There is growing evidence that susceptibility to many inflammatory and infectious diseases may be influenced by our genetic make up. Genetic variants in important immune genes may partially explain variation in susceptibility to common diseases. Interferon-γ (IFNγ) is one of the central mediators of the innate and adaptive immunity and has been implicated in a wide range of infectious and inflammatory disease processes. Severe disruptive mutations in coding regions of the IFN-γ receptor 1 gene (IFNGR1) have been found to be associated with fatal but very rare mycobacterial infections. This study looked at common polymorphisms in potentially regulatory non-coding regions of the IFNγ gene and the IFNGR1 gene and investigated their association with susceptibility to severe malaria, a disease for which there have been indications of a genetic component to susceptibility. Malaria is one of the major causes of childhood deaths in Africa. IFNγ and its receptor have been shown to be critically involved in the host response to the malaria parasites. The promoter regions of IFNGR1 and its neighbouring genes, located on chromosome 6q23, and IFNγ and its neighbours, on chromosome 12ql4, were screened for polymorphisms. Haplotypes and linkage disequilibrium maps were constructed, signatures of natural selection were investigated, haplotype tagging SNPs were dentified, and association with disease was analysed. One of these preliminary results was a putative association between the IFNGRl-470ddel allele and susceptibility to severe malaria in the Mandinka ethnic group. This allele was in strong linkage disequilibrium (LD) with markers which are a considerable distance away which might represent a signature of natural selection. To assess the potential functional significance of the IFNGR1-47Q polymorphism, its effects on DNA-protein interactions and gene expression was investigated further in various cell lines. Evidence of tissue-specific nuclear protein binding to this site which seems to be involved in transcriptional regulation was observed.

Plasmodium falciparum and Mycobacterium tuberculosis infections collectively cause as many as five million deaths world-wide each year. In the most afflicted populations, currently available drugs and vaccines appear inadequate. By offering insight into the pathophysiology of diseases, genetic studies provide options for new therapeutic approaches to major health problems. The results of case-control studies of genetic factors associated with disease outcomes in malaria and tuberculosis in an African setting are presented in this thesis. Glucose-6-Phosphate dehydrogenase (G6PD) deficiency, the commonest enzymopathy of humans, affects over 400 million people. The geographical correlation of its distribution with the historical endemicity of malaria suggests that this disorder has risen in frequency through natural selection by malaria. However, attempts to confirm that G6PD deficiency is protective in case-control studies of malaria have yielded conflicting results. Hence, for this X-linked disorder, it is unclear whether both male hemizygotes and female heterozygotes are protected or, as frequently suggested, only females. Furthermore, how much protection may be afforded is unknown. In two large case-control studies of over 2000 African children, I found that the common African form of G6PD deficiency (G6PD A-) is associated with a 46-58% reduction in risk of severe malaria for both female heterozygotes and male hemizygotes. A mathematical model incorporating the measured selective advantage against malaria suggests that a counterbalancing selective disadvantage, associated with this enzyme deficiency, has retarded its rise in frequency in malaria-endemic regions. There is some evidence that two T helper cell subsets, Thl and Th2, regulate the immune response and thus influence the course of infections in mammalian hosts. These T cell subsets are reciprocal and associated with distinct cytokine profiles. Th2 T cell differentiation is promoted mainly by interleukin-4. Analysis of an IL-4 promoter polymorphism indicates that homozygosity for a putatively upregulatory IL-4 promoter variant is associated with a signficantly increased risk for severe malaria whilst heterozygotes are protected against this condition. Epidemiological evidence implicates host genetic factors as major determinants of variable susceptibility to tuberculosis. Most attempts to define the genetic factor(s) have focused on the HLA genes but only one result, an association of HLA-DR2 with increased susceptibility to disease in Asian populations, has been reported with any consistency. The genetic component in tuberculosis is likely to be determined by multiple genes and, therefore, in this study, the role of both HLA and non-HLA candidate genes was investigated. No association was found with variants of the macrophage gene, NRAMP1, the homologue of which has been implicated in the regulation of genetic resistance in the mouse model. Examination of certain class I and class II HLA alleles as well as the -590 interleukin-4 promoter polymorphism also did not show any association with disease. However, heterozygotes for a promoter polymorphism at position -238 of the tumour necrosis factor gene and homozygotes for dysfunctional variants of the gene encoding the collectin, mannose binding protein, were both at increased risk of developing pulmonary tuberculosis.

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

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

Les moustiques anophèles sont les vecteurs du parasite Plasmodium l’agent du paludisme. Le parasite subit des pertes massives pendant son cycle de développement chez l’anophèle, ce qui suggère que les moustiques sont capables de développer une réaction immunitaire efficace contre le parasite. La connaissance de l’immunité et de la résistance des moustiques au genre Plasmodium provient principalement de systèmes de laboratoire qui utilisent des espèces de parasites de rongeurs ou d’oiseaux comme modèles du paludisme humain. Les observations présentées dans cette thèse suggèrent que certains gènes comme Tep1 et LRIM1 sont des médiateurs de réponses antiparsitiques contre différents Plasmodiums dans différents vecteurs. Cependant, le degré d'efficacité avec laquelle un moustique est capable de réduire le nombre de parasites peut être variable surtout entre combinaison de souche de moustique et de souche de parasite différentes, selon que la paire soit hautement compatible ou non.<p><p><p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished