter 2.2.3.6 --- Preparation of DH5a competent cells --- p.49 / Chapter 2.2.3.7 --- Transformation of recombinant plasmid --- p.50 / Chapter 2.2.4 --- Mammalian cell transfection --- p.50 / Chapter 2.2.4.1 --- Stable transfection --- p.50 / Chapter 2.2.4.2 --- Transient transfection --- p.51 / Chapter 2.2.5 --- Biological function studies of Sj-PIG-N gene --- p.52 / Chapter 2.2.5.1 --- Flow Cytometry (FCM) analysis --- p.52 / Chapter 2.2.5.2 --- In Vitro Mannose Labeling of Microsomes and Characterization of Glycolipids --- p.53 / Chapter 2.2.6 --- Intracellular localization assay --- p.54 / Chapter Chapter Three --- Results / Chapter 3.1 --- Random sequence analysis of cercaria EST from S. japonicum Cercaria stage cDNA library --- p.55 / Chapter 3.1.1 --- Sequencing results --- p.57 / Chapter 3.1.2 --- BlastX Search results --- p.61 / Chapter 3.2 --- The expression of Sj-PIG-N gene in both adult worms and cercaria --- p.68 / Chapter 3.2.1 --- Spectrophotometric analysis of total RNA --- p.68 / Chapter 3.2.2 --- Detection of Sj-PIG-N gene expression in both adult worm and cercaria stages --- p.70 / Chapter 3.3 --- Cloning of the full length of Sj-PIG-N gene --- p.72 / Chapter 3.3.1 --- Amplification of the full length of Sj-PIG-N gene --- p.72 / Chapter 3.3.2 --- Obtaining the full length sequence of Sj-PIG-N gene --- p.74 / Chapter 3.4 --- Sequence analysis of Ml length Sj-PIG-N cDNA --- p.80 / Chapter 3.5 --- Analysis of Sj-PIG-N protein structure by computer programs --- p.91 / Chapter 3.6 --- Construction of Sj-PIG-N gene into mammalian cell expression vector pEGFP-Nl and pEGFP-Hyg --- p.97 / Chapter 3.6.1 --- Construction of pBluescriptII KS(+)-Sj-PIG-N-B --- p.97 / Chapter 3.6.2 --- Construction of pBluescriptll KS(+)-Sj-PIG-N-E --- p.101 / Chapter 3.6.3 --- Construction of pTRE2-Sj-PIG-N --- p.106 / Chapter 3.6.4 --- Construction of pEGFP-Hyg-Sj-PIG-N --- p.109 / Chapter 3.6.4.1 --- Construction of pEGFP-Hyg --- p.109 / Chapter 3.6.4.2 --- Construction of pEGFP-Hyg-Sj-PIG-N --- p.114 / Chapter 3.7 --- Molecular analysis of Sj-PIG-N gene --- p.117 / Chapter 3.7.1 --- Functional analysis of Sj-PIG-N --- p.117 / Chapter 3.7.1.1 --- FACS analysis of surface expression of GPI-anchored protein - Thyl --- p.117 / Chapter 3.7.1.2 --- FACS analysis of surface expression of GPI-anchor protein - CD24 --- p.119 / Chapter 3.7.1.3 --- In Vitro Mannose Labeling of Microsomes and Characterization of Glycolipids --- p.121 / Chapter 3.7.2 --- Localization of Sj-PIG-N with immunofluorescent staining --- p.123 / Chapter Chapter Four --- Discussion / Chapter 4.1 --- S. japonicum cercaria EST analysis --- p.125 / Chapter 4.2 --- Structure analysis of Sj-Pig-N gene --- p.128 / Chapter 4.3 --- Molecular analysis of Sj-PIG-N --- p.131 / Chapter 4.4 --- Further study --- p.134 / Chapter 4.5 --- Conclusion --- p.136 / References --- p.137

Law Pui-ki. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 98-105). / Abstracts in English and Chinese. / Statement --- p.i / Acknowledgments --- p.ii / Abstract --- p.iii / Table of contents --- p.viii / List of figures --- p.xv / List of tables --- p.xvii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Schistosomiasis --- p.1 / Chapter 1.1.1 --- Disease burden --- p.1 / Chapter 1.1.2 --- Causative agents --- p.1 / Chapter 1.1.3 --- Transmission --- p.2 / Chapter 1.1.4 --- Pathology of the disease --- p.3 / Chapter 1.1.5 --- Control and therapy --- p.3 / Chapter 1.2 --- Schistosomiasis in China --- p.5 / Chapter 1.3 --- Schistosoma japonicum --- p.6 / Chapter 1.3.1 --- Life cycle of S. japonicum --- p.6 / Chapter 1.3.2 --- Biology of S. japonicum cercaria --- p.9 / Chapter 1.3.3 --- Transformation of cercaria to schistosomulum --- p.10 / Chapter 1.3.4 --- Cercarial stage-specific antigens --- p.12 / Chapter 1.4 --- Aim of study --- p.14 / Chapter Chapter 2 --- Materials and methodology --- p.15 / Chapter 2.1 --- Materials --- p.15 / Chapter 2.1.1 --- Schistosoma japonicum cercaria cDNA library --- p.15 / Chapter 2.1.2 --- Cercarial stage-specific clones --- p.15 / Chapter 2.1.3 --- Adult worm and cercaria RNA --- p.18 / Chapter 2.1.4 --- "Snail intermediate host, Oncomelania hupensis" --- p.18 / Chapter 2.1.5 --- Bacterial strains --- p.18 / Chapter 2.1.6 --- Chemicals --- p.19 / Chapter 2.1.7 --- Kits and reagents --- p.21 / Chapter 2.1.8 --- Nucleic acids --- p.22 / Chapter 2.1.9 --- Solutions --- p.22 / Chapter 2.1.10 --- Enzymes --- p.24 / Chapter 2.1.11 --- Primers --- p.25 / Chapter 2.1.12 --- Antibodies --- p.26 / Chapter 2.2 --- Methodology --- p.27 / Chapter 2.2.1 --- Identification of cercarial stage-specific genes --- p.27 / Chapter 2.2.1.1 --- Sequence analysis of cercarial stage-specific clones --- p.27 / Chapter 2.2.1.2 --- "Confirmation of stage-specific expression of the selected gene, 20H8 and sjCa8, by RNA dot blot" --- p.29 / Chapter 2.2.1.2.1 --- Cloning of S. japonicum glyceraldehyde-3-phosphate dehydrogenase (sjGAPDH) (GenBank accession no. U75571) --- p.29 / Chapter 2.2.1.2.1.1 --- Reverse transcription and PCR amplification of sjGAPDH --- p.29 / Chapter 2.2.1.2.1.2 --- Cloning of sjGADPH in pBluescript II KS (-) --- p.29 / Chapter 2.2.1.2.1.3 --- Sequence verification of cloned sjGAPDH --- p.30 / Chapter 2.2.1.2.2 --- Synthesis of Digoxigenin (DIG)-labeled probe --- p.31 / Chapter 2.2.1.2.2.1 --- Synthesis of DIG-labeled probe by PCR --- p.31 / Chapter 2.2.1.2.2.2 --- Estimation of concentration of DIG-labeled probe --- p.32 / Chapter 2.2.1.2.3 --- RNA dot blot --- p.33 / Chapter 2.2.1.2.3.1 --- Transferring RNA to the membrane using the BIO-RAD blotting manifold --- p.33 / Chapter 2.2.1.2.3.2 --- Hybridization of DIG-labeled probe to detect sjGAPDH --- p.33 / Chapter 2.2.1.2.3.3 --- Detection of the chemiluminescent signal --- p.33 / Chapter 2.2.1.2.3.4 --- Stripping membrane for reprobing --- p.34 / Chapter 2.2.2 --- Characterization of the cercarial stage-specific gene and gene product of 20H8 --- p.35 / Chapter 2.2.2.1 --- Cloning of full-length cDNA of 20H8 --- p.35 / Chapter 2.2.2.1.1 --- 5'RACE of 20H8 --- p.35 / Chapter 2.2.2.1.2 --- Cloning of full-length 20H8 into pBluescript II SK(-) --- p.36 / Chapter 2.2.2.2 --- Analysis of DNA sequence and deduced amino acid sequence of 20H8 --- p.37 / Chapter 2.2.2.3 --- Demonstration of the immunogenicity and antigenicity of 20H8 --- p.38 / Chapter 2.2.2.3.1 --- Expression of 20H8 in E. coli --- p.38 / Chapter 2.2.2.3.1.1 --- "Cloning of 20H8 in an E coli expression vector, pET32a+" --- p.38 / Chapter 2.2.2.3.1.2 --- Expression of recombinant 20H8 protein in E. coli --- p.39 / Chapter 2.2.2.3.2 --- Purification and concentration of recombinant 20H8 protein --- p.40 / Chapter 2.2.2.3.3 --- Production of antiserum --- p.40 / Chapter 2.2.2.3.4 --- Evaluation of immunogenicity of recombinant 20H8 protein --- p.41 / Chapter 2.2.2.3.5 --- Evaluation of antigenicity of recombinant 20H8 protein --- p.42 / Chapter 2.2.2.4 --- Immunolocalization of 20H8 in cercaria --- p.43 / Chapter 2.2.2.4.1 --- Collection of S. japonicum cercaria --- p.43 / Chapter 2.2.2.4.2 --- Immunofluorescence staining of cercaria --- p.43 / Chapter 2.2.3 --- Characterization of the cercarial stage-specific gene and gene product of sjCa8 --- p.45 / Chapter 2.2.3.1 --- Analysis of DNA sequence and deduced amino acid sequence of sjCa8 --- p.45 / Chapter 2.2.3.2 --- Demonstration of the immunogenicity and antigenicity of sjCa8 --- p.46 / Chapter 2.2.3.2.1 --- Expression of sjCa8 in E. coli --- p.46 / Chapter 2.2.3.2.2 --- Purification and concentration of recombinant sjCa8 protein --- p.46 / Chapter 2.2.3.2.3 --- Production of antiserum --- p.46 / Chapter 2.2.3.2.4 --- Evaluation of the immunogenicity of sjCa8 --- p.47 / Chapter 2.2.3.2.5 --- Evaluation of the antigenicity of sjCa8 --- p.47 / Chapter 2.2.3.3 --- Demonstration of calcium-binding property of sjCa8 --- p.48 / Chapter 2.2.3.3.1 --- Calcium-dependent electrophoretic mobility shift --- p.48 / Chapter 2.2.3.3.2 --- Ruthenium red assay --- p.48 / Chapter 2.2.3.4 --- Immunolocalization of sjCa8 in cercaria --- p.49 / Chapter Chapter 3 --- Results --- p.50 / Chapter 3.1. --- Identification of cercarial stage-specific genes --- p.50 / Chapter 3.1.1 --- Identification of cercarial stage-specific genes by microarray --- p.50 / Chapter 3.1.2 --- "Confirmation of stage-specific expression of the selected gene, 20H8 and sjCa8, by RNA dot blot" --- p.54 / Chapter 3.2. --- Characterization of the cercarial stage-specific gene and gene product of 20H8 --- p.57 / Chapter 3.2.1 --- Cloning of full-length cDNA of 20H8 --- p.57 / Chapter 3.2.2 --- Analysis of DNA sequence and deduced amino acid sequence of 20H8 --- p.59 / Chapter 3.2.3 --- Demonstration of the immunogenicity and antigenicity of 20H8 --- p.62 / Chapter 3.2.3.1 --- Expression of 20H8 in E. coli --- p.62 / Chapter 3.2.3.2 --- Purification and concentration of recombinant 20H8 protein --- p.64 / Chapter 3.2.3.3 --- Production of antiserum --- p.65 / Chapter 3.2.3.4 --- Evaluation of immunogenicity of recombinant 20H8 protein --- p.66 / Chapter 3.2.3.5 --- Evaluation of antigenicity of recombinant 20H8 protein --- p.67 / Chapter 3.2.4 --- Immunolocalization of 20H8 in cercaria --- p.68 / Chapter 3.3. --- Characterization of the cercarial stage-specific gene and gene product of sjCa8 --- p.70 / Chapter 3.3.1 --- Analysis of DNA sequence and deduced amino acid sequence of sjCa8 --- p.70 / Chapter 3.3.2 --- Demonstration of the immunogenicity and antigenicity of sjCa8 --- p.75 / Chapter 3.3.2.1 --- Expression of sjCa8 in E. coli --- p.75 / Chapter 3.3.2.2 --- Purification and concentration of recombinant sjCa8 protein --- p.76 / Chapter 3.3.2.3 --- Production of anti-sjCa8 serum --- p.77 / Chapter 3.3.2.4 --- Evaluation of immunogenicity of sjCa8 protein --- p.77 / Chapter 3.3.2.5 --- Evaluation of antigenicity of sjCa8 protein --- p.78 / Chapter 3.3.3 --- Demonstration of calcium-binding property of sjCa8 --- p.79 / Chapter 3.3.3.1 --- Electrophoretic motility shift --- p.80 / Chapter 3.3.3.2 --- Ruthenium red binding assay --- p.80 / Chapter 3.3.4 --- Immunolocalization of sjCa8 in cercaria 81 --- p.81 / Chapter Chapter 4 --- Discussion --- p.83 / Chapter 4.1 --- Identification of cercarial stage-specific genes --- p.83 / Chapter 4.1.1 --- Identification of cercarial stage-specific genes by microarray --- p.83 / Chapter 4.1.2 --- "Confirmation of stage-specific expression of the selected genes, 20H8 and sjCa8" --- p.85 / Chapter 4.2 --- Characterization of the cercarial stage-specific gene and gene product of 20H8 --- p.86 / Chapter 4.2.2 --- Analysis of DNA sequence and deduced amino acid sequence of 20H8 --- p.86 / Chapter 4.2.3 --- Demonstration of the immunogenicity and antigenicity of 20H8 --- p.88 / Chapter 4.2.4 --- Immunolocalization of 20H8 in cercaria --- p.90 / Chapter 4.3 --- Characterization of the cercarial stage-specific gene and gene product of sjCa8 --- p.91 / Chapter 4.3.1 --- Analysis of DNA sequence and deduced amino acid sequence of sjCa8 --- p.91 / Chapter 4.3.2 --- Demonstration of the immunogenicity and antigenicity of sjCa8 --- p.93 / Chapter 4.3.3 --- Demonstration of calcium-binding property of sjCa8 --- p.94 / Chapter 4.3.4 --- Immunolocalization of sjCa8 in cercaria --- p.94 / Chapter 4.4 --- Conclusions --- p.95 / References --- p.96

Thesis (M. Phil.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 180-211).

published_or_final_version / Microbiology / Master / Master of Philosophy

Lok Chui-Lin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 142-157). / Abstracts in English and Chinese. / Statement --- p.I / Acknowledgement --- p.II / Abstract --- p.IV / Chinese Abstract (摘要） --- p.VI / Abbreviation --- p.VIII / Table of Contents --- p.XIII / List of Tables --- p.XVII / List of Figures --- p.XVIII / Chapter Chapter One : --- Literature Review --- p.1 / Chapter 1.1 --- The Schistosoma Species --- p.1 / Chapter 1.1.1 --- The Schistosoma Gene Discovery --- p.3 / Chapter 1.1.2 --- Schistosome Transcriptome --- p.4 / Chapter 1.2 --- Schistosomiasis --- p.4 / Chapter 1.2.1 --- Immunopathology of Schistosomiasis --- p.5 / Chapter 1.2.2 --- Diagnosis of Schistosomiasis --- p.7 / Chapter 1.2.3 --- Treatment and Control for Schistosomiasis --- p.7 / Chapter 1.2.4 --- Vaccine Development for Schistosomiasis --- p.8 / Chapter 1.3 --- "The Species, Schistosoma japonicum" --- p.9 / Chapter 1.3.1 --- The Life Cycle of Schistosoma japonicum --- p.10 / Chapter 1.3.1.1 --- "The Egg, Miracidium Phase of the Life Cycle" --- p.12 / Chapter 1.3.1.2 --- Developmental Cycle within Mollusc Host --- p.12 / Chapter 1.3.1.3 --- The Cercaria Phase of Life Cycle --- p.13 / Chapter 1.3.1.4 --- Adult Schistosome in Definitive Host --- p.14 / Chapter 1.4 --- Invasion by Schistosome Cercariae --- p.15 / Chapter 1.5 --- "The Anti-inflammatory Protein, Sml6" --- p.16 / Chapter 1.5.1 --- Discovery of Sm 16 --- p.16 / Chapter 1.5.2 --- Cloning and Expression of Gene-encoding Sm 16 --- p.17 / Chapter 1.5.3 --- Potential Anti-inflammatory Therapy using Sm 16 --- p.18 / Chapter 1.6 --- Innate Immunity and Adaptive Immunity --- p.18 / Chapter 1.6.1 --- Macrophage --- p.18 / Chapter 1.6.2 --- Major Histocompatiblity Complex (MHC) --- p.20 / Chapter 1.6.3 --- Adaptive Immunity to Parasites --- p.20 / Chapter 1.7 --- Inflammation --- p.21 / Chapter 1.7.1 --- Cells of the Inflammatory Process --- p.23 / Chapter 1.7.2 --- Cytokines --- p.24 / Chapter 1.7.2.1 --- Interleukin-1 (IL-1) System --- p.26 / Chapter 1.7.2.2 --- Interferon (IFN) System --- p.27 / Chapter 1.7.3 --- Anti-inflammatory Therapy --- p.28 / Chapter 1.8 --- Aim of Study --- p.29 / Chapter Chapter Two : --- Materials and Methods --- p.30 / Chapter 2.1 --- Materials --- p.30 / Chapter 2.1.1 --- "Cell Lines, Mouse Strain and Bacterial Strains" --- p.30 / Chapter 2.1.2 --- Plasmids --- p.31 / Chapter 2.1.3 --- Chemicals --- p.31 / Chapter 2.1.4 --- "Kits, Nucleic Acids and Reagents" --- p.34 / Chapter 2.1.5 --- Antibodies and Immunoglobins --- p.35 / Chapter 2.1.6 --- Cell Culture Reagents --- p.35 / Chapter 2.1.7 --- Solutions --- p.36 / Chapter 2.1.8 --- Solutions of Reaction Kits --- p.39 / Chapter 2.1.9 --- Enzymes --- p.41 / Chapter 2.1.10 --- Major Equipments and Materials --- p.41 / Chapter 2.1.11 --- Primers --- p.43 / Chapter 2.1.11.1 --- Sequencing and Sj 16 Gene-coding Specific Primers --- p.43 / Chapter 2.1.11.2 --- Primers for Cytokines --- p.43 / Chapter 2.2 --- Methods --- p.45 / Chapter 2.2.1 --- Amplification of Sjl6 cDNA from Schistosoma japonicum Cercariae --- p.45 / Chapter 2.2.1.1 --- Isolation of Cercariae total RNA by Guanidinium Thiocyanate - Cesium Chloride Ultracentrifugation --- p.45 / Chapter 2.2.1.2 --- Reverse Transcription - Polymerase Chain Reaction (RT-PCR) --- p.46 / Chapter 2.2.1.2.1 --- Reverse Transcription (RT) --- p.46 / Chapter 2.2.1.2.2 --- Polymerase Chain Reaction (PCR) --- p.46 / Chapter 2.2.2 --- Cloning and Subcloning of Sj 16 --- p.47 / Chapter 2.2.2.1 --- Preparation of DH5a Competent Cells --- p.47 / Chapter 2.2.2.2 --- Purification of Plasmid DNA --- p.48 / Chapter 2.2.2.3 --- Restriction Enzyme Digestion of DNA --- p.49 / Chapter 2.2.2.4 --- Purification of DNA Fragments from Agarose Gel --- p.50 / Chapter 2.2.2.5 --- Ligation of Purified DNA Fragments --- p.51 / Chapter 2.2.2.6 --- Transformation of Recombinant Plasmid --- p.52 / Chapter 2.2.2.7 --- Selection of Transformed Clones --- p.52 / Chapter 2.2.2.7.1 --- Screening by X-gal and IPTG : a-complementation --- p.52 / Chapter 2.2.2.7.2 --- Screening by Polymerase Chain Reaction --- p.53 / Chapter 2.2.2.8 --- Cycle Sequencing --- p.53 / Chapter 2.2.3 --- Expression of the rSj 16 in Eukaryotic System --- p.55 / Chapter 2.2.3.1 --- Transfection of pSecTag2B/Sj 16 Plasmid into Animal Cells --- p.55 / Chapter 2.2.3.2 --- PCR Screening of Transfected Cells --- p.56 / Chapter 2.2.3.3 --- Analysis of mRNA Transcript by RT-PCR --- p.56 / Chapter 2.2.3.4 --- Concentration of the Condition Medium --- p.57 / Chapter 2.2.3.5 --- Western Blot analysis of rSjl6 Expression --- p.58 / Chapter 2.2.4 --- Expression of rSjl6 in Bacterial System --- p.59 / Chapter 2.2.4.1 --- Transformation of pET30a+/Sjl6 Plasmid into BL21 --- p.59 / Chapter 2.2.4.2 --- Optimization of rSj 16 Expression --- p.60 / Chapter 2.2.4.3 --- Solubility of the rSjl6 --- p.60 / Chapter 2.2.4.4 --- Estimation of rSj 16 Concentration --- p.62 / Chapter 2.2.4.5 --- Western Blot Analysis of rSj 16 --- p.62 / Chapter 2.2.5 --- Recombinant Protein Purification --- p.63 / Chapter 2.2.5.1 --- Affinity Chromatography of Recombinant Protein --- p.63 / Chapter 2.2.5.2 --- Dialysis of Eluted Recombinant Protein in PBS --- p.64 / Chapter 2.2.5.3 --- Estimation of Recombinant Protein Concentration --- p.65 / Chapter 2.2.6 --- Demonstrate the Anti-inflammatory Activity of rSj 16 --- p.65 / Chapter 2.2.6.1 --- Thioglycollate Induced Macrophage Recruitment --- p.65 / Chapter 2.2.6.2 --- Cytospin and Hemacolor Staining of PECs --- p.66 / Chapter 2.2.6.3 --- FACS Analysis of PECs --- p.67 / Chapter 2.2.6.4 --- Isolation of total RNA by TRIZOL Reagent --- p.67 / Chapter 2.2.7 --- Immunogenicity and Antigenicity of rSjl6 --- p.68 / Chapter 2.2.7.1 --- Western Blot of rSjl6 with Schistosoma japonicum infected rabbit serum --- p.69 / Chapter 2.2.7.2 --- Preparation of Anti-Sj 16 Serum --- p.69 / Chapter 2.2.7.3 --- Western Blot of rSjl6 with immunized mice serum --- p.70 / Chapter 2.2.8 --- FACS analysis of MHC (I) Expression --- p.71 / Chapter 2.2.9 --- Anti-proliferative Assay using BrdU Kit --- p.72 / Chapter Chapter Three : --- Results --- p.73 / Chapter 3.1 --- Amplification of Sj 16 cDNA from Schistosoma japonicum Cercariae total RNA --- p.73 / Chapter 3.2 --- Construction of pBluescript II SK(-) / Sjl6 --- p.75 / Chapter 3.3 --- Analysis of Sj 16 Nucleotide and Amino Acid Sequence --- p.78 / Chapter 3.3.1 --- Blastn Search Analysis --- p.80 / Chapter 3.3.2 --- Blastx Search Analysis --- p.82 / Chapter 3.3.3 --- Structural Analysis --- p.84 / Chapter 3.4 --- Subcloning of Sjl6 cDNA into pET30a+ and pSecTag2B Expression Vector --- p.88 / Chapter 3.5 --- Expression of the rSj 16 --- p.92 / Chapter 3.5.1 --- Animal Cell Expression --- p.92 / Chapter 3.5.1.1 --- Analysis of mRNA Transcript by RT-PCR --- p.93 / Chapter 3.5.1.2 --- Western Blot of Condition Medium --- p.95 / Chapter 3.5.2 --- Bacterial Cell Expression --- p.97 / Chapter 3.5.2.1 --- Optimization of rSjl6 Expression --- p.97 / Chapter 3.5.2.2 --- Estimation of rSjl6 Concentration --- p.98 / Chapter 3.5.2.3 --- Solubility of rSj16 --- p.99 / Chapter 3.5.2.4 --- Western Blot Analysis of rSjl6 --- p.100 / Chapter 3.6 --- Purification of Recombinant Protein --- p.101 / Chapter 3.6.1 --- Purification of rSj16 --- p.101 / Chapter 3.6.2 --- Purification of rSjCa8 --- p.104 / Chapter 3.7 --- Anti-inflammatory Activity of rSj 16 --- p.107 / Chapter 3.7.1 --- Analysis of PECs in Thioglycollate Induced Inflammation --- p.107 / Chapter 3.7.2 --- Hemacolor Staining of PECs --- p.110 / Chapter 3.7.3 --- FACS Analysis of PECs --- p.110 / Chapter 3.7.4 --- RT-PCR of RNA Isolated from PECs --- p.115 / Chapter 3.8 --- Immunogenicity and Antigenicity of rSjl6 --- p.117 / Chapter 3.8.1 --- Immunogenicity of rSj 16 --- p.117 / Chapter 3.8.2 --- Antigenicity of rSj16 --- p.117 / Chapter 3.9 --- Inhibitory Effect of rSj 16 on rMuIFN-a4 Induced Up-regulation of MHC(I) Expression --- p.120 / Chapter 3.9.1 --- Time Course of rMuIFN-α4 Induced Up-regulation of MHC(I) Expression --- p.120 / Chapter 3.9.2 --- Inhibitory Effect of rSjl6 on rMuIFN-α4 Induced MHC (I) Up-regulation --- p.120 / Chapter 3.9.3 --- "Anti-proliferation Effect of rMuIFN-a4, rSj 16 and rSjCa 8" --- p.124 / Chapter 3.9.4 --- Effect of Signal Transduction Inhibitors on rMuIFN-a4 Induced MHC (I) Up-regulation --- p.126 / Chapter Chapter Four : --- Discussion and Conclusion --- p.129 / Chapter 4.1 --- Discussion --- p.129 / Chapter 4.1.1 --- Overview --- p.129 / Chapter 4.1.2 --- Molecular and Structural Analysis of rSj 16 --- p.130 / Chapter 4.1.3 --- Relationship between Sml6 and Sjl6 --- p.131 / Chapter 4.1.4 --- Anti-inflammatory Activity of rSj 16 --- p.132 / Chapter 4.1.5 --- Immunogenicity and Antigenicity of rSjl6 --- p.137 / Chapter 4.1.6 --- Inhibitory Effect of rSjl6 on rMuIFN-a4 Induced Up-regulation of MHC (I) Expression --- p.138 / Chapter 4.1.7 --- Relation between Sj 16 and the Innate Immune System --- p.139 / Chapter 4.1.8 --- Further Study and Significance --- p.140 / Chapter 4.2 --- Conclusion --- p.141 / References --- p.142

Adult schistosomes depend for growth and development on hormonal signals from the mammalian host, which may include the insulin signalling pathway. In this project, I firstly used microarray analysis to demonstrate that human insulin can be utilised by adult S. japonicum in culture, resulting in the modulation of distinct metabolic effects as reflected in transcriptional levels of parasite genes. The addition of insulin resulted in the differential expression of 1,101 genes with many related to functions corresponding to the biological and metabolic effects of insulin reported for mammalian cells. Those identified genes in male or female S. japonicum worms that were up or down regulated after exposure to insulin were predominantly involved in growth and development, with significant sex-specific responses evident. Insulin appeared to play a similar role in male parasites as those seen in classical mammalian systems including an increase in protein synthesis though gene transcription and the stimulation of mRNA translation and control protein degradation via the ubiquitin proteasome pathway. Microarray analysis indicated that insulin not only leads to increased gene expression of the PI3-K pathway, which enhances parasite growth, but may also play a role in the sexual differentiation and fecundity of female worms by activating the MAPK pathway. As the insulin target proteins, two types of insulin receptors from Schistosoma japonicum were isolated, S. japonicum insulin receptors 1 (SjIR-1) and 2 (SjIR-2), with features similar to insulin receptors from other taxa. The sequences share 70% and 74% identity to S. mansoni insulin receptor 1 and 2 (SmIR-1 and SmIR-2), respectively. SjIR-1 and SjIR-2 are highly conserved in their tyrosine kinase domain to other IRs from Homo, Mus musculus and Drosophila melanogaster. SjIR-2 is located in the parenchyma in males and in the vitelline glands of female worms, which occupy most of male or female tissue and play an important role in growth or fecundity. In contrast, SjIR-1 was located in the tegument and intestinal epithelium of adult worms, representing much smaller cellular regions compared with the voluminous vitelline tissue or parenchyma. This observation was further confirmed by real time PCR showing that SjIR-2 was more abundantly expressed in S. japonicum adult worm than SjIR-1. Phylogenetic analysis showed that SjIR-2 and SmIR-2 are closer to EmIR than to SjIR-1 and SmIR-1, indicating that SjIR-1 and SmIR-1 might perform specific functions in schistosomes, while SjIR-2, SmIR-2 and EmIR might share similar roles in parasite growth and development in the three parasitic flatworms. Structure modelling recovered the conserved structure between the SjIRs and Homo sapiens IR (HIR) implying a common predicted binding mechanism in the ligand domain and the same downstream signal transduction processing in the tyrosine kinase domain as in HIR. Two-hybrid analysis was used to confirm that the ligand domains of SjIR-1 and SjIR-2 contain the insulin binding site. Incubation of adult worms in vitro, both with a specific insulin receptor inhibitor and anti-SjIRs antibodies, resulted in a significant decrease in worm glucose levels, suggesting again the same function for SjIRs in regulating glucose uptake as described for mammalian cells. Adult worms of S. japonicum possess insulin receptors that can specifically bind to insulin, indicating that the parasite can utilize host insulin for development and growth by sharing the same pathway as mammalian cells in regulating glucose uptake. In vaccination/challenge trials, there was no significant reduction in adult worm burdens with either of the SjLD vaccines. However, there were significant reductions in mean lengths of adult worms ranging from 22-25% in the SjLD1 vaccinated group to 37-42% in the SjLD2 vaccinated groups, significant reductions in faecal eggs in both the SjLD1 (66%) and SjLD2 (68%) vaccinated groups, and a reduction in liver egg numbers in the SjLD1(33%) vaccinated group. These results show that although the SjLDs vaccines were unable to reduce adult worm numbers by clearing them from the vaccinated mice, nevertheless, they significantly depressed the growth of male and female adult worms and affected female egg production. The protective efficacy obtained in terms of the substantial decrease in faecal eggs exceeded that of many of the recently available schistosome antigens and prototype vaccine formulations, which, at best, elicit 40–50% protection in animals using the standard readouts of reduced worm burden or egg production and viability. Overall, disruption of this insulin pathway leading to parasite starvation through the prevention of glucose uptake thereby affecting parasite growth, development and female fecundity, provides a new intervention target and transmission blocking approach to combat schistosomiasis and may be applicable for the control of other debilitating parasitic infections as well.

Schistosome is the causative agent of schistosomiasis which is one of the world's most prevalent tropical diseases. In the skin of infected host, significant inflammatory response to the parasite is not observed. Previous studies from Schistosoma mansoni showed that this subdued inflammatory response was due to a 16-KDa protein, Sm16, which is present abundantly in the secretions of schistosomulae. Provided that Schistosoma japonicum shares the same infective pathway as S. mansoni by penetrating the skin, it seems logical that S. japonicum has a protein with a similar role to Sm16 to down-regulate host immune responses. According to the cDNA sequence of Sm16, a corresponding gene (designated Sj16) of Sm16 has previously been amplified and cloned from the cercarial cDNA of S. japonicum. Sequence analysis showed that Sj16 shares 99% identity with Sm16 in its nucleotide sequence, and 100% identity in its protein sequence. While previous studiers reported their failure in obtaining the soluble recombinant protein of Sm16, we expressed and purified the recombinant Sj16 (rSj16) from E. coli in the present study. Western blot and ELISA analysis showed that S. japonicum-infected rabbit sera could not recognize rSj16, indicating that native Sj16 might fail to induce circulating antibodies during S. japonicum infection. In the in vivo study, rSj16 dramatically suppressed not only the recruitment of leukocytes to the peritoneal cavity of BALB/c mice injected with thioglycollate, but also the maturation of thioglycollate-induced peritoneal macrophages. The suppression effect was accompanied by a marked up-regulation of IL-10 and IL-1RA transcripts, and down-regulation of IL-12p35, IL-1beta and MIP-2 transcripts in peritoneal cells. Further analysis revealed that rSj16 also inhibited both humoral and cellular immune responses to heterologous antigens. In addition, rSj16 was found to induce macrophage differentiation of the murine myeloid leukemia WEHI-3B (JCS) cells, and regulate the differentiation of mouse hematopoietic cells towards the macrophage lineage. Although previous studies indicated the involvement of endogenous IL-1alpha, IL-1beta and TNF-alpha in the macrophage differentiation of JCS cells, the results from this study suggested that rSj16-induced JCS cell differentiation do not rely on the endogenous production of these three cytokines. This is the first study to successfully express and purify sufficient soluble rSj16, and demonstrate the anti-inflammatory and immunomodulatory effects of the rSj16. / Hu, Shaomin. / Adviser: Ming Chiu Fung. / Source: Dissertation Abstracts International, Volume: 71-01, Section: B, page: 0210. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 139-154). / 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.

Schistosomiasis, a disease of humans caused by helminth parasites of the genus Schistosoma, kills 200 to 500 thousand people annually, endangering over 600 million people world-wide with 200 million people infected in 2003 [1, 2]. Three species of schistosome are primarily responsible for human infections, namely, Schistosoma haematobium, endemic to Africa, India, and the Middle East, S. mansoni, endemic to Africa / South America, and S. japonicum endemic to China and the Philippines [3]. The major pathological effects of schistosomiasis result from the deposition of parasite ova in human tissues and the subsequent intense granulomatous response induced by these eggs. There is a high priority to provide an effective sub-unit vaccine against these schistosome flukes, using proteins encoded by cDNAs expressed by the parasites at critical phases of their development. One technique that may expedite this gene identification is the use of microarrays for expression analysis. A 22,575 feature custom oligonucleotide DNA microarray designed from public domain databases of schistosome ESTs (Expressed Sequence Tags) was used to explore differential gene expression between the Philippine (SJP) and Chinese (SJC) strains of S. japonicum, and between males and females. It was found that 593, 664 and 426 probes were differentially expressed between the two geographical strains when mix sexed adults, male worms and female worms were compared respectively. Additionally, the study revealed that 1,163 male- and 1,016 female-associated probes were differentially expressed in SJP whereas 1,047 male- and 897 female-associated probes were differentially expressed in SJC [4]. Further to this, a detailed real time PCR expression study was used to explore the differential expression of eight genes of interest throughout the SJC life cycle, which showed that several of the genes were down-regulated in different life cycle stages. The study has greatly expanded previously published data of strain and gender-associated differential expression in S. japonicum. Further, the new data will provide a stepping stone for understanding the complexities of the biology, sexual differentiation, maturation, and development of human schistosomes, signaling new approaches for identifying novel intervention and diagnostic targets against schistosomiasis [4].