by Fong Man Kim. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 127-137). / Abstracts in English and Chinese. / Thesis committee --- p.i / Acknowledgements --- p.ii / Abstract --- p.iii / List of figures --- p.viii / List of tables --- p.xi / Abbreviations --- p.xii / Table of contents --- p.xiv / Chapter CHAPTER 1 --- GENERAL INTRODUCTION --- p.1 / Chapter CHAPTER 2 --- LITERATURE REVIEW --- p.3 / Chapter 2.1 --- MIH from Penaeus vannamei --- p.3 / Chapter 2.1.1 --- General Introduction to P. vannamei --- p.3 / Chapter 2.1.1.1 --- Morphology --- p.3 / Chapter 2.1.1.2 --- Geographical distribution --- p.5 / Chapter 2.1.1.3 --- Economic value --- p.5 / Chapter 2.1.2 --- Physiology of Molting in Crustacean --- p.7 / Chapter 2.1.2.1 --- The molt cycle --- p.7 / Chapter 2.1.2.2 --- Physiological effects of ecdysone --- p.8 / Chapter 2.1.2.3 --- Regulation of the secretion of ecdysone --- p.9 / Chapter 2.1.2.4 --- Physiological effects of Molt-inhibiting hormone --- p.10 / Chapter 2.1.3 --- Cloning of MIH cDNA from P. vannamei --- p.14 / Chapter 2.1.3.1 --- Molecular identity of MIH --- p.14 / Chapter 2.1.3.2 --- Cloning of MIH cDNA --- p.15 / Chapter 2.1.3.3 --- Comparison of the cloned MIH-like cDNA with the CHH/MIH/VIH peptide family --- p.16 / Chapter 2.2 --- Plants as Bioreactors --- p.20 / Chapter 2.2.1 --- Principles & Techniques --- p.20 / Chapter 2.2.2 --- Advantages of plant bioreactors --- p.21 / Chapter 2.2.3 --- Tobacco expression system --- p.22 / Chapter 2.2.3.1 --- Tobacco as model plants --- p.22 / Chapter 2.2.3.2 --- Transformation methods --- p.23 / Chapter 2.2.4 --- Phaseolin --- p.26 / Chapter CHAPTER 3 --- EXPRESSION OF MIH IN TRANSGENIC TOBACCO --- p.28 / Chapter 3.1 --- Introduction --- p.28 / Chapter 3.2 --- Materials & Methods --- p.29 / Chapter 3.2.1 --- Chemicals --- p.29 / Chapter 3.2.2 --- Plant materials --- p.29 / Chapter 3.2.3 --- Bacterial strains and plasmid vectors --- p.30 / Chapter 3.2.4 --- Construction of chimeric genes - --- p.30 / Chapter 3.2.4.1 --- PCR amplification of MIH --- p.30 / Chapter 3.2.4.2 --- Cloning of PCR-amplified MIH into vector pET --- p.31 / Chapter 3.2.4.3 --- Cloning of MIH into vector pBK/Phas-sp and pTZ/Phas --- p.31 / Chapter 3.2.4.4 --- Cloning of MIH into binary vector pBI121 --- p.32 / Chapter 3.2.5 --- Transformation of Agrobacterium with pBI121/Phas-sp-MIH and pBI121 /Phas-MIH by electroporation --- p.39 / Chapter 3.2.6 --- Transformation of tobacco --- p.40 / Chapter 3.2.7 --- Selection of transgenic plants --- p.41 / Chapter 3.2.8 --- GUS assay --- p.42 / Chapter 3.2.9 --- Extraction of leaf genomic DNA --- p.43 / Chapter 3.2.10 --- Extraction of total RNA from developing seeds --- p.44 / Chapter 3.2.11 --- Synthesis of DIG-labeled DNA and RNA probes --- p.45 / Chapter 3.2.12 --- Southern blot analysis of genomic DNA --- p.47 / Chapter 3.2.13 --- Reverse transcriptase - polymerase chain reaction (RT-PCR) --- p.47 / Chapter 3.2.14 --- Northern blot analysis of total RNA --- p.48 / Chapter 3.2.15 --- Protein extraction and tricine-SDS-PAGE --- p.49 / Chapter 3.2.16 --- Purification of 6xHis-tag proteins --- p.50 / Chapter 3.2.17 --- Western blot analysis --- p.50 / Chapter 3.2.18 --- In vitro transcription & translation --- p.52 / Chapter 3.2.18.1 --- Construction of transcription vector containing the chimeric MIH gene --- p.52 / Chapter 3.2.18.2 --- In vitro transcription --- p.56 / Chapter 3.2.18.3 --- In vitro translation --- p.56 / Chapter 3.2.19 --- Particle bombardment --- p.57 / Chapter 3.2.19.1 --- Construction of MIH-GUSN fusion chimeric genes --- p.57 / Chapter 3.2.19.2 --- Conditions of particle bombardment --- p.63 / Chapter 3.2.20 --- Codon modification of MIH gene --- p.63 / Chapter 3.3 --- Results --- p.73 / Chapter 3.3.1 --- Construction of chimeric MIH genes --- p.73 / Chapter 3.3.2 --- "Tobacco transformation, selection and regeneration" --- p.73 / Chapter 3.3.3 --- Detection of GUS activity --- p.74 / Chapter 3.3.4 --- Southern blot analysis --- p.79 / Chapter 3.3.5 --- Detection of MIH transcript in transgenic tobacco --- p.83 / Chapter 3.3.5.1 --- RT-PCR --- p.83 / Chapter 3.3.5.2 --- Northern blot analysis --- p.86 / Chapter 3.3.6 --- Detection of MIH protein by Tricine-SDS-PAGE --- p.86 / Chapter 3.3.7 --- Detection of MIH protein by western blot analysis --- p.88 / Chapter 3.3.7.1 --- Western blot analysis using Anti-MIH antibody --- p.88 / Chapter 3.3.7.2 --- Western blot analysis using Anti-His antibody --- p.90 / Chapter 3.3.7.3 --- Western blot analysis using Anti-MIHA & Anti-MIHB antibodies --- p.90 / Chapter 3.3.8 --- Purification of 6xHis-tag proteins by Ni-NTA column --- p.94 / Chapter 3.3.8.1 --- Western blot analysis of proteins purified by Ni-NTA column --- p.97 / Chapter 3.3.9 --- In vitro transcription and translation --- p.100 / Chapter 3.3.9.1 --- In vitro transcription --- p.100 / Chapter 3.3.9.2 --- In vitro translation --- p.100 / Chapter 3.3.10 --- Particle bombardments --- p.103 / Chapter 3.3.10.1 --- Transient expression of MIH in soybean & tobacco leaves --- p.103 / Chapter CHAPTER 4 --- DISCUSSION --- p.107 / Chapter 4.1 --- Transient expression of MIH genes --- p.109 / Chapter 4.1.1 --- In vitro transcription and translation --- p.109 / Chapter 4.1.2 --- Particle bombardments --- p.220 / Chapter 4.2 --- Post-transcriptional gene silencing (PTGS) --- p.114 / Chapter 4.2.1 --- Post-transcriptional cis-inactivation --- p.114 / Chapter 4.2.2 --- Post-transcriptional trans-inactivation --- p.116 / Chapter 4.2.3 --- MIH gene and PTGS --- p.118 / Chapter 4.3 --- Codon usage --- p.119 / Chapter 4.3.1 --- Codon usage of MIH in plants --- p.120 / Chapter 4.3.2 --- Codon modification of MIH and further study on MIH expression in plants --- p.122 / Chapter 4.4 --- Post-translational protein degradation --- p.123 / Chapter 4.4.1 --- Construction of LRP-MIH fusion proteins --- p.123 / CONCLUSION --- p.125 / REFERENCES --- p.127

by Lee Juon Kiu. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 139-152). / Abstracts in English and Chinese. / Thesis committee --- p.i / Statement --- p.ii / Acknowledgements --- p.iii / Abstract --- p.v / Table of contents --- p.ix / List of figures --- p.xv / List of tables --- p.xvii / List of graphs --- p.xviii / List of abbreviations --- p.xix / Chapter Chapter 1: --- General Introduction --- p.1 / Chapter Chapter 2: --- Literature Review --- p.4 / Chapter 2.1 --- Human granulocyte colony-stimulating factor (hG-CSF) --- p.4 / Chapter 2.1.1 --- Physiological roles --- p.4 / Chapter 2.1.2 --- Molecular properties --- p.8 / Chapter 2.1.3 --- Biochemical properties --- p.9 / Chapter 2.1.4 --- Comparison to G-CSF of other specie --- p.10 / Chapter 2.1.5 --- Clinical application --- p.11 / Chapter 2.1.6 --- Economic value --- p.13 / Chapter 2.2 --- Expression systems producing recombinant hG-CSF --- p.15 / Chapter 2.2.1 --- Bacteria --- p.15 / Chapter 2.2.2 --- Yeasts --- p.17 / Chapter 2.2.3 --- Animal cell lines --- p.18 / Chapter 2.2.4 --- Transgenic animals --- p.19 / Chapter 2.2.5 --- Transgenic plants --- p.20 / Chapter 2.3 --- Plant as bioreactors --- p.21 / Chapter 2.3.1 --- Characteristics of using plant as bioreactors --- p.22 / Chapter 2.3.2 --- Transgenic plants producing hematopoietic growth factors --- p.24 / Chapter 2.3.2.1 --- Granulocyte-macrophage colony-stimulating factor (GM-CSF) --- p.24 / Chapter 2.3.2.2 --- Erythropoietin (Epo) --- p.26 / Chapter 2.3.3 --- Arabidopsis and tobacco as model plants --- p.27 / Chapter 2.3.3.1 --- Arabidopsis --- p.28 / Chapter 2.3.3.2 --- Tobacco --- p.28 / Chapter 2.3.4 --- Phaseolin and its regulatory sequences --- p.29 / Chapter 2.4 --- Plant transformation methods --- p.31 / Chapter 2.4.1 --- Agrobacterium-mediated transformation --- p.31 / Chapter 2.4.1.1 --- Tissue culture methods --- p.31 / Chapter 2.4.1.2 --- Non-tissue culture (In planta) methods --- p.32 / Chapter 2.4.2 --- Direct DNA uptake transformation --- p.33 / Chapter 2.4.2.1 --- Chemical methods --- p.33 / Chapter 2.4.2.2 --- Electrical methods --- p.34 / Chapter 2.4.2.3 --- Physical methods --- p.34 / Chapter Chapter 3: --- Materials and Methods --- p.36 / Chapter 3.1 --- Introduction --- p.36 / Chapter 3.2 --- Chemicals --- p.37 / Chapter 3.3 --- Bacterial strains --- p.37 / Chapter 3.4 --- Chimeric gene construction --- p.37 / Chapter 3.4.1 --- Cloning of pTZ/Phas/His/EK/hG-CSF --- p.41 / Chapter 3.4.2 --- Cloning of pBK/Phas/SP/His/EK/hG-CSF --- p.44 / Chapter 3.4.3 --- Cloning of pBK/Phas/SP/hG-CSF --- p.47 / Chapter 3.4.4 --- Confirmation of sequence fidelity of chimeric genes --- p.50 / Chapter 3.4.5 --- Cloning of chimeric genes into Agrobacterium binary vector --- p.51 / Chapter 3.5 --- Expression in Arabidopsis --- p.52 / Chapter 3.5.1 --- Agrobacterium GV3101/pMP90 transformation --- p.52 / Chapter 3.5.2 --- Arabidopsis transformation --- p.53 / Chapter 3.5.2.1 --- Plant materials --- p.53 / Chapter 3.5.2.2 --- Vacuum infiltration --- p.54 / Chapter 3.5.3 --- Screening of successful R1 transformants --- p.55 / Chapter 3.5.4 --- Screening of hemizygous and homozygous transgenic Arabidopsis --- p.56 / Chapter 3.5.5 --- GUS assay --- p.57 / Chapter 3.5.6 --- Genomic DNA extraction --- p.57 / Chapter 3.5.7 --- Southern blot analysis --- p.58 / Chapter 3.5.8 --- Total RNA extraction from developing siliques --- p.59 / Chapter 3.5.9 --- Northern blot analysis --- p.60 / Chapter 3.5.10 --- Protein extraction and Tricine SDS-PAGE --- p.61 / Chapter 3.5.11 --- Western blot analysis --- p.62 / Chapter 3.5.12 --- Functional analysis --- p.63 / Chapter 3.5.12.1 --- Culture ofNFS-60 cells --- p.64 / Chapter 3.5.12.2 --- MTT assay --- p.65 / Chapter 3.6 --- Expression in tobacco --- p.67 / Chapter 3.6.1 --- Agrobacterium LBA4404/pAL4404 transformation --- p.67 / Chapter 3.6.2 --- Tobacco transformation --- p.68 / Chapter 3.6.2.1 --- Plant materials --- p.68 / Chapter 3.6.2.2 --- Tobacco transformation using leaf-disc technique --- p.68 / Chapter 3.6.3 --- Regeneration of transgenic tobacco --- p.69 / Chapter 3.6.4 --- GUS assay --- p.70 / Chapter 3.6.5 --- Genomic DNA extraction --- p.70 / Chapter 3.6.6 --- Southern blot analysis --- p.70 / Chapter 3.6.7 --- Total RNA extraction from immature seeds --- p.70 / Chapter 3.6.8 --- Northern blot analysis --- p.71 / Chapter 3.6.9 --- Protein extraction and Tricine SDS-PAGE --- p.71 / Chapter 3.6.10 --- Western blot analysis --- p.71 / Chapter 3.6.11 --- Functional analysis --- p.71 / Chapter 3.6.11.1 --- Culture of NFS-60 cells --- p.72 / Chapter 3.6.11.2 --- MTT assay --- p.72 / Chapter Chapter 4: --- Results --- p.73 / Chapter 4.1 --- Chimeric gene construction --- p.73 / Chapter 4.1.1 --- Cloning of pTZ/Phas/His/EK/hG-CSF --- p.73 / Chapter 4.1.2 --- Cloning of pBK/Phas/SP/His/EK/hG-CSF --- p.75 / Chapter 4.1.3 --- Cloning of pBK/Phas/SP/hG-CSF --- p.77 / Chapter 4.1.4 --- Cloning of chimeric genes into Agrobacterium binary vector --- p.79 / Chapter 4.2 --- Expression in Arabidopsis --- p.81 / Chapter 4.2.1 --- Agrobacterium GV3101/pMP90 transformation --- p.81 / Chapter 4.2.2 --- Arabidopsis transformation and screening of R1 transformants --- p.83 / Chapter 4.2.3 --- Screening of hemizygous transgenic R1 Arabidopsis --- p.84 / Chapter 4.2.4 --- Screening of homozygous transgenic R2 Arabidopsis --- p.86 / Chapter 4.2.5 --- GUS assay --- p.88 / Chapter 4.2.6 --- Genomic DNA extraction --- p.89 / Chapter 4.2.7 --- Southern blot analysis --- p.91 / Chapter 4.2.8 --- Total RNA extraction from developing siliques --- p.93 / Chapter 4.2.9 --- Northern blot analysis --- p.94 / Chapter 4.2.10 --- Protein extraction and Tricine SDS-PAGE --- p.96 / Chapter 4.2.11 --- Western blot analysis --- p.99 / Chapter 4.2.12 --- Functional analysis --- p.103 / Chapter 4.3 --- Expression in tobacco --- p.108 / Chapter 4.3.1 --- Agrobacterium LBA4404/pAL4404 transformation --- p.108 / Chapter 4.3.2 --- Tobacco transformation and regeneration of transformants --- p.109 / Chapter 4.3.3 --- GUS assay --- p.111 / Chapter 4.3.4 --- Genomic DNA extraction --- p.112 / Chapter 4.3.5 --- Southern blot analysis --- p.114 / Chapter 4.3.6 --- Total RNA extraction from immature seeds --- p.116 / Chapter 4.3.7 --- Northern blot analysis --- p.116 / Chapter 4.3.8 --- Protein extraction and Tricine SDS-PAGE --- p.118 / Chapter 4.3.9 --- Western blot analysis --- p.120 / Chapter 4.3.10 --- Functional analysis --- p.123 / Chapter Chapter 5: --- Discussion --- p.126 / Chapter 5.1 --- Introduction --- p.126 / Chapter 5.2 --- Successful in producing biologically active rhG-CSF from transgenic plants --- p.128 / Chapter 5.2.1 --- Production level --- p.129 / Chapter 5.2.2 --- O-glycosylation --- p.130 / Chapter 5.2.3 --- Phaseolin signal peptide --- p.131 / Chapter 5.2.4 --- Functional analysis --- p.131 / Chapter 5.3 --- Comparison of the productivity of other expression systems producing rhG-CSF --- p.132 / Chapter 5.4 --- Comparison of the productivity of plants producing different human proteins --- p.135 / Chapter 5.5 --- Future perspectives --- p.137 / Chapter Chapter 6: --- Conclusion --- p.138 / References --- p.139

Notre thèse est une contribution pour repenser les rapports entre sciences et éthiques, et avancer vers une démocratie épistémique. Qu'il s'agisse de démontrer l'insoutenabilité d'une science contre l'Homme ou d'identifier les conditions d'une remontée de l'Homme dans les sciences, la visée nous semble la même : il s'agit de réunir-sans-unifier ce qui, dans la science, est de l'ordre de l'épistémique, du technique et de l'éthique. Pour ce faire, il nous faut préalablement travailler en profondeur sur deux espaces - épistémologique et éthique -, et ceci sans d'abord les mélanger ou les recouvrir l'un sur l'autre. Car si les sciences nous sont effectivement données dans leurs mélanges (avec le technique, le politique, l'économique, le social ou le philosophique), rendant à la mode les thèmes de technoscience, de nouveau régime de production des savoirs ou encore de science post-normale, il ne s'agit pas pour nous d'un symptôme de la fin de l'épistémologie mais de la nécessité de son renouvellement. Celui-ci passera, et il s'agit là de notre thèse principale, par de nouveaux rapports avec l'éthique. Nous donnons à cette thèse le nom d'intégrité épistémique et éthique des sciences. Afin de définir les conditions et la portée de celle-ci, nous proposons deux hypothèses, respectivement au sein de l'espace épistémologique et éthique : celle d'un pluralisme épistémique ordonné et celle d'une éthique générique. Nous défendons ces hypothèses à la lumière d'un long travail d'instruction d'un objet des sciences et techniques contemporaines, le poisson génétiquement modifié. In fine, notre travail permet de ré-interroger les postulats classiques de l'évaluation et de proposer de nouvelles pistes de recherches. / The current crisis of the concept of science invites us to renew the links between epistemology and ethics. In this context, we make the assumption of epistemic and ethics integrity of science. To defend this thesis we advance two main assumptions : (i) that of an epistemic pluralism : in this regard, we suggest five hypotheses : pluralism as epistemic posture, pluralism as a non-epistemological description of science, pluralism as a form of common sense, pluralism as a new thought of the uncertainty and pluralism as a indisciplinaire approach. (ii) that of a generic ethics : to do this, we proceed in three levels : in the space of ethics, the mode of action and scope of ethics in science. With the support of this work in the areas epistemologies and ethical, the conditions for epistemic and ethics integrity of science are, in our opinion, the following : a pluralistic attitude, a democracy epistemic and an thinking of integrative objects. We give the characteristics of these conditions, then we put them in perspectives with the specific case of GM fish.

La progression tumorale est un processus multi-étapes dépendant notamment des interactions entre les cellules cancéreuses et le stroma environnant. Le développement du cancer du sein implique une communication étroite entre les cellules épithéliales mammaires, les cellules inflammatoires, les myofibroblastes et les cellules endothéliales. Ainsi, le microenvironnement tumoral apparaît comme une cible de choix dans le traitement anti-tumoral. L’utilisation de modèles précliniques est une étape clé dans le développement et la validation de nouvelles thérapies. Néanmoins, peu d’études sont disponibles sur le rôle du stroma péri-tumoral dans ces modèles.Dans le but d’étudier le stroma péri-tumoral des modèles précliniques de cancers du sein, nous avons combiné une analyse par cytométrie en flux à une analyse par immunohistochimie afin d’identifier, puis de quantifier, les différentes populations stromales hématopoïétiques (lymphocytes, monocytes/macrophages, polynucléaires) et non hématopoïétiques (myofibroblastes, cellules endothéliales). Vingt et un modèles de xénogreffe de tumeurs humaines de cancers du sein ainsi que 2 modèles transgéniques (MMTV-PyMT et MMTV-ErbB2), ainsi que leurs allogreffes respectives, furent utilisés lors de ce travail.Les analyses des tumeurs humaines et murines ont montré un infiltrat stromal très hétérogène d’une tumeur à l’autre, avec pour composante majoritaire les macrophages. Un infiltrat important en polynucléaires a également été détecté dans les modèles de PDX, caractéristique d’une inflammation locale importante dans ces modèles. L’analyse phénotypique de macrophages a montré une expression variable de marqueurs M1 et M2 dans les modèles de PDX. Les macrophages issus de tumeurs murines transgéniques, spontanées ou allogreffées, présentaient quant à eux un profil majoritairement M1. L’étude transcriptomique de macrophages triés, a permis à la fois de valider les résultats obtenus au niveau protéique mais a également mis en évidence des différences majeures dans l’expression de nombreux gènes, impliqués dans des voies de signalisation variées telles que la croissance tumorale, l’invasion et la métastase.Cette étude nous a permis de mettre en évidence le rôle de la tumeur sur son microenvironnement. En effet, celle-ci est à la fois capable d’attirer un panel de cellules stromales qui lui et propre et ensuite de l’activer de façon spécifique. / Tumor development is a multi-step process influencing by interactions between tumor cells and surrounding stroma. Breast cancer development involves a high level of communication between mammary epithelial cells, inflammatory cells, myofibroblasts and endothelial cells. So, the tumoral microenvironment appears as a prime target for anti-tumoral treatment. The use of preclinical models is a critical step in development and validation processes of new therapies. Nevertheless, the role of stroma in these models is poorly understood.In order to evaluate stromal cell populations in breast cancer preclinical models, we combined flow cytometry analysis and immunohistochemistry to identify, and then quantify, various stromal populations as hematopoietic cells (lymphocytes, monocytes/macrophages, polymorphonuclear leukocytes) and non-hematopoietic cells (myofibroblasts, endothelial cells). Twenty-one breast cancer patient-derived xenografts as well as 2 transgenic mouse models (MMTV-PyMT and MMTV-ErbB2), and their respective allografts, were studied.Analysis of human and murine tumors showed a strong heterogeneity between tumors regarding infiltrating stroma-cells, with a high proportion of macrophages. A significant amount of polymorphonuclear leukocytes was also detected in PDXs, indicating a local inflammation in these models. The phenotypic analysis of macrophages showed a variable expression of M1 and M2 markers in PDXs. Macrophages infiltrating transgenic mouse tumors, spontaneous or allografted, were mainly M1. Transcriptomic analyses of sorted macrophages, allowed us to validate previous results but also highlighted major differences in the expression of numerous genes implicated in various pathways as tumor growth, invasion and metastasis.Finally, this study highlighted the impact of tumor cells on their surrounding stroma. Indeed, we demonstrate that cancer cells are able to attract a specific panel of stromal cells and activate them in a specific way.

A soja geneticamente modificada tolerante ao herbicida glifosato tem sido a cultura derivada da engenharia genética mais cultivada atualmente no mundo. Como todo alimento GM a soja tem sido alvo de investigação em relação a sua Biossegurança. Novas estratégias têm sido desenvolvidas e aplicadas neste campo de pesquisa, sendo que métodos rápidos e eficientes de análise proteômica têm sido utilizados para avaliação e monitoramento da segurança e inocuidade alimentar, indicando mudanças no perfil protéico entre variedades convencionais e GM. O objetivo do presente trabalho foi avaliar os mapas protéicos de amostras de soja convencionais e suas derivadas geneticamente modificadas tolerantes ao herbicida glifosato, utilizando técnicas de análise proteômica com ênfase para inocuidade alimentar. Foram utilizadas seis amostras de soja, sendo três convencionais parentais e três derivadas GM, cultivadas entre 2004-2005, em Goiás. O extrato bruto protéico foi submetido à análise por eletroforese unidimensional e bidimensional. A eletroforese 2D, foi realizada utilizando tiras com gradiente de pH de 3-10 e 4-7. As imagens dos mapas protéicos das seis variedades, produzidas em replicatas, foram analisadas pelo software ImageMaster 2D Platinum. O potencial alergênico do extrato protéico bruto foi avaliado para todas as variedades utilizando soro de pacientes alérgicos à soja através de immunoblotting. Nos resultados obtidos observou-se a presença das principais frações protéicas da soja pela eletroforese unidimensional sem alteração significativa entre as amostras parentais e GM, exceto para uma banda de 115 kDa presente nas amostras parentais, mas ausente nas amostras GM. A partir da análise por eletroforese 2D foram identificadas as formas peptídicas correspondentes às frações de β-conglicinina e glicinina bem como diversas outras proteínas encontradas na soja como o inibidor de tripsina e a lipoxigenase. Através do software foi possível observar que um spot apresentou diferença estatística entre as amostras analisadas, expresso em maior concentração nas amostras GM do que nas parentais. Nos testes de alergenicidade, os extratos protéicos das variedades GM demonstraram reatividade similar em relação as suas respectivas variedades parentais. A proteína de 115 kDa foi sequenciada e identificada como a proteína precursora da cadeia α da β-conglicinina e o spot das amostras GM que apresentou diferença estatística significativa foi identificado como a proteína precursora de G4 glicinina. A diferença observada entre as variedades parentais e GM para as subunidades α de β-conglicinina e G4 glicinina pode ter ocorrido devido a variações normais observadas entre diferentes variedades de soja. Os resultados demonstram a viabilidade de aplicação das ferramentas proteômicas na identificação de alterações de perfis protéicos de amostras de soja parentais e GM. Pelos dados obtidos podemos concluir que as diferenças apresentadas não comprometem a inocuidade alimentar das amostras de soja GM em relação a suas respectivas variedades parentais. / Genetically modified soya-tolerant to the herbicide glyphosate culture has been derived from the more cultivated genetic engineering in the world today. As GM soya beans whole food has been investigated in relation to your biosafety. New strategies have been developed and applied research in this field, and fast and efficient methods of analysis proteomics have been used for assessment and monitoring of food security and safety, indicating changes in own protein profile between conventional and GM varieties. The aim of this work was to assess the maps soy protein samples of conventional and genetically modified their derived to the herbicide glyphosate-tolerant, using Proteomics analysis techniques with emphasis on food safety. Six samples were used for conventional soya, three and three derived from GM parental, grown between 2004-2005. The crude protein extract own was subjected to analysis by electrophoresis one-dimensional and two-dimensional. 2D electrophoresis using Strip was held with pH gradient of 3-10 and 4-7. Protein maps images of six varieties produced in replicates have been analysed by the 2D Platinum software ImageMaster. The potential allergenic in crude protein extracts was evaluated for all varieties using allergic patient serum soya by immunoblotting. In the results obtained noted the presence of the main protein fractions of soya by one-dimensional electrophoresis without significant change between parental and GM samples, except for a band of 115 parental kDa present in the sample, but absent in GM samples. From the analysis by 2D electrophoresis peptides forms were identified corresponding to fractions of β-conglicinina and glicinina as well as several other proteins found in soy as trypsin inhibitor and lipoxygenase. Through the software has been possible to observe that a spot presented statistical difference between the samples tested, expressed in greater concentration in the samples GM in parenting. In tests of allergenicity, GM varieties protein extracts showed similar reactivity in respect of their parental varieties. 115 KDa protein was sequenced and identified as the protein precursor of α subunit of β-conglicinina and the spot that GM samples presented significant statistical difference was identified as the G4 glicinina protein precursor. The difference between parental and GM varieties for subunits α of β-conglicinina and G4 glicinina may have occurred due to normal variation between different varieties of soy. The results demonstrate the viability of applying the tools Proteomics in identification of protein profiles changes of soya samples parental and GM. By data obtained can be concluded that the differences do not compromise the safety of food GM soybean samples with regard to their parental varieties.

O objetivo foi avaliar níveis de nitrogênio (N), fósforo (P) e potássio (K) em milho (Zea mays L.) com alto potencial genético para produtividade, sendo convencional e geneticamente modificado, visando recomendações de manejo da nutrição sob os pontos de vista técnico e econômico. Foram conduzidos seis experimentos, estudando cinco níveis de fornecimento dos nutrientes N, P e K, sendo T1= omissão completa do N; T2= 50 kg ha-1 de N; T3= 100 kg ha-1 de N; T4= 150 kg ha-1 de N; T5= 200 kg ha-1de N; T6= omissão completa do P; T7= 40 kg ha-1 de P2O5; T8= 80 kg ha-1 de P2O5; T9= 120 kg ha-1 de P2O5; T10= 160 kg ha-1 de P2O5; T11= omissão completa do K; T12= 50 kg ha-1 de K2O; T13= 100 kg ha-1 de K2O; T14= 150 kg ha-1 de K2O; T15= 200 kg ha-1 de K2O, e dois híbridos, o DKB390 convencional e o DKB390PRO, totalizando 20 unidades experimentais para cada ensaio. Avaliou-se índice de coloração verde com clorofilometro no estádio R1, altura de planta, rendimento de grãos, teor foliar de N, P e K no estádio R1 e teor de P e K no solo em pós-colheita em 3 profundidades (0-10 cm, 10-20 cm e 20-40 cm). Também foi determinado os custos de produção relacionando o híbrido convencional e o geneticamente modificado. Para o híbrido convencional DKB390 a aplicação de N provocou aumentos lineares do teor de N na folha, implicando em resposta quadrática para o rendimento de grãos atingindo produção máxima de 10718 kg ha-1 para dose de 135 kg de N ha-1. Para os níveis aplicados de P, a resposta foi de forma linear crescente no solo, na folha e no rendimento de grãos para o intervalo avaliado (0 a 160 kg ha-1 de P2O5). Para o K, o rendimento expressou resposta linear crescente, por outro lado os níveis de K na folha e no solo apresentaram respostas quadrática. Para o híbrido geneticamente modificado DKB390PRO, os níveis de N aplicados provocaram resposta quadrática para o teor de N na folha com correlação positiva para o índice de coloração verde, refletindo sobre o rendimento de grãos, cuja a produção máxima foi de 11.082 kg ha-1 para a dose de 164 kg ha-1de N. Para o P, os níveis utilizados proporcionaram resposta linear crescente para o teor foliar de P, sendo que o rendimento de grãos teve resposta quadrática produzindo 9.791 kg ha-1 na dose de 95 kg ha-1de P2O5. No solo obteve-se resposta linear crescente para as profundidades 0-10 cm e 20-40 cm sendo quadrática para 10-20 cm. Para o K a resposta foi linear crescente na folha e quadrática no rendimento de grãos com produção máxima de 9.401 kg ha-1 na dose de 93 kg ha-1 K2O. Para o solo, as respostas alcançadas tiveram comportamento quadrático para as profundidades de 0-10 cm e 10-20 cm sendo linear crescente na de 20-40cm. Para produtividades maiores que 10.041kg ha-1, o custo esperado de DKB390PRO foi menor, ou seja, o produtor tem que ter o compromisso de atingir elevadas produtividades quando se utiliza híbridos de alta tecnologia. / The goal of this research was to analyses levels of nitrogen, phosphorus and potassium in corn (Zea mays L.) for conventional and genetically modified productivity, with the goal providing recommendation for nutrients applications from a technical and economic perspective. Six experiments were conducted: two hybrids were used, one conventional, DKB390, and one genetically modified, DKB390PRO. Each hybrid was tested for 3 different nutrients, N, P, and K. Five application rates were tested for each nutrient: T1= complete omission of the nutrient N; T2= 50 kg ha-1 of N; T3= 100 kg ha-1 of N; T4= 150 kg ha-1 of N; T5= 200 kg ha-1 of N; T6= complete omission of the nutrient P; T7= 40 kg ha-1 of P2O5; T8= 80 kg ha-1 of P2O5; T9= 120 kg ha-1 of P2O5; T10= 160 kg ha-1 of P2O5; T11= complete omission of the nutrient K; T12= 50 kg ha-1 of K2O; T13= 100 kg ha-1 of K2O; T14= 150 kg ha-1 of K2O; T15= 200 kg ha-1 of K2O, Green coloring index with SPAD in R1 stage, plant height, grain yield, N, P and K amount in leaf in R1 stage, and P and K amount in soil post harvest in three depths 0-10 cm, 10-20 cm and 20-40 cm were evaluated. Production costs comparing conventional and genetically modified hybrids were also studied. Nitrogen application for the conventional DKB390 caused a linear increase in the amount of N in the leaves, resulting in a quadratic response for grain yield reaching maximum production of 10718 kg ha-1 for an application of 135 kg ha-1. The response to P levels used was an increasing linear relationship in the leaves as well as ingrain yield for the interval studied (0 to 160 kg ha-1 of P2O5). K applications produced an increasing linear response for yield and a quadratic response in the leaves and soil. N application used produced a quadratic response in the leaves for the genetically modified hybrid, DKB390PROwith positive correlation for the green coloring index, reflected in grain yield, whose maximum production was 11082 kg ha-1 for the application rate of 164 kg ha-1 of N. The rates of P applied induced a linear response in P concentrations in the leaves, while grain yield had a quadratic response producing 9791 kg ha-1 for the application rate of 95 kg ha-1 of P2O5 and in soil the response was increasingly linear for the depths 0-10 and 20-40 cm and quadratic for 10-20 cm. Responses in the leaves were increasingly linear for K , quadratic in grain yield with maximum production of 9401 kg ha-1 in the application rate of 93 kg ha-1 K2O, and for soil the responses were quadratic for the depths of 0-10 and 10-20 cm being increasingly linear in the 20-40cm. The expected cost of DKB390PRO is less for yields greater than 10, 041 kg ha-1, and producers should be committed to these higher yields when using high technology hybrids.

Cheung Chun Kai. / Thesis submitted in: December 2003. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 133-146). / Abstracts in English and Chinese. / Acknowledgements --- p.ii / Abstract --- p.iv / 摘要 --- p.vii / Table of Contents --- p.ix / List of Tables --- p.xv / List of Figures --- p.xvi / List of Abbreviations --- p.xxi / Chapter Chapter 1 --- Overview --- p.1 / Chapter Chapter 2 --- Literature Review --- p.3 / Chapter 2.1 --- Historical background --- p.3 / Chapter 2.2 --- Insulin-like growth factor --- p.5 / Chapter 2.2.1 --- Structure and synthesis --- p.5 / Chapter 2.2.2 --- Physiologic role and biological actions --- p.6 / Chapter 2.3 --- Insulin-like growth factor binding protein-3 --- p.8 / Chapter 2.3.1 --- Structure and synthesis --- p.8 / Chapter 2.3.2 --- Physiologic role and biological actions --- p.8 / Chapter 2.4 --- Clinical aspects --- p.10 / Chapter 2.4.1 --- Metabolic effects of IGF-1 --- p.10 / Chapter 2.4.1.1 --- Similarities between IGF-I and insulin --- p.11 / Chapter 2.4.1.2 --- Differences between IGF-I and insulin --- p.13 / Chapter 2.4.2 --- Glucose and protein metabolism --- p.14 / Chapter 2.4.3 --- Therapeutic use of IGF-I --- p.15 / Chapter 2.4.3.1 --- Type 1 diabetes mellitus --- p.16 / Chapter 2.4.3.2 --- Type 2 diabetes mellitus --- p.17 / Chapter 2.4.4 --- Side effects --- p.19 / Chapter 2.5 --- World demands --- p.21 / Chapter 2.5.1 --- Significance of large-scale production --- p.21 / Chapter 2.5.2 --- IGF-I production --- p.21 / Chapter 2.6 --- Plants as bioreactors --- p.24 / Chapter 2.6.1 --- Medical molecular farming --- p.24 / Chapter 2.6.2 --- Advantages of plant bioreactor --- p.24 / Chapter 2.6.3 --- Commercial biopharmaceutical protein --- p.25 / Chapter 2.7 --- Tobacco expression system --- p.26 / Chapter 2.7.1 --- Tobacco model plant --- p.26 / Chapter 2.7.2 --- Transformation methods --- p.26 / Chapter 2.8 --- Hypotheses and aims of study --- p.28 / Chapter Chapter 3 --- Expression of Human IGF-I and IGFBP-3 in Transgenic Tobacco --- p.30 / Chapter 3.1 --- Introduction --- p.30 / Chapter 3.2 --- Materials and methods --- p.31 / Chapter 3.2.1 --- Chemicals --- p.31 / Chapter 3.2.2 --- Plant materials --- p.31 / Chapter 3.2.3 --- Bacterial strains --- p.32 / Chapter 3.2.4 --- Codon modification of IGF-I and IGFBP-3 cDNAs --- p.32 / Chapter 3.2.5 --- Transient assay to study IGF-I or IGFBP-3 translatability --- p.39 / Chapter 3.2.5.1 --- Construction of chimeric genes for particle bombardment --- p.39 / Chapter 3.2.5.2 --- Particle bombardment of GUS fusion constructs --- p.42 / Chapter 3.2.6 --- Construction of chimeric genes for tobacco transformation --- p.44 / Chapter 3.2.6.1 --- Construction of chimeric genes with different promoters --- p.44 / Chapter 3.2.6.1.1 --- Construction of chimeric gene with CaMV 35S promoter --- p.44 / Chapter 3.2.6.1.2 --- Construction of chimeric genes with phaseolin promoter --- p.46 / Chapter 3.2.6.2 --- Construction of fusion constructs --- p.48 / Chapter 3.2.6.2.1 --- Construction of GUS fusion constructs --- p.48 / Chapter 3.2.6.2.2 --- Construction of LRP fusion constructs --- p.51 / Chapter 3.2.6.3 --- Construction of phaseolin targeting constructs --- p.56 / Chapter 3.2.6.3.1 --- Construction of phaseolin targeting constructs without AFVY --- p.56 / Chapter 3.2.6.3.2 --- Construction of phaseolin targeting constructs with AFVY --- p.60 / Chapter 3.2.6.4 --- Cloning of chimeric genes into Agrobacterium binary vector pBI 121 --- p.64 / Chapter 3.2.7 --- Confirmation of sequencing fidelity of chimeric genes --- p.66 / Chapter 3.2.8 --- Transformation of Agrobacterium by electroporation --- p.66 / Chapter 3.2.9 --- Transformation of tobacco --- p.67 / Chapter 3.2.10 --- Selection and regeneration of transgenic tobacco --- p.67 / Chapter 3.2.11 --- GUS assay --- p.68 / Chapter 3.2.12 --- Extraction of leaf genomic DNA --- p.68 / Chapter 3.2.13 --- PCR of genomic DNA --- p.69 / Chapter 3.2.14 --- Synthesis of DIG-labeled double-stranded DNA probe --- p.69 / Chapter 3.2.15 --- Southern blot analysis --- p.70 / Chapter 3.2.16 --- Extraction of total RNA from leaves or developing seeds --- p.70 / Chapter 3.2.17 --- Northern blot analysis --- p.71 / Chapter 3.2.18 --- Extraction of total protein --- p.71 / Chapter 3.2.19 --- Tricine SDS-PAGE --- p.72 / Chapter 3.2.20 --- Western blot analysis --- p.72 / Chapter 3.2.21 --- Enterokinase digestion of fusion protein --- p.73 / Chapter Chapter 4 --- Results --- p.74 / Chapter 4.1 --- Particle bombardment for transient assay --- p.74 / Chapter 4.1.1 --- Construction of GUS fusion genes for particle bombardment --- p.74 / Chapter 4.1.2 --- Transient expression of GUS fusion genes in soybean cotyledons and tobacco leaves --- p.76 / Chapter 4.2 --- Construction of chimeric genes for tobacco transformation --- p.78 / Chapter 4.3 --- "Tobacco transformation, selection and regeneration" --- p.81 / Chapter 4.4 --- Detection of GUS activity --- p.83 / Chapter 4.5 --- Detection of transgene integration --- p.84 / Chapter 4.5.1 --- Extraction of genomic DNA and PCR --- p.84 / Chapter 4.5.2 --- Southern blot analysis --- p.88 / Chapter 4.6 --- Detection of transgene transcription --- p.92 / Chapter 4.6.1 --- Extraction of total RNA --- p.92 / Chapter 4.6.2 --- Northern blot analysis --- p.92 / Chapter 4.7 --- Detection of transgene translation --- p.99 / Chapter 4.7.1 --- Extraction of total protein and Tricine SDS-PAGE --- p.99 / Chapter 4.7.2 --- Western blot analysis --- p.102 / Chapter 4.7.3 --- Enterokinase digestion of fusion protein --- p.109 / Chapter Chapter 5 --- Discussion --- p.111 / Chapter 5.1 --- Codon modification of IGF-I and IGFBP-3 cDNAs --- p.114 / Chapter 5.2 --- Transient expression of IGF-I and IGFBP-3 cDNAs --- p.116 / Chapter 5.3 --- Fusion of IGF-I and IGFBP-3 cDNA with LRP gene --- p.118 / Chapter 5.4 --- Enterokinase digestion --- p.120 / Chapter 5.5 --- Phaseolin targeting signal --- p.122 / Chapter 5.6 --- Gene silencing --- p.124 / Chapter 5.7 --- Future perspectives --- p.128 / Chapter Chapter 6 --- Conclusion --- p.131 / References --- p.133

by Ng Wing Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 156-174). / Abstracts in English and Chinese. / Acknowledgements --- p.iii / Abstract --- p.v / 摘要 --- p.vii / Table of Contents --- p.ix / List of Figures --- p.xiii / List of Tables --- p.xvi / List of Graphs --- p.xvii / List of Abbreviations --- p.xviii / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter Chapter 2 --- Literature Review --- p.3 / Chapter 2.1 --- Human granulocyte colony-stimulating factor (hG-CSF) --- p.3 / Chapter 2.1.1 --- Historical background --- p.3 / Chapter 2.1.2 --- Physiological Roles --- p.5 / Chapter 2.1.3 --- Molecular properties --- p.8 / Chapter 2.1.4 --- Biochemical properties --- p.9 / Chapter 2.1.5 --- Comparison to G-CSF of other species --- p.11 / Chapter 2.1.6 --- Biological Activities --- p.12 / Chapter 2.1.7 --- Clinical Applications --- p.14 / Chapter 2.1.7.1 --- Clinical use in myelosuppressive chemotherapy and neutropenic fever --- p.14 / Chapter 2.1.7.2 --- Clinical use in bone marrow transplantation (BMT) and peripheral blood progenitor cell (PBPC) transplantation --- p.14 / Chapter 2.1.7.3 --- Clinical use in HIV infection --- p.16 / Chapter 2.1.7.4 --- Clinical use in diabetes mellitus --- p.17 / Chapter 2.1.7.5 --- Clinical use in severe chronic neutropenia --- p.18 / Chapter 2.1.7.6 --- Future prospects --- p.18 / Chapter 2.1.7.7 --- Dosages and adverse effects --- p.19 / Chapter 2.1.8 --- Economic value --- p.20 / Chapter 2.2 --- Plant as bioractor --- p.20 / Chapter 2.2.1 --- Medical molecular farming --- p.20 / Chapter 2.2.2 --- Commercial biopharmaceutical proteins --- p.25 / Chapter 2.2.3 --- Transgenic plants producing hematopoietic growth factors --- p.25 / Chapter 2.2.3.1 --- Granulocyte-macrophage colony-stimulating factor (GM-CSF) --- p.26 / Chapter 2.2.3.2 --- Interleukin-2 (IL-2) --- p.28 / Chapter 2.3 --- Rice as expression system --- p.29 / Chapter 2.3.1 --- Characteristics --- p.29 / Chapter 2.3.2 --- Advantages of using rice as bioreactor --- p.30 / Chapter 2.3.3 --- Previous studies --- p.31 / Chapter 2.3.4 --- Transformation method --- p.33 / Chapter 2.3.5 --- Super-binary vector --- p.34 / Chapter 2.4 --- Strategies for enhancing protein expression level --- p.36 / Chapter 2.4.1 --- Vacuolar targeting --- p.36 / Chapter 2.4.1.1 --- Protein targeting signals --- p.38 / Chapter 2.4.1.2 --- Binding protein of 80kDa (BP-80) --- p.39 / Chapter 2.4.1.3 --- a-Tonoplast intrinsic protein (α-TIP) --- p.39 / Chapter 2.4.1.4 --- Receptor homology region-transmembrane domain-Ring H2 motif (RMR) --- p.40 / Chapter 2.4.2 --- Fusion with glutelin in rice --- p.41 / Chapter 2.5 --- Hypotheses and aims of this study --- p.43 / Chapter Chapter 3 --- Materials and Methods --- p.45 / Chapter 3.1 --- Introduction --- p.45 / Chapter 3.2 --- Chemicals --- p.45 / Chapter 3.3 --- Bacterial strains --- p.46 / Chapter 3.4 --- Chimeric genes construction --- p.46 / Chapter 3.4.1 --- Protein targeting constructs --- p.51 / Chapter 3.4.2 --- Enterokinase site constructs --- p.60 / Chapter 3.4.3 --- Glutein signal peptide constructs --- p.65 / Chapter 3.4.4 --- Glutelin fusion constructs --- p.70 / Chapter 3.4.5 --- Sequence fidelity of chimeric genes --- p.77 / Chapter 3.4.6 --- Cloning of chimeric genes into rice super-binary vector --- p.77 / Chapter 3.5 --- Rice transformation --- p.79 / Chapter 3.5.1 --- Plant materials --- p.79 / Chapter 3.5.2 --- Agrobacterium transformation --- p.79 / Chapter 3.5.3 --- A grobacterium-mediated transformation of rice --- p.79 / Chapter 3.6 --- Transgenic expression --- p.81 / Chapter 3.6.1 --- Extraction of leaf genomic DNA --- p.81 / Chapter 3.6.2 --- Synthesis of DIG-labeled double-stranded DNA probe --- p.82 / Chapter 3.6.3 --- Southern blot analysis --- p.83 / Chapter 3.6.4 --- Extraction of total RNA from immature rice seeds --- p.84 / Chapter 3.6.5 --- Northern blot analysis --- p.85 / Chapter 3.6.6 --- Protein extraction --- p.86 / Chapter 3.6.7 --- Tricine SDS-PAGE --- p.86 / Chapter 3.6.8 --- Western blot analysis --- p.87 / Chapter 3.6.9 --- Enterokinase digestion of EK fusion proteins --- p.88 / Chapter 3.7 --- Confocal immunoflorescence studies of rhG-CSF in rice grain --- p.89 / Chapter 3.7.1 --- Preparation of sample sections --- p.89 / Chapter 3.7.2 --- Double-labeling of fluorescence probes --- p.89 / Chapter 3.7.3 --- Image collection --- p.90 / Chapter 3.8 --- Functional analysis of rhG-CSF --- p.91 / Chapter 3.8.1 --- Culture of NFS-60 cells --- p.91 / Chapter 3.8.2 --- MTT cell proliferation assay --- p.92 / Chapter 3.9 --- Bacterial expression of anti-hG-CSF --- p.93 / Chapter 3.9.1 --- pET expression in E. coli --- p.93 / Chapter 3.9.2 --- Purification of His-hG-CSF --- p.97 / Chapter 3.9.3 --- Immunization of rabbits --- p.97 / Chapter Chapter 4 --- Results --- p.99 / Chapter 4.1 --- Construction of chimeric genes for rice transformation --- p.99 / Chapter 4.2 --- "Rice transformation, selection and regeneration" --- p.103 / Chapter 4.3 --- Southern blot analysis --- p.105 / Chapter 4.4 --- Northern blot analysis --- p.109 / Chapter 4.5 --- Western blot analysis --- p.114 / Chapter 4.6 --- Enterokinase digestion of EK fusion proteins --- p.125 / Chapter 4.7 --- Confocal immunofluorescence studies of rhG-CSF in transgenic rice grain --- p.128 / Chapter 4.8 --- Functional analysis of rhG-CSF --- p.132 / Chapter 4.9 --- Bacterial expression of anti-hG-CSF --- p.135 / Chapter 4.9.1 --- Expression and purification of recombinant His-hG-CSF in E. coli --- p.135 / Chapter 4.9.2 --- Titer and specificity of the anti-serum --- p.137 / Chapter Chapter 5 --- Discussion --- p.139 / Chapter 5.1 --- Introduction --- p.139 / Chapter 5.2 --- Fusion of hG-CSF with protein sorting determinants --- p.141 / Chapter 5.3 --- Fusion of hG-CSF with rice glutelin --- p.145 / Chapter 5.4 --- Glutelin signal peptide --- p.146 / Chapter 5.5 --- O-glycosylation --- p.148 / Chapter 5.6 --- Enterokinase digestion --- p.148 / Chapter 5.7 --- Expression level of rhG-CSF --- p.149 / Chapter 5.8 --- Functional analysis of rhG-CSF --- p.151 / Chapter 5.9 --- Future perspectives --- p.151 / Chapter Chapter 6 --- Conclusion --- p.155 / References --- p.156

Genetically modified organisms (GMOs) are confined physically in order to contain their spread in nature and to minimise chances of horizontal gene transfer. However, with the potential that GMOs hold as cheap, reliable and efficient micro-machines, their eventual uncontrolled release into the wider space is becoming more likely. Indeed, their application as environmental sensors is largely increasing. Nevertheless, the field of synthetic biology may also afford solutions to the problem. A major potential application of GMOs is the delivery of antigens to human and animal hosts, through the utilization of live, engineered microbes. Recombinant technology is promising for several reasons including their capacity to be less reactogenic, more potent, safer and genetically definable. Also, they have the potential to provide protection against multiple targets simultaneously, are relatively inexpensive and can be eradicated with antibiotics, as the need arises. Besides, delivery of vaccines to mucosal surfaces is more efficient. Mutant Salmonella expressing heterologous antigens have been shown to induce protection against a variety of pathogens. Nevertheless, limited containment systems are available that can be applicable for bacterial antigen carriers. This project aims to design safeguards for the bacterial antigen delivery systems that limit ORF translatability and self-inactivates/destructs upon exit from the host. In this work, double quadruplet codons were suppressed by orthogonal tRNAs, providing a barrier for gene translation in the recipient cells when antigen is horizontally transferred. Furthermore, three kill switches were designed that are activated by a decrease in temperature from 37 °C. First, Sau3AI endonuclease was activated by protein self-splicing at low temperature mediated by Mtu recA intein. The activation of the endonuclease led to three-fold logarithmic decrease in the number of viable cells within two hours of gene expression. Second, RNA-dependent activation of RNase 7 showed a reduction in the number of viable cells at low temperature of three logarithmic folds. RNase 7 was controlled by the cspA 5’UTR, which sequesters ribosome binding site at 37 °C and allows translation at low temperature. Third, CspA 5’UTR was shown to regulate expression of TEV protease at 37 °C and low temperature. This led to bacterial cellular inhibition within two hours of TEV induction and five-fold logarithmic reduction in the number of viable cells at low temperature. In addition, for the first time and contrary to previous studies, the TEV protease was shown to inhibit cellular growth. It was also shown that biofilm formation was drastically impaired by the TEV activity. The three killing switches and the quadruplet translation system are poised to function as robust safeguards for bacterial antigen delivery systems.

To date, few studies have focused on the effects of genetically modified trees (GM trees) on the environment. One concern with GM trees is that they may have unanticipated effects on non-target organisms, i.e. effects on organisms that are not direct targets of the genetically modified trait. The main objective of this thesis was to study potential non-target effects from the interaction between GM trees and natural enemies, including phytopathogens and herbivorous insects. To study this I used a system consisting of GM trees featuring changes in growth-related characteristics, and naturally occurring enemies. The GM trees used were the aspen hybrids Populus tremula x tremuloides: one unmodified wild type clone T89 (control) and transgenic lines with altered expression of gibberellin (GA 20-oxidase), sucrose (SPS) or pectin (PME); and Populus tremula x alba: one unmodified wild type clone INRA 717-1-B4 (control) and lines modified to suppress the activity of the enzymes in the lignin biosynthetic pathway, i.e. CAD, COMT, CCR or CCoAOMT. The natural enemies used were the parasitic phytopathogens Melampsora pinitorqua, M. populnea and Venturia tremulae, and the herbivorous leaf-beetle Phratora vitellinae. To address this question inoculation experiments, feeding preference experiments, analyses of secondary chemistry and field inventories were performed. The results of the studies showed that the GM trees significantly affected the interaction with the natural enemies, both in the laboratory as well as in the field. For instance, both M. pinitorqua and V. tremulae showed an altered disease incidence on the GM trees of P. tremula x tremuloides compared to the unmodified wild type T89, where all tested transgenic lines exhibited altered susceptibility to the pathogens. However, there were also differences in aggressiveness to the aspens depending on pathogen population. The results from the field inventory showed that lines within all tested transgenic construct, COMT, CAD, CCoAOMT and CCR of P. tremula x alba differed significantly from the wild type INRA 717-1-B4 in susceptibility to M. populnea. In addition, the susceptibility to the rust also differed significantly between lines carrying the same transgenic constructs. Furthermore, we found that overexpression of SPS in P. tremula x tremuloides, unintentionally induced changes in plant secondary chemistry, where the GM-line SPS33A exhibited the largest deviation from the wild type T89 in contents of plant phenolics and nitrogen, and that these changes coincide with a concurrent decrease in herbivory by P. vitellinae on this line. I argue that the altered interactions are the result of physiological changes in the trees. They can originate from direct effects i.e. altered expression of the modified trait, indirect effects of the genetic modification process e.g. pleiotropy, or effects from the transformation process e.g. position effects, to which the tested natural enemies respond. The result stresses the importance of further research on the causes and mechanisms responsible for the altered interaction between GM trees and non-target organisms, as well as evaluating the potential environmental effects of cultivation of GM trees in the field. Such research will require collaboration between researchers from different disciplines, such as plant ecology and physiology, functional genomics, proteomics and metabolomics.