Malaria has been a major driving force in the evolution of the human genome. In sub-Saharan African populations, two neighbouring polymorphisms in the Complement Receptor 1 (CR1) gene, named Swain-Langley (Sl2) and McCoy (McCb), occur at high frequencies, consistent with selection by malaria. This thesis investigates the association between these two polymorphisms and severe malaria. Previous studies into this area have produced conflicting findings. Using a large case-control study of severe malaria in Kenyan children and statistical models adjusted for confounders, I found that the Sl2 polymorphism was associated with markedly reduced odds of cerebral malaria and death, while the McCb polymorphism was associated with increased odds of cerebral malaria. I also identified an interaction between Sl2 and α+thalassaemia, with the protective association of Sl2 greatest in children with normal α-globin. Following these epidemiological findings, I explored potential biological hypotheses which might explain them. The first approach examined whether the Sl2 and McCb polymorphisms affected how CR1 forms clusters on erythrocyte membranes, a process which is key in the binding and transfer of immune complexes from erythrocytes to macrophages. Using erythrocytes from Kenyan children, I performed immunofluorescence assays (IFAs) with confocal microscopy to quantify CR1 cluster number and volume. I found no association between the Sl2 and McCb polymorphisms and either the number or volume of CR1 clusters formed. The second approach investigated whether the cerebral malaria-specific associations seen with Sl2 and McCb might be due to expression of CR1 by human brain endothelial cells (HBEC). The immortalised cell line HBEC-5i was investigated for expression of CR1 using IFA, flow cytometry, western blotting, functional C3b degradation assays, mass spectrometry, immunoprecipitation and siRNA knockdown experiments. A pool of α-CR1 monoclonal antibodies recognised an intracellular antigen in permeabilised HBEC-5i cells which was a similar molecular weight to CR1 on western blotting. However, when the α-CR1 monoclonal antibodies were tested individually, only E11 recognised an HBEC-5i antigen. Further investigative approaches did not support the presence of CR1 on HBEC-5i cells, instead suggesting that E11 was not specific for CR1 and was instead recognising a protein in the Golgi apparatus. The final approach was to examine whether the Sl2 and McCb polymorphisms might influence the binding of the complement components mannose binding lectin, C1q and L-ficolin to the LHR-D region of CR1. I aimed to generate recombinant proteins of the LHR-D region which included the polymorphisms. Site-directed mutagenesis of the region was successful and subcloning and expression of the mutant amplicons will be performed at a later date. In summary, I have identified opposing associations between the Sl2 and McCb polymorphisms and cerebral malaria, which do not appear to be due to differences in CR1 clustering or expression of CR1 by human brain endothelial cells. My investigation into whether the polymorphisms might influence complement component binding is ongoing.

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

by Ng Wang Kit. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 131-139). / Abstracts in English and Chinese. / Acknowledgements --- p.iii / Abstract --- p.v / List of Tables --- p.ix / List of Figures --- p.x / List of Abbreviations --- p.xiii / Table of Contents --- 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 picture --- p.3 / Chapter 2.1.2 --- Malaria mechanics --- p.4 / Chapter 2.1.3 --- Life cycle of malaria parasite --- p.4 / Chapter 2.2 --- Treatment of malaria ´ؤ malaria drugs --- p.5 / Chapter 2.2.1 --- Antimalarial drugs --- p.5 / Chapter 2.2.2 --- Drug resistance --- p.6 / Chapter 2.3 --- Treatment of malaria - malarial vaccines --- p.7 / Chapter 2.3.1 --- Malarial vaccine developments --- p.7 / Chapter 2.3.2 --- Transmission blocking vaccines --- p.7 / Chapter 2.3.3 --- Pre-erythrocytic vaccines --- p.9 / Chapter 2.3.4 --- Blood stage vaccines --- p.10 / Chapter 2.4 --- The major merozoite protein - gpl95 --- p.11 / Chapter 2.5 --- Plants as bioreactors --- p.12 / Chapter 2.5.1 --- Products of transgenic plants --- p.13 / Chapter 2.6 --- Transgenic plants for production of subunit vaccines --- p.14 / Chapter 2.6.1 --- Norwalk virus capsid protein production --- p.15 / Chapter 2.6.2 --- Hepatitis B surface antigen production --- p.15 / Chapter 2.7 --- Tobacco and Arabidopsis as model plants --- p.16 / Chapter 2.7.1 --- Arabidopsis --- p.16 / Chapter 2.7.2 --- Tobacco --- p.17 / Chapter 2.8 --- Transformation methods --- p.17 / Chapter 2.8.1 --- Direct DNA uptake --- p.17 / Chapter 2.8.1.1 --- Plant protoplast transformation --- p.17 / Chapter 2.8.1.2 --- Biolistic transformation --- p.18 / Chapter 2.8.2 --- Agrobacterium-mediated transformation --- p.18 / Chapter 2.8.2.1 --- Leaf-disc technique --- p.18 / Chapter 2.8.2.2 --- In planta transformation --- p.19 / Chapter 2.9 --- Phaseolin --- p.20 / Chapter 2.10 --- Detection and purification of recombinant products - Histidine tag --- p.21 / Chapter 2.11 --- Aims of study and hypotheses --- p.22 / Chapter Chapter 3: --- Materials and Methods --- p.24 / Chapter 3.1 --- Introduction --- p.24 / Chapter 3.2 --- Chemicals --- p.24 / Chapter 3.3 --- Expression in tobacco system --- p.24 / Chapter 3.3.1 --- Plant materials --- p.24 / Chapter 3.3.2 --- Bacterial strains --- p.25 / Chapter 3.3.3 --- Chimeric gene construction for tobacco transformation --- p.25 / Chapter 3.3.3.1 --- The cloning of pTZPhasp/flgp42-His/Phast (F1) --- p.26 / Chapter 3.3.3.2 --- The cloning of pBKPhasp-sp/flgp42-His/Phast (P9) --- p.30 / Chapter 3.3.3.3 --- The cloning of pHM2Ubip/flgp42-His/Nost (C2) --- p.30 / Chapter 3.3.4 --- Confirmation of sequence fidelity of chimeric gene by DNA sequencing --- p.33 / Chapter 3.3.5 --- Cloning of chimeric gene into binary vector --- p.34 / Chapter 3.3.6 --- Triparental mating of Agrobacterium tumefaciens LBA4404/pAL4404 --- p.35 / Chapter 3.3.7 --- Tobacco transformation and regeneration --- p.36 / Chapter 3.3.8 --- GUS assay --- p.37 / Chapter 3.3.9 --- Genomic DNA isolation --- p.37 / Chapter 3.3.10 --- PCR amplification and detection of transgene --- p.38 / Chapter 3.3.11 --- Southern blot analysis --- p.38 / Chapter 3.3.12 --- Total seeds RNA isolation --- p.39 / Chapter 3.3.13 --- RT-PCR --- p.39 / Chapter 3.3.14 --- Northern blot analysis --- p.40 / Chapter 3.3.15 --- Protein extraction and SDS-PAGE --- p.40 / Chapter 3.3.16 --- Western blot analysis --- p.41 / Chapter 3.4 --- Expression in Arabidopsis system --- p.42 / Chapter 3.4.1 --- Plant materials --- p.42 / Chapter 3.4.2 --- Bacterial strains --- p.42 / Chapter 3.4.3 --- Chimeric gene construction --- p.42 / Chapter 3.4.3.1 --- The cloning of pBKPhasp-sp/His/EK/p42/Phast (DH) --- p.43 / Chapter 3.4.3.2 --- The cloning of pTZPhaSp/His/EK/p42/Phast (EH) --- p.45 / Chapter 3.4.3.3 --- The cloning of pBKPhasp-sp/His/EK/flgp42/Phast (DHF) and pTZPhasp/His/EK/flgp42/Phast (EHF) --- p.45 / Chapter 3.4.4 --- Confirmation of sequence fidelity of chimeric genes --- p.45 / Chapter 3.4.5 --- Cloning of chimeric gene into Agrobacterium binary vector --- p.49 / Chapter 3.4.6 --- Transformation of Agrobacterium tumefaciens GV3101/pMP90 with chimeric gene constructs --- p.49 / Chapter 3.4.7 --- Arabidopsis Transformation --- p.49 / Chapter 3.4.8 --- Vacuum infiltration transformation --- p.50 / Chapter 3.4.9 --- Selection of successful transformants --- p.51 / Chapter 3.4.10 --- Selection for homozygous plants with single gene insertion --- p.51 / Chapter 3.4.11 --- GUS assay --- p.52 / Chapter 3.4.12 --- Genomic DNA isolation --- p.52 / Chapter 3.4.13 --- PCR amplification and detection of transgenes --- p.52 / Chapter 3.4.14 --- Southern Blot analysis --- p.52 / Chapter 3.4.15 --- Total siliques RNA isolation --- p.53 / Chapter 3.4.16 --- RT-PCR --- p.53 / Chapter 3.4.17 --- Northern blot analysis --- p.53 / Chapter 3.4.17 --- Protein extraction and SDS-PAGE --- p.54 / Chapter 3.4.18 --- Western blot analysis --- p.54 / Chapter 3.5 --- In vitro transcription and translation --- p.54 / Chapter 3.5.1 --- In vitro transcription --- p.54 / Chapter 3.5.2 --- In vitro translation --- p.55 / Chapter 3.6 --- Particle bombardment of GUS fusion gene --- p.56 / Chapter 3.6.1 --- Chimeric gene constructs --- p.56 / Chapter 3.6.2 --- Particle bombardment using snow bean cotyledon --- p.61 / Chapter Chapter 4: --- Results --- p.63 / Chapter 4.1 --- Tobacco system --- p.63 / Chapter 4.1.1 --- Chimeric gene constructs --- p.63 / Chapter 4.1.2 --- Tobacco transformation and regeneration --- p.65 / Chapter 4.1.3 --- GUS activity assay --- p.67 / Chapter 4.1.4 --- Molecular analysis of transgene integration --- p.68 / Chapter 4.1.4.1 --- Genomic DNA extraction and PCR --- p.68 / Chapter 4.1.4.2 --- Southern blot analysis --- p.70 / Chapter 4.1.5 --- Molecular analysis of transgene expression --- p.72 / Chapter 4.1.5.1 --- Total RNA isolation and RT-PCR --- p.72 / Chapter 4.1.5.2 --- Northern blot analysis --- p.75 / Chapter 4.1.6 --- Genomic PCR to confirm whole gene transfer --- p.76 / Chapter 4.1.7 --- Biochemical analysis of transgene expression --- p.78 / Chapter 4.1.7.1 --- Protein extraction and SDS-PAGE --- p.78 / Chapter 4.1.7.2 --- Western blot analysis --- p.78 / Chapter 4.2 --- Arabidopsis system --- p.83 / Chapter 4.2.1 --- Chimeric gene constructs --- p.83 / Chapter 4.2.2 --- Arabidopsis transformation and selection --- p.85 / Chapter 4.2.3 --- Selection of transgenic plants --- p.87 / Chapter 4.2.4 --- Assay of GUS activity --- p.91 / Chapter 4.2.5 --- Molecular analysis of transgene integration --- p.92 / Chapter 4.2.5.1 --- Genomic DNA extraction and PCR --- p.92 / Chapter 4.2.5.2 --- Southern blot analysis --- p.96 / Chapter 4.2.6 --- Molecular analysis of transgene expression --- p.99 / Chapter 4.2.6.1 --- Total RNA isolation and RT-PCR --- p.99 / Chapter 4.2.6.2 --- Northern blot analysis --- p.106 / Chapter 4.2.7 --- Genomic PCR for confirmation of whole gene transfer --- p.107 / Chapter 4.2.8 --- Biochemical analysis of transgene expression --- p.108 / Chapter 4.2.8.1 --- Protein extraction and SDS-PAGE --- p.108 / Chapter 4.2.8.2 --- Western blot analysis --- p.108 / Chapter 4.3 --- In vitro transcription and translation --- p.112 / Chapter 4.4 --- Particle bombardment of p42/ GUS fusion gene --- p.115 / Chapter Chapter 5: --- Discussion and Future perspectives --- p.117 / Chapter 5.1 --- Failure in detecting transgene expression --- p.117 / Chapter 5.2 --- Poor transgene expression --- p.120 / Chapter 5.2.1 --- Bacillus thuringiensis toxin and green fluorescent protein --- p.120 / Chapter 5.2.2 --- AT-richness --- p.121 / Chapter 5.2.3 --- Deleterious sequence - AUUUA --- p.123 / Chapter 5.2.4 --- Presence of AAUAAA or AAUAAA-like motifs --- p.125 / Chapter 5.2.5 --- Codon usage --- p.126 / Chapter 5.3 --- Future perspectives --- p.127 / Chapter Chapter 6: --- Conclusion --- p.129 / References --- p.131