Fu Lai Hong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 106-110). / Abstracts in English and Chinese. / Thesis/Assessment Committee --- p.ii / Statement --- p.iii / Acknowledgements --- p.iv / Abstract --- p.v / 摘要 --- p.vi / Lists of Figures --- p.x / Lists of Tables --- p.xiii / List of Abbreviations --- p.xiv / Amino acid abbreviation --- p.xvi / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Human α-L-iduronidase (hIDUA) --- p.2 / Chapter 1.1.1 --- Lysosomal storage disease --- p.2 / Chapter 1.1.2 --- Treatments of MPS 1 --- p.4 / Chapter 1.2 --- Plant cells as bioreactors --- p.5 / Chapter 1.3 --- The Plant secretary pathway --- p.7 / Chapter 1.3.1 --- Transport of soluble proteins --- p.9 / Chapter 1.3.2 --- Transport of integral membrane proteins --- p.10 / Chapter 1.4 --- Differences between plant and human proteins --- p.11 / Chapter 1.5 --- Reducing the differences between plant and human proteins --- p.12 / Chapter 1.6 --- Previous study: Expression of IDUA in transgenic tobacco plant --- p.13 / Chapter 1.7 --- Project objectives --- p.14 / Chapter 1.8 --- Long term significance --- p.14 / Chapter Chapter 2 --- Materials and Methods --- p.15 / Chapter 2.1 --- Introduction --- p.16 / Chapter 2.2 --- Materials --- p.18 / Chapter 2.2.1 --- Chemical --- p.18 / Chapter 2.2.2 --- Plant materials --- p.18 / Chapter 2.2.3 --- Plasmid vectors and bacterial strains --- p.18 / Chapter 2.2.4 --- Human a-iduronidase (hIDUA) cDNA --- p.19 / Chapter 2.2.5 --- Primers --- p.20 / Chapter 2.3 --- Methods --- p.22 / Chapter 2.3.1 --- Generation of IDUA antibodies --- p.22 / Chapter 2.3.1.1 --- Synthetic peptide raised IDUA antibodies --- p.23 / Chapter 2.3.1.1.1 --- Design of synthetic peptides --- p.23 / Chapter 2.3.1.1.2 --- Immunization of rabbits --- p.25 / Chapter 2.3.1.2 --- E. coli-derived rhIDUA protein --- p.25 / Chapter 2.3.1.2.1 --- Cloning and expression of rhIDUA --- p.25 / Chapter 2.3.1.2.2 --- Western analysis of E. coli-derived rhIDUA --- p.29 / Chapter 2.3.1.2.3 --- MS/MS analysis of rhIDUA protein --- p.29 / Chapter 2.3.1.2.4 --- Immunization of rabbits --- p.31 / Chapter 2.3.2 --- Affinity-purified antibodies --- p.33 / Chapter 2.3.3 --- Characterization of affinity-purified IDUA antibodies --- p.33 / Chapter 2.3.4 --- Construction of chimeric gene constructs --- p.34 / Chapter 2.3.5 --- Expression of IDUA in tobacco BY-2 cells --- p.39 / Chapter 2.3.5.1 --- Electropoartion of Agrobacteria --- p.39 / Chapter 2.3.5.2 --- Agrobacterium-mediated transformation --- p.39 / Chapter 2.3.5.3 --- Screening of positive trans formants --- p.40 / Chapter 2.3.6 --- Characterization of transgenic BY-2 cell expressing IDUA fusion --- p.40 / Chapter 2.3.6.1 --- Genomic DNA polymerase chain reaction (Genomic DNA PCR) --- p.40 / Chapter 2.3.6.1.1 --- Genomic DNA extraction from BY-2 callus --- p.40 / Chapter 2.3.6.1.2 --- Genomic DNA PCR of tobacco BY-2 callus --- p.41 / Chapter 2.3.6.2 --- Reverse transcription-PCR (RT-PCR) --- p.42 / Chapter 2.3.6.2.1 --- Total RNA extraction from BY-2 cell --- p.42 / Chapter 2.3.6.2.2 --- RT-PCR of BY-2 cell --- p.42 / Chapter 2.3.6.3 --- Western blot analysis of BY-2 cell and medium --- p.43 / Chapter 2.3.6.3.1 --- Protein extraction from tobacco BY-2 cells and culture medium --- p.43 / Chapter 2.3.6.3.2 --- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) --- p.44 / Chapter 2.3.6.3.3 --- Immunodetection and Coomassie blue stain --- p.44 / Chapter 2.3.7 --- Purification of IDUA from culture media --- p.46 / Chapter Chapter 3 --- Results --- p.47 / Chapter 3.1 --- Generation of IDUA antibodies --- p.48 / Chapter 3.1.1 --- Cloning and expression of rhIDUA in E. coli --- p.48 / Chapter 3.1.2 --- Characterization of IDUA antibodies --- p.51 / Chapter 3.1.2.1 --- Specificity of IDUA antibodies towards hIDUA protein. --- p.51 / Chapter 3.1.2.2 --- Cross-reactivity of IDUA antibodies with wild type tobacco BY-2 cell --- p.55 / Chapter 3.2 --- Chimeric gene constructs construction and confirmation --- p.58 / Chapter 3.3 --- Screening of transformed tobacco BY-2 callus with kanamycin-resistance --- p.66 / Chapter 3.4 --- Genomic DNA PCR screening of transformed tobacco BY-2 callus . --- p.67 / Chapter 3.5 --- RT-PCR screening of transformed BY-2 cells --- p.70 / Chapter 3.6 --- Western blot analysis of transformed tobacco BY-2 cells and culture media --- p.72 / Chapter 3.6.1 --- Tobacco BY-2 cells --- p.72 / Chapter 3.6.2 --- Tobacco BY-2 cell culture media --- p.76 / Chapter 3.7 --- Purification of IDUA protein in culture media --- p.81 / Chapter Chapter 4 --- Discussion --- p.82 / Chapter Chapter 5 --- Summary and Future Perspectives --- p.89 / Chapter 5.1 --- Summary --- p.90 / Chapter 5.2 --- Future perspectives --- p.92 / Appendix Identification and Characterization of an Unknown Protein by 1B Antibody --- p.93 / Chapter 6.1 --- Introduction --- p.94 / Chapter 6.2 --- Objectives --- p.94 / Chapter 6.3 --- Materials and Methods --- p.95 / Chapter 6.3.1 --- Western blot analysis of different plant species --- p.95 / Chapter 6.3.2 --- Subcellular localization of the unknown protein --- p.95 / Chapter 6.3.3 --- Affinity-purification of the unknown protein --- p.95 / Chapter 6.4 --- Results --- p.97 / Chapter 6.4.1 --- Western blot analysis of different plant species --- p.97 / Chapter 6.4.2 --- Subcellular localization of an unknown protein --- p.98 / Chapter 6.4.3 --- Affinity-purification of 1B protein --- p.104 / Chapter 6.5 --- Summary and Future Perspectives --- p.105 / Chapter 6.5.1 --- Summary --- p.105 / Chapter 6.5.2 --- Future Perspectives --- p.105 / References --- p.106

Seto Tai Chi. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 122-138). / Abstracts in English and Chinese. / Thesis Committee --- p.ii / Statement --- p.iii / Acknowledgements --- p.iv / Abstract (in English) --- p.v / Abstract (in Chinese) --- p.vii / Table of Contents --- p.ix / List of Tables --- p.xvi / List of Figures --- p.xv / Chapter Chapter 1 --- General Introduction and Literature Review --- p.1 / Chapter 1.1 --- Introduction --- p.2 / Chapter 1.2 --- Tobacco seed as bioreactor --- p.4 / Chapter 1.2.1 --- Advantages of using tobacco seed to produce bioactive human lysosomal enzyme --- p.4 / Chapter 1.2.2 --- Disadvantages and potential problems of using tobacco seed to produce bioactive human lysosomal enzyme --- p.5 / Chapter 1.2.2.1 --- Difference of asparagine-linked N-glycosylation between plant and human protein --- p.8 / Chapter 1.2.2.2 --- Immunogenicity of recombinant protein with plant-derived N-glycan to human --- p.10 / Chapter 1.2.2.3 --- "Strategy to ""humanize"" plant-derived recombinant human lysosomal enzyme" --- p.10 / Chapter 1.2.2.4 --- Lack of specific glycan structure一mannose-6-phosphate (M6P) tag addition --- p.11 / Chapter 1.2.2.5 --- Strategy for M6P tag addition on plant-derived human lysosomal enzyme --- p.12 / Chapter 1.3 --- The plant secretory pathway --- p.13 / Chapter 1.3.1 --- Plant vacuole in tobacco seed --- p.16 / Chapter 1.3.2 --- Soluble protein trafficking in plant cell --- p.17 / Chapter 1.3.3 --- Integral membrane protein trafficking in plant cell --- p.17 / Chapter 1.3.4 --- Components involved in integral membrane protein trafficking to PSV crystalloid --- p.19 / Chapter 1.3.4.1 --- BP-80 (80-kDa binding protein) --- p.19 / Chapter 1.3.4.2 --- α-TIP (α-tonoplast intrinsic protein) --- p.20 / Chapter 1.3.5 --- Using specific integral membrane protein trafficking system to target recombinant human lysosomal enzyme to tobacco seed PSV --- p.21 / Chapter 1.4 --- Homo sapiens α-L-iduronidase (hIDUA) --- p.21 / Chapter 1.4.1 --- Global situation of lysosomal storage disease一hIDUA deficiency --- p.21 / Chapter 1.4.2 --- Physiological role --- p.22 / Chapter 1.4.3 --- Molecular property --- p.24 / Chapter 1.4.3.1 --- Mutation and polymorphism --- p.24 / Chapter 1.4.4 --- Lysosomal secretory pathway --- p.24 / Chapter 1.4.5 --- Biochemical property --- p.25 / Chapter 1.4.6 --- Clinical application --- p.27 / Chapter 1.4.6.1 --- Enzyme replacement therapy (ERT) --- p.27 / Chapter 1.4.6.2 --- Clinical trial --- p.28 / Chapter 1.4.6.3 --- Economic value --- p.29 / Chapter 1.4.7 --- Expression system --- p.29 / Chapter 1.4.7.1 --- Production (overexpression) of rhIDUA in CHO cell system --- p.30 / Chapter 1.4.7.2 --- Production of rhIDUA in tobacco plant leaf --- p.30 / Chapter 1.5 --- Project objective and long-term significance --- p.30 / Chapter 1.5.1 --- Project objective --- p.30 / Chapter 1.5.2 --- Long-term significance --- p.31 / Chapter Chapter 2 --- Generation and Characterization of Anti-IDUA Antibodies --- p.32 / Chapter 2.1 --- Introduction --- p.33 / Chapter 2.2 --- Materials --- p.33 / Chapter 2.2.1 --- Chemical --- p.33 / Chapter 2.3 --- Methods --- p.35 / Chapter 2.3.1 --- Generation of polyclonal anti-IDUA antibody --- p.35 / Chapter 2.3.1.1 --- Design of synthetic peptide --- p.35 / Chapter 2.3.1.2 --- Conjugation of synthetic peptide to carrier protein --- p.39 / Chapter 2.3.1.3 --- Immunization of rabbit --- p.39 / Chapter 2.3.2 --- Characterization of polyclonal anti-IDUA antibody in rabbit serum --- p.40 / Chapter 2.3.2.1 --- Dot-blot analysis --- p.40 / Chapter 2.3.3 --- Purification of polyclonal anti-IDUA antibody --- p.42 / Chapter 2.3.3.1 --- Construction of anti-IDUA antibody affinity column --- p.42 / Chapter 2.3.3.2 --- Affinity-purification of anti-IDUA antibody --- p.42 / Chapter 2.3.4 --- Western blot detection of denatured rhIDUA --- p.42 / Chapter 2.4 --- Results --- p.43 / Chapter 2.4.1 --- Characterization of polyclonal anti-IDUA antibody --- p.43 / Chapter 2.5 --- Discussion --- p.51 / Chapter 2.6 --- Conclusion --- p.51 / Chapter Chapter 3 --- Generation and Characterization of Transgenic Tobacco Plants Expressing rhIDUA Fusions --- p.52 / Chapter 3.1 --- Introduction --- p.53 / Chapter 3.1.1 --- Signal peptide of hIDUA (hIDUA SP) --- p.54 / Chapter 3.1.2 --- Signal peptide of proaleurain (Pro. SP) --- p.54 / Chapter 3.1.3 --- Hypothesis to be tested in this study --- p.54 / Chapter 3.2 --- Materials --- p.55 / Chapter 3.2.1 --- Chemical --- p.55 / Chapter 3.2.2 --- Primers --- p.55 / Chapter 3.2.3 --- Bacterial strain --- p.58 / Chapter 3.2.4 --- The insert-Homo sapiens α-L-iduronidase (hIDUA) cDNA used in this study --- p.58 / Chapter 3.2.5 --- The vector-pLJ526 used in this study --- p.59 / Chapter 3.3 --- Methods --- p.61 / Chapter 3.3.1 --- Construction of chimeric gene construct --- p.61 / Chapter 3.3.1.1 --- Restriction endonuclease´ؤPfIMIl --- p.61 / Chapter 3.3.1.2 --- Recombinant DNA and molecular cloning techniques used in this study --- p.61 / Chapter 3.3.1.3 --- Cloning of pSPIDUA-FLAG --- p.62 / Chapter 3.3.1.4 --- Cloning of pSPIDUA-control --- p.62 / Chapter 3.3.1.5 --- Cloning of a universal construct (pUniversal) --- p.62 / Chapter 3.3.1.6 --- Cloning of pSP-IDUA-T7 --- p.66 / Chapter 3.3.1.7 --- Cloning of pSP-IDUA-control --- p.66 / Chapter 3.3.1.8 --- Cloning of chimeric gene construct into Agrobacterium binary vector --- p.66 / Chapter 3.3.2 --- Expression of chimeric gene construct in tobacco plant --- p.73 / Chapter 3.3.2.1 --- Tobacco plant --- p.73 / Chapter 3.3.2.2 --- Electroporation of Agrobacterium --- p.73 / Chapter 3.3.2.3 --- Agrobacterium-mediated transformation of tobacco plant --- p.74 / Chapter 3.3.2.4 --- Selection and regeneration of tobacco transformant --- p.75 / Chapter 3.3.3 --- Characterization of transgenic tobacco plant expressing rhIDUA fusion --- p.75 / Chapter 3.3.3.1 --- Genomic DNA polymerase chain reaction (PCR) --- p.75 / Chapter 3.3.3.2 --- Southern blot analysis --- p.76 / Chapter 3.3.3.3 --- Total RNA reverse transcription-PCR (RT-PCR) --- p.77 / Chapter 3.3.3.4 --- Northern blot analysis of tobacco leaf --- p.78 / Chapter 3.3.3.5 --- Western blot analysis --- p.79 / Chapter 3.3.4 --- Purification of plant-derived rhIDUA fusion --- p.81 / Chapter 3.3.4.1 --- Construction of affinity column with anti-IDUA antibody --- p.81 / Chapter 3.3.4.2 --- Affinity-purification of rhIDUA fusion from tobacco mature seed --- p.81 / Chapter 3.3.5 --- Confocal immunoflorescence study --- p.82 / Chapter 3.3.5.1 --- Preparation of paraffin section --- p.82 / Chapter 3.3.5.2 --- Single immunocytochemical labeling --- p.82 / Chapter 3.3.5.3 --- Double labeling with one monoclonal and one polyclonal antibodies --- p.83 / Chapter 3.3.5.4 --- Double labeling with two polyclonal antibodies --- p.83 / Chapter 3.3.5.5 --- Image collection --- p.84 / Chapter 3.4 --- Results --- p.85 / Chapter 3.4.1 --- Chimeric gene construction and confirmation --- p.85 / Chapter 3.4.2 --- Selection and regeneration of tobacco transformant with kanamycin- resistance --- p.86 / Chapter 3.4.3 --- Genomic DNA PCR screening of tobacco transformant --- p.88 / Chapter 3.4.4 --- Southern blot analysis of tobacco transformant --- p.91 / Chapter 3.4.5 --- Total RNA RT-PCR screening of tobacco transformant --- p.93 / Chapter 3.4.6 --- Northern blot analysis of tobacco transformant --- p.93 / Chapter 3.4.7 --- Western blot analysis --- p.96 / Chapter 3.4.7.1 --- Western blot analysis of pSP-IDUA-T7-121 transformant leaf --- p.96 / Chapter 3.4.7.2 --- Western blot analysis of pSP-IDUA-T7-121 transformant mature seed --- p.98 / Chapter 3.4.8 --- Affinity-purification of rhIDUA fusion --- p.98 / Chapter 3.4.9 --- Expression level of rhIDUA fusion --- p.102 / Chapter 3.4.10 --- Subcellular localization of rhIDUA fusion --- p.102 / Chapter 3.5 --- Discussion --- p.111 / Chapter Chapter 4 --- Summary and Future Perspectives --- p.117 / References --- p.122 / Appendix 1 --- p.139 / Appendix II (List of Abbreviations) --- p.141

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

Devido aos problemas de deficiência de Mn relatados em soja Roundup Ready, à tendência de aumento de cultivo da soja Intacta RR2 PRO® no Brasil e à possibilidade de aumento de rendimentos desta soja relacionado à resposta a adubação foliar com Mn, realizou-se o presente trabalho. Este foi dividido em dois estudos em casa de vegetação (estudos I e II) e um em campo (estudo III). Cada estudo foi realizado em dois solos (um com alto teor de Mn e outro com baixo teor de Mn), avaliando-se os resultados de cada um separadamente. Estudo I: Dois experimentos foram realizados em delineamento em blocos aleatorizados, com quatro repetições e em esquema fatorial 2 × 6 × 4 com parcela subdividida no tempo. Formaram-se 48 tratamentos pela combinação de dois níveis do fator soja (cultivada sem ou com glifosato) e seis do fator fonte do nutriente (sem Mn ou Controle, Cloreto, Sulfato, Carbonato, EDTA e Citrato) alocados nas parcelas principais, e de quatro níveis do fator tempo (4, 24, 48 e 72 h após a aplicação do fertilizante) alocados nas subparcelas. Cada tratamento foi aplicado com uma haste flexível de algodão nas folhas e nos três primeiros trifólios (trifólios tratados) da planta de soja em estádio V4. Avaliou-se a absorção foliar de Mn através da determinação de massa de matéria seca, teor e conteúdo de Mn dos trifólios tratados e da haste de plantas de soja ainda em estádio V4. Estudo II: Dois experimentos foram realizados em delineamento em blocos aleatorizados, com quatro repetições e em esquema fatorial 2 × 6. Formaram-se 12 tratamentos pela combinação de dois níveis do fator soja e seis níveis do fator fonte do nutriente. Cada tratamento foi aplicado nos trifólios tratados da planta de soja em estádio V4. Avaliou-se a distribuição foliar de Mn através da determinação de massa de matéria seca, teor e conteúdo de Mn dos trifólios tratados, hastes, trifólios formados após a aplicação dos tratamentos (trifólios não tratados), vagens e grãos de plantas de soja em estádio R8. Estudo III: Realizaram-se dois experimentos em delineamento similar ao do estudo II, mas com seis blocos. Cada tratamento foi aplicado com pulverizador de pressão constante sobre a parte aérea de plantas de soja em estádio V4. Avaliou-se a massa de matéria seca, teor e conteúdo de Mn das hastes, vagens e grãos de plantas de soja em estádio R8. Foram avaliados também componentes de produção e rendimento de grãos. A quantidade absorvida de Mn é dependente da fonte utilizada e a fonte Cloreto foi a que proporcionou maior absorção de Mn, enquanto a fonte EDTA, apresentou maior eficiência em aumentar o conteúdo de Mn das hastes logo após a aplicação. O Mn aplicado nos trifólios pode ser redistribuído desta parte para outras da planta, embora aparentemente em pequenas quantidades, e até o final do ciclo da soja estará em maior proporção nos trifólios tratados. A soja tratada com Mn não apresentou grãos com maior acúmulo deste, mas na soja cultivada no \"solo -Mn\" e sem glifosato o conteúdo de Mn foi maior do que na soja com glifosato. Em termos de produtividade de grãos, a adubação foliar com Mn em aplicação única na soja no estádio V4 recebendo ou não aplicação de glifosato e cultivada em solo originalmente com alto teor de Mn não proporcionou diferenças. / Due to Mn deficiency problems related to Roundup Ready soybean, the tendency to increase cultivation of Intacta RR2 PROTM soybeans in Brazil and to the possibility of increased yield of this related to the response to Mn foliar fertilization, this work was carried out. It was divided into two greenhouse studies (I and II) and one in the field (study III). Each study was performed in two soils (one with high content of Mn and the other with low content), evaluating the results of each one separately. Study I: the two trials carried out by using factorial split-plot design, with three factors in four replications in randomized complete block design (RCBD). Soybean factor with two levels (without and with glyphosate) and Mn source factor with six levels (Control, Chloride, Sulphate, Carbonate, EDTA and Citrate), both distributed in factorial form into main plots and time factor (4, 24, 48 and 72 h after fertilizer application) distributed in the sub-plots. Each treatment was applied with a swab in the unifoliate leaves and the first three trifoliates (treated trifoliates) of soybean in V4 stage. Mn foliar absorption was determined by dry matter mass, concentration and content of Mn of treated trifoliates and stem of soybean plants in the V4 stage. Study II: The two trials carried out by using 2 × 6 factorial with four replications in RCBD. Soybean factor with two levels and Mn fertilizer source factor with six levels. Each treatment was applied to the treated trifoliates of the V4 soybean plant. the leaf distribution of Mn was determined by the dry matter mass, concentration and Mn content of the treated trifoliates, stems, trifoliates formed after the application of the treatments (untreated trifoliates), pods and grains of soybean plants in the R8 stage. Study III: Two experiments were carried out in a similar design of study II, but with six replications. Each treatment was applied with a constant pressure sprayer on the above ground part of V4 soybean plants. The foliar Mn was evaluated by determining the dry matter mass, content and Mn content of the stems, pods and grains of soybean plants at stage R8. Production components and grain yield were also evaluated. The absorbed amount of Mn is dependent on the source used and the Chloride is the one that provided the highest Mn absorption, but sources such as EDTA showed a higher efficiency in increasing the Mn content of the stems soon after application. The Mn applied in the trifoliates can be redistributed from this part to others of the plant, although apparently in small amounts, and will be in greater proportion in the treated trifoliates until the end of the soybean cycle. Mn-treated soybean did not present grains with higher accumulation, but in soybean cultivated grown in soil with low Mn concentration and without glyphosate the Mn content was higher than in soybean with glyphosate. In terms of grain yield, the foliar fertilization with Mn in single application in the soybean V4 stage without or with glyphosate grown in soil with high Mn content did not present significant differences.

Esta pesquisa tem como objetivo principal analisar o efeito da adoção das tecnologias utilizadas pelos produtores típicos de grãos (soja, milho e trigo) da região oeste do Paraná sobre a rentabilidades de culturas e sistemas entre os anos-safras 2007/08 e 2016/17, período que corresponde à mudança entre a baixa adoção de tecnologias modificadas geneticamente para um cenário de grande dependência e utilização. Este período também corresponde a uma mudança significativa na destinação das áreas e das culturas dentro da propriedade típica, com prioridade para o cultivo da soja no verão e incremento do cultivo de milho na 2ª safra, assim como maior utilização da área total na 2ª safra. A propriedade típica de Cascavel (PR) foi utilizada como base das informações analisadas. O ferramental utilizado se apoiou no trabalho realizado por Paiva (1975), comparando os resultados em termos de receita líquida de dois cenários produtivos específicos, um tradicional e outro moderno. A premissa é que o agricultor escolherá ou adotará a atividade e a técnica que apresentar melhor resultado e vantagem econômica. O período tradicional levou em consideração os resultados produtivos e de custos de produção das safras 2007/08, 2008/09 e 2009/10, em que a propriedade típica de Cascavel cultivava ainda um percentual alto de variedades de soja e híbridos de milho convencionais, assim como no portfólio de culturas semeava o milho na 1ª safra. A 2ª safra foi semeada com milho e trigo, mas sem ocuparem a totalidade da área disponível para o cultivo. O período moderno abrangeu os anos safras 2014/15, 2015/16 e 2016/17. Nestas safras, a propriedade típica de Cascavel (PR) passou a cultivar toda a área com soja e milho modificados geneticamente. A soja preencheu toda a área da 1ª safra e o milho a maior parte da área da 2ª safra. O trigo completou o cultivo da 2ª safra. Milho 2ª safra e trigo passaram a ocupar uma parcela maior da área disponível em 2ª safra. No geral, os resultados aqui apresentados indicaram que as receitas líquidas dos anos mais recentes (2014/15, 2015/16 e 2016/17) superaram as registradas nos anos bases de análise (2007/08, 2008/09 e 2009/10), direcionando para um contexto em que o produtor adotou técnicas com melhor benefício econômico/financeiro. / The research main objective is analyze the impact of technology\'s adoption on typical grain producers (soybean, corn and wheat) profitability on the western region of Paraná and between the years 2007/08 and 2016/17, a period that corresponds a change from low genetically modified technologies adoption to a scenario of high dependence and utilization. This period also corresponds to a significant change in the allocation of areas and crops within the typical farm, with priority for summer soybean production and increment of corn cultivation as second crop, as well as greater utilization of the total area in the second crop. The typical farm of Cascavel (PR) was used as the basis to the analyzed information. The tool used was based on Paiva (1975), comparing results in terms of net revenue of two specific production scenarios, one traditional and other modern. The premise was that the farmer will choose or adopt the activity and technique that present the best result and economic advantage. The traditional period considers productivity and production costs of the 2007/08, 2008/09 and 2009/10 harvests where Cascavel\'s typical farm still cultivated a high percentage of conventional soybean varieties and corn hybrids as well as portfolio of crops sowed corn in the first crop. The second crop was sown with maize and wheat, but did not occupy the entire area available for cultivation. The modern period covered the 2014/15, 2015/16 and 2016/17 season. In these harvests, the typical property of Cascavel began to cultivate the entire area with genetically modified soy and corn. Soybean filled the entire area of the 1st crop and maize most of the area of the 2nd crop. The wheat completed the cultivation of the 2nd crop. Over the seasons it has been found more intensive cultivation system in the 2nd crop area, increasing corn sowing and reducing wheat. In addition, the typical farm no longer sowed maize in the first crop and began to sow only genetically modified soybeans and corn. In general, the results presented here indicate that most recent years (2014/15, 2015/16 and 2016/17) net revenues exceed those recorded in the base analysis years (2007/08, 2008/09 and 2009/10), leading to a context in which the producer has adopted better techniques over time.

by Chan, Yuk Wah. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 166-175). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.iii / ABSTRACT --- p.v / 摘要 --- p.vii / LIST OF CONTENTS --- p.viii / LIST OF FIGURES --- p.xiv / LIST OF TABLES --- p.xvii / LIST OF ABBREVIATIONS --- p.xviii / Chapter CHAPTER 1. --- GENERAL INTRODUCTION --- p.1 / Chapter CHAPTER 2. --- LITERATURE REVIEW --- p.5 / Chapter 2.1 --- Vitamin A --- p.5 / Chapter 2.1.1 --- General and properties --- p.5 / Chapter 2.1.2 --- Biological importance of vitamin A --- p.6 / Chapter 2.1.3 --- Deficiency symptoms --- p.9 / Chapter 2.1.4 --- Dietary source of vitamin A --- p.10 / Chapter 2.1.5 --- Metabolism of dietary vitamin A and provitamin A in human --- p.12 / Chapter 2.1.5.1 --- Digestion and absorption --- p.12 / Chapter 2.1.5.2 --- Bioconversion --- p.12 / Chapter 2.1.5.2.1 --- "β, β-carotene-15,15'-monooxygenase (BCMO)" --- p.13 / Chapter 2.1.5.3 --- "Transport, uptake and storage" --- p.15 / Chapter 2.2 --- Vitamin A deficiency (VAD) --- p.19 / Chapter 2.2.1 --- Present situation --- p.19 / Chapter 2.2.2 --- Global efforts in dealing with VAD --- p.21 / Chapter 2.2.2.1 --- Vitamin A supplementation --- p.21 / Chapter 2.2.2.2 --- Food fortification --- p.22 / Chapter 2.2.2.3 --- Biofortification --- p.23 / Chapter 2.2.2.3.1 --- Conventional selective breeding --- p.23 / Chapter 2.2.2.3.2 --- Biosynthesis of provitamin A in plants --- p.25 / Chapter 2.2.2.3.3 --- Carotenoids enhancement in major plants and food crops --- p.31 / Chapter 2.3 --- Inherent problems of the present carotenoid enhancement --- p.34 / Chapter 2.3.1 --- Recommended Dietary Amount of vitamin A --- p.34 / Chapter 2.3.2 --- Factors affecting the bioefficacy of provitamin A in human body --- p.35 / Chapter 2.3.2.1 --- Bioavailability --- p.36 / Chapter 2.3.2.2 --- Bioconvertibility --- p.38 / Chapter 2.3.2.3 --- Health and nutrition status --- p.39 / Chapter 2.4 --- Previous study in our lab --- p.41 / Chapter 2.4.1 --- Overexpression of rice PSY1 --- p.41 / Chapter 2.4.2 --- Introduction of carotenoid genes and BCMOs into rice --- p.44 / Chapter 2.5 --- Overview of the project --- p.50 / Chapter CHAPTER 3. --- MATERIALS AND METHODS --- p.52 / Chapter 3.1 --- Chemicals --- p.52 / Chapter 3.2 --- Bacterial strains in regular cloning --- p.52 / Chapter 3.3 --- BCMO genes and carotenogenic genes --- p.53 / Chapter 3.4 --- Expression of BCMOs in bacterial system --- p.54 / Chapter 3.4.1 --- lac promoter system --- p.54 / Chapter 3.4.2 --- pBAD-TOPO® system --- p.56 / Chapter 3.5 --- Construction of gene cassettes for plant transformation --- p.58 / Chapter 3.5.1 --- Gene cassettes for carrot transformation --- p.58 / Chapter 3.5.1.1 --- Construction of gene cassettes for chicken or zebrafish bcmo driven by CaMV 35S promoter --- p.58 / Chapter 3.5.1.2 --- Construction of gene cassettes for chicken or zebrafish bcmo driven by lycopene-β-cyclase promoter --- p.63 / Chapter 3.5.2 --- Gene cassettes for Arabidopsis transformation --- p.67 / Chapter 3.5.2.1 --- Construction of gene cassettes expressing Dcpsy --- p.67 / Chapter 3.5.2.2 --- Construction of gene cassettes expressing mbcmos --- p.69 / Chapter 3.5.3 --- Gene cassettes for Rice transformation --- p.72 / Chapter 3.5.3.1 --- Construction of gene cassettes expressing mbcmos --- p.72 / Chapter 3.5.3.2 --- Construction of gene cassettes expressing Ospsyl and mbcmos --- p.74 / Chapter 3.5.4 --- Confirmation of sequence fidelity --- p.76 / Chapter 3.6 --- Carrot transformation --- p.76 / Chapter 3.6.1 --- Plant materials --- p.76 / Chapter 3.6.2 --- Preparation of Agrobacterium --- p.76 / Chapter 3.6.3 --- Agrobacterium mediated transformation --- p.77 / Chapter 3.6.3.1 --- Seed germination --- p.78 / Chapter 3.6.3.2 --- Co-cultivation with hypocotyls --- p.78 / Chapter 3.6.3.3 --- Callus induction and selection --- p.78 / Chapter 3.6.3.4 --- Liquid cell culture preparation and embryogenesis induction --- p.79 / Chapter 3.6.3.5 --- Regeneration --- p.80 / Chapter 3.7 --- Arabidopsis Transformation --- p.80 / Chapter 3.7.1 --- Plant materials --- p.80 / Chapter 3.7.2 --- Preparation of Agrobacterium --- p.81 / Chapter 3.7.3 --- Agrobacterium mediated transformation --- p.81 / Chapter 3.7.3.1 --- Co-cultivation --- p.81 / Chapter 3.7.3.2 --- Selection --- p.82 / Chapter 3.8 --- Rice transformation --- p.83 / Chapter 3.8.1 --- Plant materials --- p.83 / Chapter 3.8.2 --- Preparation of Agrobacterium --- p.83 / Chapter 3.8.3 --- Agrobacterium mediated transformation --- p.83 / Chapter 3.8.3.1 --- Callus induction from mature rice seeds --- p.84 / Chapter 3.8.3.2 --- Co-cultivation and selection --- p.84 / Chapter 3.9 --- Detection of transgene expression --- p.86 / Chapter 3.9.1 --- Detection at DNA level --- p.86 / Chapter 3.9.1.1 --- Genomic DNA extraction --- p.86 / Chapter 3.9.1.2 --- PCR screening --- p.86 / Chapter 3.9.1.3 --- Synthesis of DIG-labelled DNA probes --- p.86 / Chapter 3.9.1.4 --- Southern blot analysis --- p.87 / Chapter 3.9.2 --- Detection at RNA level --- p.88 / Chapter 3.9.2.1 --- Total RNA extraction --- p.88 / Chapter 3.9.2.2 --- Northern blot analysis --- p.89 / Chapter 3.9.2.3 --- RT-PCR --- p.89 / Chapter 3.9.3 --- Detection at protein level --- p.89 / Chapter 3.9.3.1 --- Antibody production --- p.89 / Chapter 3.9.3.1.1 --- B.CMO protein induction in pET30a-bacterial system --- p.90 / Chapter 3.9.3.1.2 --- Immunization of rabbit and serum collection --- p.93 / Chapter 3.9.3.2 --- Protein extraction and Tricine SDS-PAGE --- p.93 / Chapter 3.9.3.3 --- Western blot analysis --- p.94 / Chapter 3.9.4 --- Detection at final product level --- p.95 / Chapter 3.9.4.1 --- UPLC analysis --- p.95 / Chapter 3.9.4.1.1 --- Extraction of total carotenoids and retinoids --- p.95 / Chapter 3.9.4.1.2 --- UPLC identification --- p.96 / Chapter CHAPTER 4. --- RESULTS --- p.97 / Chapter 4.1 --- Modified bcmo genes --- p.97 / Chapter 4.2 --- Expression of BCMOs in bacterial system --- p.102 / Chapter 4.2.1 --- lac promoter system --- p.104 / Chapter 4.2.2 --- pBAD-TOPO® system --- p.106 / Chapter 4.2.3 --- UPLC detection --- p.108 / Chapter 4.3 --- Carrot transformation --- p.110 / Chapter 4.3.1 --- Construction of gene cassettes for carrot transformation --- p.110 / Chapter 4.3.2 --- Seed germination and co-cultivation --- p.112 / Chapter 4.3.3 --- Callus induction and selection --- p.113 / Chapter 4.3.4 --- Embryogenesis induction and regeneration --- p.113 / Chapter 4.3.5 --- Callus induction in the dark --- p.115 / Chapter 4.3.6 --- Detection of native BCMO --- p.116 / Chapter 4.3.6.1 --- Genomic PCR screening of 35Spro - zebcmo transgenic lines --- p.116 / Chapter 4.3.6.2 --- Southern blot analysis of 35Spro - zebcmo transgenic lines --- p.117 / Chapter 4.3.6.3 --- RT-PCR of 35Spro - zebcmo transgenic lines --- p.118 / Chapter 4.3.6.4 --- Detection at protein level --- p.119 / Chapter 4.3.6.4.1 --- Antibody production --- p.119 / Chapter 4.3.6.5 --- Western blot analysis of 35Spro - zebcmo transgenic lines --- p.123 / Chapter 4.3.6.6 --- Genomic PCR screening of later transgenic lines --- p.123 / Chapter 4.3.6.7 --- Western blot analysis of later transgenic lines --- p.125 / Chapter 4.3.6.8 --- UPLC analysis of later transgenic lines --- p.127 / Chapter 4.3.7 --- Detection of modified BCMO --- p.130 / Chapter 4.3.7.1 --- Genomic PCR screening --- p.130 / Chapter 4.3.7.2 --- Northern blot analysis --- p.132 / Chapter 4.3.7.3 --- Western blot analysis --- p.134 / Chapter 4.3.8 --- UPLC analysis --- p.136 / Chapter 4.4 --- Arabidopsis transformation --- p.138 / Chapter 4.4.1 --- Construction of gene cassettes for Arabidopsis transformation --- p.138 / Chapter 4.4.2 --- Selection --- p.139 / Chapter 4.4.3 --- Genmoic PCR screening of Arabidopsis transformants --- p.140 / Chapter 4.4.4 --- UPLC analysis for Arabidopsis transformants --- p.142 / Chapter 4.5 --- Rice transformation --- p.144 / Chapter 4.5.1 --- Construction of gene cassettes for rice transformation --- p.144 / Chapter 4.5.2 --- "Callus induction from mature rice seeds, co-cultivation and selection" --- p.146 / Chapter 4.5.3 --- Genomic PCR screening of Rice transformants --- p.147 / Chapter 4.5.4 --- UPLC analysis of rice transformants --- p.149 / Chapter CHAPTER 5. --- DISCUSSION --- p.151 / Chapter 5.1 --- Bacterial expression of BCMO --- p.151 / Chapter 5.2 --- Analysis of BCMO in plants --- p.153 / Chapter 5.2.1 --- Carrot --- p.154 / Chapter 5.2.1.1 --- Expression of BCMO in carrot transformants --- p.154 / Chapter 5.2.1.2 --- UPLC analysis of carrot transformants --- p.155 / Chapter 5.2.2 --- Arabidopsis --- p.156 / Chapter 5.2.3 --- Rice --- p.158 / Chapter 5.3 --- Proposed explanation for the failure of retinal production --- p.159 / Chapter 5.3.1 --- Retinal sequestration --- p.160 / Chapter 5.3.2 --- Localization of BCMO --- p.161 / Chapter 5.4 --- Future prospects --- p.163 / Chapter CHAPTER 6. --- CONCLUSIONS --- p.165 / REFERENCES --- p.166 / APPENDICES --- p.176

The National Bioengineered Food Disclosure Standard (PL 114-216) will require nearly all foods sold in the U.S. to bear a statement disclosing whether they contain genetically modified (GM) material. Past studies suggest the presence of such a statement could have profound effects on consumers; however, research comparing consumer response towards different GM-disclosure statements is scarce. PL 114-216 states that GM foods shall not be considered more or less safe than their non-bioengineered counterparts, nevertheless it would benefit regulators and food manufacturers to be aware of the possible effects such disclosures might have on consumers. In a nationwide survey, multiple disclosure statements with varying degrees of public familiarity were compared to evaluate consumer perceptions and attitudes associated with each statement. Average consumer knowledge level of GM processes was also measured. The statements were then paired with actual food items to determine whether specific product categories influenced consumer responses. A select few of these statements and foods were included in a taste panel, allowing researchers to analyze if disclosure statements affected a consumer's sensorial experience. Results suggested that consumers were most favorable towards statements indicating the absence of GM-material, however they also responded less negatively towards new disclosure statements that do not have negative connotations. Additionally, consumers may react differently depending on the food accompanying a particular disclosure, although the taste panel data found no evidence that statements affected actual eating experience. Importantly, data from both surveys and taste panel suggested a disclosure statement may affect consumer willingness to buy a product.

As economies develop, novel products are created and markets for these products arise. Genetically modified food (GMF) is an example of such a novel product and provides economists with the opportunity to investigate an infant market. Of particular interest with GMF is the impact of consumer reactions on the market. The response of consumers to GMF and their willingness to pay for it has emerged as an important factor in the development of this technology. This research investigates these consumer responses. Prior research suggests that two aspects of consumer behaviour may be relevant for the GMF market. First, consumers may react differently to different types of GMF, so that some products are potentially more economically viable. Secondly, some consumers appear to prefer not having GMF at all. Consumer behaviour is often framed according to neoclassical economic theory. Consumer preferences over goods and the attributes of those goods are generally held to have certain properties. The aspects of consumers' reactions to GMF noted above, however, may be in conflict with two properties of preferences in neoclassical theory. First, preferences over food attributes are not separable, but may interact with each other. Secondly, some consumers may have preferences regarding GMF that are not continuous. As a result, aggregate impacts of introducing GMF may be difficult to measure, which raises a third issue for investigation, aggregation. Finally, an alternative model of consumer behaviour is bounded rationality, which theorises that choices may be discontinuous as a result of specific protocols. It also suggests that consumers seek to make good-enough choices, rather than attempting to maximise their satisfaction. Thus, optimisation or maximisation is the fourth issue considered in this thesis. In order to investigate these properties of consumers' preferences, a choice experiment survey was developed. The strength of a choice experiment for examining these issues is its focus on the impact of each product attribute on a respondent's choices. Thus, it may be possible to identify potentially discontinuous choice patterns and to identify choices affected by interactions between GM technology and other food attributes. Results from a neoclassical analysis of the survey data suggest that some consumers consider the type of benefit created with GM technology in making their choices. In addition, one-quarter to one-half of respondents may have had discontinuous preferences with respect to GMF. Reactions to GMF appear related to respondents' attitudes, but not to socio-economic or demographic descriptors. As a result, aggregate measures of the impact of GMF may not fully account for consumers' responses. A boundedly rational model also has reasonable goodness of fit, and may provide a different perspective on consumer behaviour. It is hoped that the results of this research provide a better understanding of consumer behaviour regarding GMF and, by extension, of the process of consumer adoption of novel products. It is further hoped that this attempt to incorporate choice protocols into discrete choice analysis will provide a useful example for further research.

No description available.

<p>This study examines the possible incompatibility between environmental conventions and agreements promoting international trade. More specifically, it aims at discussing the Precautionary Principle and the WTO agreements on trade and how they might contradict each other on the international arena. The study focuses on the international trade with genetically modified organisms and discusses probable needs of reformulating the principles and/or conventions concerned if these are to function as tools of equal importance when handling environmental problems through policy making. The controversy is illustrated through a case-study of a trade dispute where the conflicting principles: the Precautionary Principle and WTO agreements on trade are involved, namely a dispute where a formal complaint to the WTO by the US, Canada and Argentina about the European Community supposedly applying a de facto moratorium on genetically modified organisms had the final outcome of the European Community being found to contravene international trade rules. The study concludes that actors on the international arena are still opinions apart concerning the way to perceive possible risks connected to biotechnology and genetically modified organisms. This incongruence is also found to apply to the status of the Precautionary Principle as a rule of international law as well as to which of the conventions that were applicable to the concerned dispute.</p>