Diss. (sammanfattning) Stockholm : Karolinska institutet, 2001. / Härtill 6 uppsatser.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from PDF title page (viewed on July 19, 2010). Includes bibliographical references.

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

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 4 uppsatser.

by Yu Wing Sze. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 119-126). / Abstracts in English and Chinese. / Thesis Committee --- p.ii / Statement --- p.iii / Acknowledgements --- p.iv / Abstract --- p.vi / 摘要 --- p.viii / Table of Contents --- p.x / List of Tables --- p.xvi / List of Figures --- p.xvii / List of Abbreviations --- p.xx / Chapter CHAPTER 1 --- General Introduction --- p.1 / Chapter CHAPTER 2 --- Literature Review --- p.3 / Chapter 2.1 --- Toxoplasma gondii --- p.3 / Chapter 2.1.1 --- Morphology and Life Cycle of T. gondii --- p.3 / Chapter 2.1.2 --- Routes of Transmission --- p.7 / Chapter 2.2 --- Toxoplasmosis --- p.8 / Chapter 2.2.1 --- Influences and Symptoms --- p.8 / Chapter 2.2.2 --- Treatment of Toxoplasmosis --- p.10 / Chapter 2.2.2.1 --- Antitoxoplasma Drugs --- p.10 / Chapter 2.2.2.2 --- Toxoplasma Vaccines --- p.12 / Chapter 2.3 --- Major T. gondii Surface Antigen - P30 --- p.16 / Chapter 2.4 --- Plants as Bioreactors --- p.19 / Chapter 2.4.1 --- Advantages of Plant Bioreactors --- p.19 / Chapter 2.4.2 --- Plant-based Vaccines --- p.20 / Chapter 2.4.2.1 --- VP2 Capsid Protein of Mink Enteritis Virus --- p.21 / Chapter 2.4.2.2 --- Hepatitis B Surface Antigen --- p.21 / Chapter 2.4.2.3 --- Norwalk Virus Capsid Protein --- p.22 / Chapter 2.5 --- Tobacco Expression System --- p.23 / Chapter 2.5.1 --- Transformation Methods --- p.23 / Chapter 2.5.1.1 --- Agrobacterium-mediated Transformation --- p.23 / Chapter 2.5.1.2 --- Direct DNA Uptake --- p.24 / Chapter 2.6 --- Phaseolin and Its Regulatory Sequences --- p.26 / Chapter CHAPTER 3 --- Expression of P30 in Transgenic Tobacco --- p.28 / Chapter 3.1 --- Introduction --- p.28 / Chapter 3.2 --- Materials and Methods --- p.29 / Chapter 3.2.1 --- Chemicals --- p.29 / Chapter 3.2.2 --- Oligos: Primers and Adapters --- p.29 / Chapter 3.2.3 --- Plant Materials --- p.31 / Chapter 3.2.4 --- Bacterial Strains --- p.31 / Chapter 3.2.5 --- Construction of Chimeric Genes --- p.31 / Chapter 3.2.5.1 --- Modification of pET-ASP30ΔPI --- p.32 / Chapter 3.2.5.2 --- Cloning of P30 into Vectors with Different Promoters --- p.38 / Chapter 3.2.5.2.1 --- Cloning ofP30 into Vector with CaMV 35S Promoter --- p.38 / Chapter 3.2.5.2.2 --- Cloning of P30 into Vector with Maize Ubiquitin 1 Promoter --- p.38 / Chapter 3.2.5.2.3 --- Cloning of P30 into Vector with Phaseolin Promoter --- p.38 / Chapter 3.2.5.2.4 --- Cloning of P30 into Vector with Phaseolin Promoter and Phaseolin SP --- p.39 / Chapter 3.2.5.3 --- Cloning of P30 into Agrobacterium Binary Vector pBI121 --- p.44 / Chapter 3.2.6 --- Transformation of Agrobacterium by Electroporation --- p.49 / Chapter 3.2.7 --- "Transformation, Selection and Regeneration of Tobacco " --- p.50 / Chapter 3.2.8 --- GUS Assay --- p.51 / Chapter 3.2.9 --- Synthesis of Single-stranded DIG-labeled DNA Probe --- p.51 / Chapter 3.2.10 --- Extraction of Genomic DNA from Leaves --- p.52 / Chapter 3.2.11 --- PCR of Genomic DNA with P30 Specific Primers --- p.53 / Chapter 3.2.12 --- Southern Blot Analysis of Genomic DNA --- p.53 / Chapter 3.2.13 --- Extraction of Total RNA from Leaves or Developing Seeds --- p.54 / Chapter 3.2.14 --- Reverse Transcription-Polymerase Chain Reaction of Total RNA --- p.55 / Chapter 3.2.15 --- Sequencing of RT-PCR Product --- p.56 / Chapter 3.2.16 --- Northern Blot Analysis of Total RNA --- p.56 / Chapter 3.2.17 --- Extraction of Total Protein from Leaves or Mature Seeds --- p.57 / Chapter 3.2.18 --- Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.58 / Chapter 3.2.19 --- Purification of 6xHis-tagged Proteins --- p.58 / Chapter 3.2.20 --- Western Blot Analysis of Total Protein --- p.59 / Chapter 3.2.21 --- In vitro Transcription and Translation --- p.60 / Chapter 3.2.21.1 --- Construction of Transcription Vector Containing Chimeric P30 Gene --- p.60 / Chapter 3.2.21.2 --- In vitro Transcription --- p.60 / Chapter 3.2.21.3 --- In vitro Translation --- p.60 / Chapter 3.3 --- Results --- p.65 / Chapter 3.3.1 --- Construction of Chimeric P30 Genes --- p.65 / Chapter 3.3.2 --- "Tobacco Transformation, Selection and Regeneration " --- p.65 / Chapter 3.3.3 --- Detection of GUS Activity --- p.67 / Chapter 3.3.4 --- Detection of P30 Gene in Transgenic Plants --- p.69 / Chapter 3.3.4.1 --- PCR of Genomic DNA --- p.69 / Chapter 3.3.4.2 --- Southern Blot Analysis --- p.72 / Chapter 3.3.5 --- Detection of P30 Transcript in Transgenic Plants --- p.75 / Chapter 3.3.5.1 --- RT-PCR --- p.75 / Chapter 3.3.5.2 --- Sequencing of RT-PCR Product --- p.79 / Chapter 3.3.5.3 --- Northern Blot Analysis --- p.79 / Chapter 3.3.6 --- Detection of P30 Protein in Transgenic Plants --- p.83 / Chapter 3.3.6.1 --- Western Blot Analysis of Total Protein and Ni-NTA Purified Proteins --- p.83 / Chapter 3.3.7 --- In vitro Transcription and Translation --- p.92 / Chapter 3.3.7.1 --- In vitro Transcription --- p.92 / Chapter 3.3.7.2 --- In vitro Translation --- p.92 / Chapter CHAPTER 4 --- Discussion --- p.97 / Chapter 4.1 --- General Conclusion --- p.97 / Chapter 4.2 --- Further Speculations and Investigations --- p.100 / Chapter 4.2.1 --- Other Protein Detection Procedures --- p.100 / Chapter 4.2.2 --- In vitro Transcription and Translation --- p.100 / Chapter 4.2.3 --- Gene Silencing at Transcription and/or Post-transcription Levels --- p.101 / Chapter 4.2.4 --- Gene Silencing at Translation and/or Post-translation Levels --- p.102 / Chapter (A) --- AUG Context Sequence --- p.102 / Chapter (B) --- Codon Usage --- p.103 / Chapter (C) --- N-end Rule --- p.107 / Chapter (D) --- Phaseolin Sorting Signal --- p.107 / Chapter CHAPTER 5 --- Future Perspectives --- p.109 / Chapter 5.1 --- Codon Modification of the P30 Gene --- p.110 / Chapter 5.2 --- Fusion of the P30 Gene with the LRP Gene --- p.117 / Chapter CHAPTER 6 --- Conclusion --- p.118 / References --- p.119

Os principais marcadores sorológicos para o diagnóstico da toxoplasmose aguda ou recente são os anticorpos IgM específicos e anticorpos IgG de baixa avidez. Entretanto em alguns pacientes, anticorpos IgM e baixa avidez de anticorpos IgG podem persistir, ultrapassando o período da fase recente aguda contribuindo para erros de interpretação diagnóstica. No presente estudo, a eficiência diagnóstica do ensaio imunoenzimático foi avaliada, com o uso de frações antigênicas ou peptídeos sintéticos originados do antígeno ESA de T.gondii, denominados de SAG-1, GRA-1 e GRA-7. Foram estudadas frações isoladas e combinadas em múltiplos peptídeos antigênicos (MAP), visando estabelecer um perfil confiável para definição sorológica de toxoplasmose recente aguda em amostra única de soro. A melhor eficiência diagnóstica do ensaio foi encontrada com o uso da combinação de peptídeos SAG- 1,GRA-1 e GRA-7, denominada MAP1. A detecção de anticorpos IgG e IgM anti- MAP1 apresentou a melhor definição entre a fase recente aguda da fase recente não aguda na toxoplasmose. Nossos resultados mostraram que IgM anti-MAP1 poderá se constituir um marcador sorológico importante no aumento da eficiência diagnóstica da toxoplasmose recente aguda / The main serological marker for the diagnosis of recent toxoplasmosis is the specific IgM antibody, along with IgG antibodies of low avidity. However, in some patients these antibodies may persist long after the acute/recent phase, contributing to misdiagnosis in suspected cases of toxoplasmosis. In the present study, the diagnostic efficiency of ELISA was evaluated, with the use of peptides derived from T. gondii ESA antigens, named SAG-1, GRA-1 and GRA-7. In the assay referred to, we studied each of these peptides individually, as well as in four different combinations, as Multiple Antigen Peptides (MAP), aiming to establish a reliable profile for the acute/recent toxoplasmosis with only one patient serum sample. The diagnostic performance of the assay using MAP1, with the combination of SAG-1, GRA-1 and GRA-7 peptides, demonstrated better discrimination of the acute/recent phase from non acute/recent phase of toxoplasmosis. Our results show that IgM antibodies to MAP1 may be useful as a serological marker, enhancing the diagnostic efficiency of the assay for acute/recent phase of toxoplasmosis

Os principais marcadores sorológicos para o diagnóstico da toxoplasmose aguda ou recente são os anticorpos IgM específicos e anticorpos IgG de baixa avidez. Entretanto em alguns pacientes, anticorpos IgM e baixa avidez de anticorpos IgG podem persistir, ultrapassando o período da fase recente aguda contribuindo para erros de interpretação diagnóstica. No presente estudo, a eficiência diagnóstica do ensaio imunoenzimático foi avaliada, com o uso de frações antigênicas ou peptídeos sintéticos originados do antígeno ESA de T.gondii, denominados de SAG-1, GRA-1 e GRA-7. Foram estudadas frações isoladas e combinadas em múltiplos peptídeos antigênicos (MAP), visando estabelecer um perfil confiável para definição sorológica de toxoplasmose recente aguda em amostra única de soro. A melhor eficiência diagnóstica do ensaio foi encontrada com o uso da combinação de peptídeos SAG- 1,GRA-1 e GRA-7, denominada MAP1. A detecção de anticorpos IgG e IgM anti- MAP1 apresentou a melhor definição entre a fase recente aguda da fase recente não aguda na toxoplasmose. Nossos resultados mostraram que IgM anti-MAP1 poderá se constituir um marcador sorológico importante no aumento da eficiência diagnóstica da toxoplasmose recente aguda / The main serological marker for the diagnosis of recent toxoplasmosis is the specific IgM antibody, along with IgG antibodies of low avidity. However, in some patients these antibodies may persist long after the acute/recent phase, contributing to misdiagnosis in suspected cases of toxoplasmosis. In the present study, the diagnostic efficiency of ELISA was evaluated, with the use of peptides derived from T. gondii ESA antigens, named SAG-1, GRA-1 and GRA-7. In the assay referred to, we studied each of these peptides individually, as well as in four different combinations, as Multiple Antigen Peptides (MAP), aiming to establish a reliable profile for the acute/recent toxoplasmosis with only one patient serum sample. The diagnostic performance of the assay using MAP1, with the combination of SAG-1, GRA-1 and GRA-7 peptides, demonstrated better discrimination of the acute/recent phase from non acute/recent phase of toxoplasmosis. Our results show that IgM antibodies to MAP1 may be useful as a serological marker, enhancing the diagnostic efficiency of the assay for acute/recent phase of toxoplasmosis

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