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The serological and chemical properties of antigens extracted from strains of Bacteroides fragilis and related species belonging to several different DNA homology groups were investigated. Antisera prepared against formalin-treated whole cells suspensions of representative strains were tested against cell suspensions, cell wall preparations, and extracts of homologous and heterologous strains using agglutination, immunodiffusion, and hemagglutination techniques. Serological results indicated that the species were antigenically distinct, although minor cross reactions were observed across species boundaries. However, the serological properties did not appear to distinguish genetic heterogeneity at levels down to approximately 65% homology. Homology groups, including the two B. fragilis subgroups, were relatively homogeneous, although the presence of serotypes within each homology group was suggested. Immunodiffusion tests demonstrated, however, that the "homogeneity" was not always represented by a single "common" antigen but rather implied a mosaic antigen composition for each strain. A minimum of ten and six different antigenic factors were demonstrated on B. fragilis 2393 and 2553, respectively. Similar mosaics were observed with members of the other species. Hemagglutination patterns using crude antigen extracts were also consistent with the antigenic mosaic. Many of the strains were found to be capsulated. Preliminary studies demonstrated a similar sugar composition in the capsular material to that in lipopolysaccharide extracted from the same strain with aqueous phenol. Studies also suggested that the capsule influenced the serological properties of the cell. The chemical make-up of all of the Bacteroides LPS was found to be similar to that of LPS from facultative organisms, although KDO and heptose were not detected in any of the Bacteroides preparations. Electron microscopy of Bacteroides LPS demonstrated a trilaminar structure characteristic of LPS from other gram negative organisms. However, gel-filtration experiments suggested that the Bacteroides LPS may be of a different architecture, particularly regarding the core region. When hydrolyzed by weak acid, Bacteroides LPS behaved differently to LPS from facultative bacteria yielding large amounts of high molecular weight polymers and very little core-type material. B. distasonis and B. thetaiotaomicron displayed lower levels of high molecular weight material, as compared with the other strains, and may represent semi-rough strains. A comparison of the sugar patterns of the antigen extracts from one member of each homology group implied distinct differences among the strains. However, the sugar patterns of two B. fragilis strains which are genetically more related to each other than to the other species, were very similar. The constituents identified in the Bacteroides LPS were glucosamine, galactosamine, glucose, galactose, mannose, fucose, and rhamnose. An unidentified aniline pthalate reacting compound with similar mobility to colitose in chromatography studies was observed in the B. thetaiotaomicron 5482 LPS. It was not thought justified to assign definite chemotypes to individual homology groups due to the possible contamination of LPS preparation with non-LPS material, e.g. capsules or a glycan polymer. / Ph. D.

The antigenic relationship between T. denticola, T. socranskii subsp. Socranskii, T. socranskii subsp. Buccale, T. socranskii subsp. paredis, Treponeme D, T. vincentii, T. phagedenis biotype Reiter, T. minutum, and T. refringens was studied using fluorescent antibody staining and those organisms were found to contain a group of common antigens. The cross-reactivity was removed when the antisera was absorbed with Reiter cells. There was a second group of common antigens that were shared by the subspecies of T. socranskii, Treponeme D, and T. pectinvorum. Triton-extracts from different treponemal species were found to contain very few cross-reacting antigens as demonstrated by immunodiffusion, crossed immunoelectrophoresis, ELISA, and Western blots. An immunodiffusion test using treponemal Triton-extracts and antisera against five treponeme species was developed for the routine identification of oral treponemes that were in either pure or mixed cultures. T. denticola cells were found to bind to fibronectin-coated microscope slides. Proteins, that bind fibronectin, in the Triton-extracts of T. denticola, were detected in a fibroectin-captured ELISA. The Triton-extracts also reacted with rabbit fibronectin antiserum and rabbit albumin antiserum in immunodiffusion and ELISA assays. Three proteins with molecular weight of 60K, 47K, and 21K, were eluted from a agarose-gelatin-fibronectin column and reacted in Wester blots with T. denticola antiserum. / M.S.

No / Isogenic mutants of Streptococcus mutans LT11, deficient in the production of the wall-associated protein antigens A and B, were generated by recombinant DNA technology. The hydrophobicity, adherence, and aggregation of the mutants were compared with those of the parent strain. These studies indicated that hydrophobicity, adherence, and saliva- or sucrose-induced aggregation were unaltered in the A- mutant but that hydrophobicity and adherence to saliva-coated hydroxylapatite were greatly reduced in the B- mutant whilst sucrose-dependent adherence and aggregation were increased. To determine whether these changes correlated with changes in the mutated gene product alone, the levels of a number of cell wall antigens were determined in each of the mutants. The loss of antigen A resulted in significantly reduced levels of wall-associated lipoteichoic acid, and loss of antigen B resulted in reductions in both antigen A and lipoteichoic acid. Data presented here thus suggest that changes in the expression of one wall antigen can have a dramatic effect on the levels of others.

This thesis describes an experiment involving guinea pigs' blood and the changes that occurred in the serum with the injection of an antigen. The serum was analyzed in complement titration tests.

A research report submitted to the faculty of Medicine, University of the Witwatersrand, Johannesburg, in part fulfillment of the requirements for the degree of Master of Medicine (in the branch of Haematology) Johannesburg 1992. / The observation by Haldane in 1949 that the distribution of malaria and certain thalassaemias were similar and that the former disease must be a selective force tor the continued existence of the latter by preservation of the heterozygotes. This theory which later became known as lithe malaria hypothesis" has been applied to other inherited conditions such as G6PD deficiency, membranopathies, certain blood group polymorphisms, other heamoglobinopathies such as sickle cell disease, blood group polymorphisms and more recently HLA phenotypes. It has been shown that the Duffy blood group antigens are the receptors for. Plasmodium vivax and since these antigens are lacking in most black Africans this species of malaria is virtually absent in Africa. It has also been shown that the glycophorins are at least in part the receptors for Pfalciparum. Several variants of the glycophorins exist and the biochemistry and, where known, the molecular mechanisms by which these arise is reviewed. Experimental work is carried out to establish the growth characteristics of Pfalciparum in an in vitro culture system using cells with glycophorin variants on their membranes. Three such variants were compared to normal cells and two (S~s-U-and Dantu) were found to be partially resistant to invasion by Pfalciparum merozoites whereas the third (Henshaw) was found to be no different to controls. / MT2018

by Chan Lik-yuen, Henry. / Thesis (M.D.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (p. 192-217). / 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.

Chan Wan Lui Wendy. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 158-162). / Abstracts in English and Chinese. / Acknowledgements --- p.iii / Abstract --- p.v / List of Abbreviations --- p.ix / List of Figures --- p.xii / List of Tables --- p.xvi / Table of Contents --- p.xvii / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter Chapter 2 --- Literature review --- p.3 / Chapter 2.1 --- Malaria --- p.3 / Chapter 2.2 --- History of malaria --- p.4 / Chapter 2.3 --- Malaria parasites --- p.4 / Chapter 2.4 --- Life cycle --- p.5 / Chapter 2.5 --- Potential use of malaria vaccine --- p.6 / Chapter 2.6 --- Merozoite surface protein 1 (MSP1) --- p.7 / Chapter 2.7 --- Potential use of MSPl --- p.8 / Chapter 2.8 --- Significance of MSPl C-terminal fragments --- p.9 / Chapter 2.8.1 --- Significance of MSP142 --- p.9 / Chapter 2.8.2 --- Significance of MSP119 --- p.11 / Chapter 2.9 --- Production of MSPl C-terminal fragments --- p.12 / Chapter 2.10 --- Plants as bioreactors --- p.12 / Chapter 2.11 --- Expression of MSPl C-terminal fragments in transgenic plants --- p.14 / Chapter 2.12 --- Phaseolin and its sorting signal --- p.19 / Chapter 2.13 --- Protein targeting signals --- p.20 / Chapter Chapter 3 --- Material and methods --- p.23 / Chapter 3.1 --- Introduction --- p.23 / Chapter 3.2 --- Chemical and enzymes --- p.23 / Chapter 3.3 --- Cloning --- p.24 / Chapter 3.3.1 --- MSP142 and MSP119 constructs --- p.24 / Chapter 3.3.2 --- Protein targeting fusion constructs --- p.24 / Chapter 3.3.3 --- GUS fusion Constructs --- p.30 / Chapter (a) --- Particle bombardment --- p.30 / Chapter (b) --- GUS fusion constructs for plant transformation --- p.32 / Chapter (c) --- Modified GUS fusion constructs --- p.38 / Chapter 3.4 --- Cloning of chimeric gene into Agrobacterium binary vector --- p.39 / Chapter 3.4.1 --- Cloning of pSUNl --- p.40 / Chapter 3.4.2 --- Primer sequence --- p.45 / Chapter 3.5 --- Bacterial strains --- p.46 / Chapter 3.6 --- Particle bombardment --- p.46 / Chapter 3.6.1 --- Plant materials --- p.46 / Chapter 3.6.2 --- Microcarrier preparation and coating DNA onto microcarrier --- p.46 / Chapter 3.6.3 --- GUS assay --- p.48 / Chapter 3.7 --- Transgenic expression in Arabidopsis thaliana --- p.49 / Chapter 3.7.1 --- Plant materials --- p.49 / Chapter 3.7.2 --- Agrobacterium transformation --- p.49 / Chapter 3.7.3 --- Vacuum infiltration Arabidopsis transformation --- p.49 / Chapter 3.7.4 --- Selection of successful transformants --- p.50 / Chapter 3.7.5 --- Selection for homozygous plants --- p.51 / Chapter 3.8 --- Transgenic expression in tobacco --- p.51 / Chapter 3.8.1 --- Plant materials --- p.51 / Chapter 3.8.2 --- Agrobacterium transformation --- p.52 / Chapter 3.8.2.1 --- Preparation of Agrobacterium tumefaciens LBA4401 competent cells --- p.52 / Chapter 3.8.3 --- Leaf discs method for tobacco transformation --- p.53 / Chapter 3.8.4 --- GUS staining --- p.54 / Chapter 3.9 --- DNA analysis --- p.55 / Chapter 3.9.1 --- Genomic DNA extraction --- p.55 / Chapter 3.9.2 --- Genomic PCR --- p.55 / Chapter 3.9.3 --- Southern blot --- p.55 / Chapter 3.10 --- RNA analysis --- p.56 / Chapter 3.10.1 --- RNA extraction --- p.56 / Chapter 3.10.2 --- Northern blot --- p.56 / Chapter 3.11 --- Protein analysis --- p.57 / Chapter 3.11.1 --- Protein extraction --- p.57 / Chapter 3.11.2 --- Western blot --- p.58 / Chapter 3.11.3 --- Western blot analysis --- p.58 / Chapter Chapter 4 --- Results --- p.60 / Chapter 4.1 --- Transient assay of gene expression of MSP142 and MSPl19 --- p.60 / Chapter 4.1.1 --- Construction of the GUS fusion constructs --- p.60 / Chapter 4.1.2 --- Particle Bombardment --- p.63 / Chapter 4.2 --- Transgenic analysis of MSP142 and MSPl19 expression --- p.70 / Chapter 4.2.1 --- MSPl42 and MSPl19 constructs and transformation --- p.70 / Chapter 4.2.2 --- Selection of transgenic plants --- p.71 / Chapter 4.2.3 --- Southern analysis --- p.75 / Chapter 4.2.4 --- Northern analysis --- p.77 / Chapter 4.2.5 --- Western analysis --- p.79 / Chapter 4.3 --- Expression of the protein-targeting and GUS fused modified MSP1 constructs --- p.81 / Chapter 4.3.1 --- Construction of the fusion constructs --- p.81 / Chapter (A) --- Protein-targeting constructs --- p.81 / Chapter (B) --- GUS fusion constructs --- p.90 / Chapter B1. --- Constructs for transient assay --- p.90 / Chapter B2. --- Modification of GUS sequence --- p.96 / Chapter B3. --- Constructs for tobacco transformation --- p.100 / Chapter 4.4 --- Transient assay of GUS fused MP42 and MP19 constructs by particle Bombardment --- p.107 / Chapter 4.4.1 --- The GUS fusion constructs --- p.107 / Chapter 4.4.2 --- Modification of GUS --- p.112 / Chapter 4.5 --- Generation of transgenic tobacco --- p.116 / Chapter 4.6 --- Southern analysis --- p.120 / Chapter 4.7 --- Northern analysis --- p.126 / Chapter (A) --- Protein-targeting constructs --- p.126 / Chapter (B) --- GUS fusion constructs --- p.130 / Chapter 4.8 --- Western analysis --- p.133 / Chapter (A) --- Protein-targeting constructs --- p.133 / Chapter (B) --- GUS fusion constructs --- p.139 / Chapter Chapter 5 --- Discussion --- p.146 / Chapter Chapter 6 --- Conclusion --- p.157 / References --- p.158

Xu Xuequn. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 100-125). / Abstracts in English and Chinese. / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Epstein-Barr (EBV) Virus --- p.1 / Chapter 1.1.1 --- Virus Structure and Genome Structure --- p.1 / Chapter 1.1.2 --- Virus Types --- p.2 / Chapter 1.2 --- EBV Infection and malignancies --- p.3 / Chapter 1.2.1 --- In Vitro Infection --- p.3 / Chapter 1.2.2 --- Infection in the Natural Host --- p.8 / Chapter 1.2.3 --- Malignancies Associated with EBV --- p.11 / Chapter 1.3 --- T Cell-Mediated Immune Response to EBV --- p.16 / Chapter 1.3.1 --- The Pathway of Cell-Mediated Immune Response in Viral Infection --- p.16 / Chapter 1.3.2 --- Cell-Mediated Immune Response to EBV --- p.18 / Chapter 1.3.3 --- The Feature of CTLs Response to EBV --- p.20 / Chapter 1.4 --- CTLs to EBV Relevant MalignancieśؤApplications and Challenges --- p.21 / Chapter 1.5 --- HLA Polymorphisms and Strategy of Epitope-Based CTLs Therapy --- p.24 / Chapter 1.6 --- The Effect of HLA Polymorphism on EBV-Specific CTL Epitope Choice in Southern Chinese --- p.27 / Chapter 1.7 --- ELISPOT Assay 226}0ؤ Detection of CTLs Response --- p.32 / Chapter 1.8 --- Aim of This Study --- p.37 / Chapter Chapter 2: --- Material and Methods: --- p.39 / Chapter 2.1 --- Peptides --- p.39 / Chapter 2.2 --- PBMCs Preparations --- p.43 / Chapter 2.3 --- PBMC Counting and Cells Dilution --- p.43 / Chapter 2.4 --- Elispot Assay --- p.44 / Chapter 2.5 --- Counting the Spots --- p.45 / Chapter 2.6 --- Spots Forming Cells (SFC/106) and Positive Standard --- p.46 / Chapter Chapter 3: --- Results --- p.47 / Chapter 3.1 --- Validation of ELISPOT assay methodology --- p.47 / Chapter 3.2 --- CTLs Response to Each Epitope in the Population --- p.55 / Chapter 3.2.1 --- Positive Response to A11 Restricted and Mutant Epitopes in the Population --- p.55 / Chapter 3.2.2 --- Positive Frequencies of A2 Restricted Epitopes in the Population --- p.63 / Chapter 3.2.3 --- Positive Frequencies of Other HLA Allele Restriction Peptides --- p.70 / Chapter 3.3 --- CTLs Response Frequencies Categorized by Proteins --- p.74 / Chapter 3.3.1 --- "CTLs Response to LMP1, LMP2, EBNA1 Epitopes" --- p.74 / Chapter 3.3.2 --- "CTLs Response to EBNA2, EBNA-LP Epitopes, EBNA3 Epitopes" --- p.75 / Chapter 3.3.3 --- CTLs Response to LYTIC Epitopes --- p.79 / Chapter 3.4 --- Summary --- p.80 / Chapter Chapter 4: --- Discussion --- p.82 / Chapter 4.1 --- Discussion of A11 Restricted Epitopes --- p.82 / Chapter 4.2 --- Discussion of A2 Restricted Epitopes --- p.86 / Chapter 4.3 --- Discussion of Other HLA Restricted Epitopes --- p.89 / Chapter 4.4 --- "Discussion ofLMPl, LMP2, EBNA1 Epitopes" --- p.92 / Chapter 4.5 --- "Discussion of EBNA2, EBNA3, and EBNA-LP epitopes" --- p.96 / Chapter 4.6 --- Discussion of LYTIC Epitopes --- p.96 / Chapter 4.7 --- Discussion of Summary --- p.98 / Chapter Chapter 5 --- Conclusion --- p.99 / Chapter 6 --- Reference --- p.100 / Chapter 7 --- Appendix --- p.126 / Chapter 7.1 --- "Appendix 1, raw data of Elispot assay on CTLs response to EBV relevant epitopes m Hong Kong donors" --- p.126 / Chapter 7.2 --- "Appendix 2, frequencies from highest cell number wells of the peptides (SFC/106)" --- p.126 / Chapter 7.3 --- "Appendix 3, typical Elispot assay figure " --- p.126

by Lau On Sun. / Thesis submitted in: November 2003. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 117-127). / Abstracts in English and Chinese. / Acknowledgements --- p.iii / Abstract --- p.v / List of Abbreviations --- p.viii / Table of Contents --- p.x / List of Figures --- p.xiii / List of Tables --- p.xv / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter Chapter 2 --- Literature Review --- p.3 / Chapter 2.1 --- Malaria --- p.3 / Chapter 2.1.1 --- Global situation --- p.3 / Chapter 2.1.2 --- Malaria parasite and its life cycle --- p.4 / Chapter 2.1.3 --- Need for a malarial vaccine --- p.5 / Chapter 2.2 --- Merozoite surface protein 1 and its fragments - the advanced malaria vaccine candidate --- p.7 / Chapter 2.2.1 --- Basic research on MSP1 --- p.7 / Chapter 2.2.2 --- Vaccine research on MSP1 --- p.8 / Chapter 2.3 --- Transgenic plants as recombinant protein production systems --- p.11 / Chapter 2.3.1 --- Characteristics --- p.11 / Chapter 2.3.2 --- Plant-based vaccine --- p.13 / Chapter 2.4 --- Expression of MSP 1 C-terminal fragments in transgenic plants --- p.15 / Chapter 2.4.1 --- Previous studies --- p.15 / Chapter 2.4.2 --- Plant-optimized MSP142 cDNA --- p.18 / Chapter 2.5 --- Phaseolin: its promoter and vacuolar-sorting signal --- p.20 / Chapter 2.6 --- Sorting of soluble protein to vacuoles in plants --- p.22 / Chapter 2.7 --- Winged bean lysine-rich protein and translational fusion strategy --- p.24 / Chapter 2.8 --- Hypotheses and aims of study --- p.26 / Chapter Chapter 3: --- Materials and Methods --- p.28 / Chapter 3.1 --- Introduction --- p.28 / Chapter 3.2 --- Chemicals --- p.28 / Chapter 3.3 --- Bacterial strains --- p.28 / Chapter 3.4 --- Chimeric gene construction --- p.29 / Chapter 3.4.1 --- Construction of the lysine-rich protein fusion constructs --- p.33 / Chapter 3.4.2 --- Construction of the phaseolin-targeting constructs --- p.37 / Chapter 3.4.3 --- Confirmation of sequence fidelity of chimeric genes --- p.42 / Chapter 3.4.4 --- Cloning of chimeric genes into Agrobacterium binary vector --- p.42 / Chapter 3.5 --- Transgenic expression in Arabidopsis and tobacco --- p.44 / Chapter 3.5.1 --- Plant materials --- p.44 / Chapter 3.5.2 --- Agrobacterium transformation --- p.44 / Chapter 3.5.3 --- Arabidopsis transformation and selection --- p.45 / Chapter 3.5.4 --- Tobacco Transformation and Selection --- p.47 / Chapter 3.5.5 --- Genomic DNA isolation --- p.49 / Chapter 3.5.6 --- Southern blot analysis --- p.49 / Chapter 3.5.7 --- Total silique RNA isolation --- p.50 / Chapter 3.5.8 --- Northern blot analysis --- p.50 / Chapter 3.5.9 --- Protein extraction and SDS-PAGE --- p.51 / Chapter 3.5.10 --- Western blot analysis --- p.52 / Chapter 3.5.11 --- Enterokinase digestion of recombinant LRP fusion protein --- p.53 / Chapter 3.5.12 --- Deglycosylation studies of recombinant MSP142-AFVY --- p.54 / Chapter 3.6 --- Confocal immunoflorescence studies of MSPl42-AFVY in tobacco --- p.55 / Chapter 3.6.1 --- Preparation of sections --- p.55 / Chapter 3.6.2 --- Labeling of fluorescence probes --- p.55 / Chapter 3.6.3 --- Image collection --- p.56 / Chapter 3.7 --- Bacterial expression of MSP 142 and anti-serum production --- p.57 / Chapter 3.7.1 --- pET expression in E. coli --- p.57 / Chapter 3.7.2 --- Purification of recombinant His-MSPl42 --- p.58 / Chapter 3.7.3 --- Immunization of rabbits --- p.59 / Chapter Chapter 4: --- Results --- p.60 / Chapter 4.1 --- Transgenic analysis of lysine-rich protein fusion constructs --- p.60 / Chapter 4.1.1 --- Construction of the lysine-rich protein fusion constructs --- p.60 / Chapter 4.1.2 --- Selection of transgenic plants --- p.62 / Chapter 4.1.3 --- Southern analysis --- p.65 / Chapter 4.1.4 --- Northern analysis --- p.69 / Chapter 4.1.5 --- Western analysis --- p.71 / Chapter 4.1.6 --- Western analysis with anti-LRP --- p.75 / Chapter 4.1.7 --- Enterokinase digestion of recombinant LRP fusion protein --- p.76 / Chapter 4.2 --- Transgenic analysis of phaseolin vacuolar-sorting signal constructs --- p.80 / Chapter 4.2.1 --- Construction of the phaseolin vacuolar-sorting signal constructs --- p.80 / Chapter 4.2.2 --- Selection of transgenic plants --- p.82 / Chapter 4.2.3 --- Southern analysis --- p.85 / Chapter 4.2.4 --- Northern analysis --- p.89 / Chapter 4.2.5 --- Western analysis --- p.91 / Chapter 4.2.6 --- Deglycosylation studies of recombinant MSPl42-AFVY --- p.96 / Chapter 4.2.7 --- Human serum detection of MSP142-AFVY --- p.100 / Chapter 4.3 --- Confocal immunofluorescence studies of MSP142-AFVY in tobacco --- p.102 / Chapter 4.4 --- Bacterial expression of MSPl42 and anti-serum production --- p.105 / Chapter 4.4.1 --- Expression and purification of recombinant His-MSPl42 in E. coli --- p.105 / Chapter 4.4.2 --- Titer and specificity of the anti-serum --- p.107 / Chapter Chapter 5 --- Discussion --- p.109 / Chapter Chapter 6 --- Conclusion --- p.116 / References --- p.117