Submitted by admin tede (tede@pucgoias.edu.br) on 2016-09-02T12:27:50Z No. of bitstreams: 1 CECY PEREIRA FIGUEIRA DA SILVA NETA MELLO.pdf: 1451626 bytes, checksum: 863ab02a5e133691994a8b778ef75e6e (MD5) / Made available in DSpace on 2016-09-02T12:27:50Z (GMT). No. of bitstreams: 1 CECY PEREIRA FIGUEIRA DA SILVA NETA MELLO.pdf: 1451626 bytes, checksum: 863ab02a5e133691994a8b778ef75e6e (MD5) Previous issue date: 2016-03-14 / With the biotechnology advancement and development, with special focus on genetic engineering, also rises the requirement of ethical fundamentals in a scenario of constant modifications. When it comes to transgenic food, there‟s controversy in the philosophical, ethical, environmental, political, legal and economic field because it‟s a subject which is inside of the daily reality, with very fast changes and in that context, the Brazilian and international legislation seems to doesn‟t follow the technology velocity and Science becomes a political and economical instrument. In this article, international law and national law related to the using of genetically modified organism are analyzed, pointing out the 11.105/05 law, considering the origin, shape and evolution of genetically modified organism, the importance of obeying the environmental law when handling and distributing products resulting of these genetic modifications to the public consumption, this law analysis, going through the environmental impacts, giving importance to the cost-benefit and the risk expressly taken by the scientific community, besides the own concept of consumer, proving the need of genetically modified food labelling and the reality of what really happens in the process of patenting acquisition in the international law and in the Brazilian law and jurisprudence decisions. / Com o avanço e o desenvolvimento da biotecnologia, com enfoque especial na engenharia genética, surge também a necessidade da inserção da ética e de seus princípios em um cenário de modificações constantes. No que tange aos alimentos transgênicos, polêmicas são levantadas no campo filosófico, ético, ambiental, político, social, jurídico e econômico, pois o tema está inserido em uma realidade do nosso dia a dia, com mudanças muito rápidas e diante deste contexto, as legislações brasileiras e internacionais parecem não acompanhar a velocidade da tecnologia e a ciência passa a ser instrumento da política e da economia. No presente trabalho são analisadas leis internacionais, legislações relativas à utilização dos organismos geneticamente modificados, ressaltando-se a Lei 11.105/05, considerando a origem, a forma e a evolução do organismo geneticamente modificado, a importância da obediência às leis de Direito Ambiental no manuseio e distribuição dos produtos oriundos destas modificações genéticas para o consumo da população, a análise desta legislação, passando pelos impactos ambientais, dando ênfase ao custo benefício e ao risco assumido expressamente pela comunidade científica, discorrendo ainda sobre o direito do consumidor, além do próprio conceito de consumidor, demonstrando também a necessidade da rotulagem dos alimentos geneticamente modificados e a forma como acontece o processo para obtenção de patentes tanto na legislação exterior quanto na legislação brasileira e as decisões jurisprudenciais.

O recente crescimento da produção de organismos geneticamente modificados (OGM) no mundo tem demandado novas políticas de controle de plantio e comercialização de produtos alimentícios produzidos com ingredientes GM. Vários aspectos influenciam a análise de detecção e quantificação de OGM em alimentos, e em última instância, o monitoramento e atendimento à legislação e rotulagem. Este estudo se propôs a avaliar três destes aspectos, através da metodologia de análise de PCR em tempo real: a degradação de DNA e a presença adventícia de culturas GM e não-GM, ambas decorrentes da produção e processamento dos grãos em matérias-primas e produtos para consumo, bem como os planos de amostragem existentes para coleta de material alimentício destinado às análises de OGMs. Resultados demonstraram que os processos de fabricação degradam o material genético em diferentes graus em algumas matrizes de alimentos, viabilizando ou não, a análise por PCR em tempo real. Na cadeia de manufatura de subprodutos de soja, milho, arroz e trigo, 45% das amostras apresentaram detecção para uma cultura diferente da principal, sendo 44% deste total, GM. A adoção de metodologias de análise que se restringem à detecção de poucos genes-alvo, ou aplicadas somente a amostras compostas de soja ou milho, já não são mais suficientes para o rastreamento e quantificação dos alimentos contendo matérias-primas geneticamente modificadas. O plano de amostragem proposto foi representativo e delineado sob medida para avaliação de bebida à base de soja produzida em escala industrial, porém mais matrizes necessitam ser testadas para uma avaliação global das estratégias de amostragem. / The recent increase in genetically modified organisms (GMO) production is requiring new control policies for cultivation and commercialization of food products containing GM ingredients. There are many factors that can influence detection and quantification of GMO ingredients in food products, and these can ultimately influence the monitoring, labeling and legislation observance. In this work, we intended to evaluate three of these factors, using real-time PCR analysis method: DNA degradation; adventitious presence of GM and non-GM cultures, both caused by grain production and raw materials and finished products processing; and the available sampling plans for the collection of food material for GM analysis. Results in some food matrices showed that the manufacturing processes can degrade the genetic material in different degrees, allowing or not, the real-time PCR analysis. Regarding the soya beans, maize, rice and wheat manufacturing chains, 45% of the samples presented positive detection for a secondary crop, of which 44% were GM. The adoption of analysis methodologies restricted to a few target-genes, or applied solely to samples composed by soya or maize is simply not enough for tracking and quantification of food containing GM raw material. The sampling plan was representative and fit-for-purpose for one tested soya-based beverage and produced in industrial scale, however, more lots and matrices need to be analyzed for a global evaluation of the sampling strategies.

O presente trabalho tem por objetivo analisar o potencial de conflito entre normas internacionais aplicáveis ao comércio transfronteiriço de alimentos transgênicos, tendo como foco as tensões entre o Protocolo de Cartagena sobre Segurança Biológica e os acordos da OMC relevantes (SPS, TBT, GATT-94 e TRIPs), assim como as fórmulas existentes para seu encaminhamento no âmbito desses acordos e da Convenção de Viena sobre Direito dos Tratados. A fim de demonstrar a complexidade do marco regulatório internacional aplicável a OGMs, são analisadas as determinações do painel da OMC na disputa relativa à moratória européia na aprovação de novos produtos biotecnológicos, com relação aos seguintes pontos: (a) aplicabilidade da Convenção sobre Diversidade Biológica, do Protocolo de Cartagena e do princípio da precaução na interpretação dos acordos abrangidos; (b) aplicação concorrente dos Acordos SPS e TBT à disputa; (c) aplicação de dispositivos excludentes do SPS. Da análise dessas determinações, pode-se depreender que o potencial de conflito entre normas internacionais aplicáveis a produtos GMs não se limita às tensões entre o Protocolo de Cartagena e os acordos da OMC, estando presente também entre esses acordos. A fim de ilustrar o paralelismo de desafios na frente multilateral e doméstica em relação à regulamentação de OGMs, parte do trabalho foi dedicada à análise de um conjunto de ações perante o Judiciário brasileiro envolvendo conflitos entre normas nacionais de distintos níveis hierárquicos aplicáveis a produtos transgênicos. Essas ações envolveram desde divergências quanto ao dispositivo constitucional aplicável para determinar a esfera de competência da União e dos Estados para legislar sobre distintos aspectos da regulamentação de OGMs até questionamentos à constitucionalidade e à legalidade de determinados diplomas legais infra-constitucionais. A crescente judicialização no Brasil dos conflitos de normas que disciplinam a liberação de produtos transgênicos ilustra as limitações do marco regulatório nacional, também presentes na frente multilateral, para acomodar satisfatoriamente os objetivos de proteção da biodiversidade e da saúde humana de um lado, e da livre comercialização de alimentos geneticamente modificados, de outro. / The main goal of this work is to analyse the potencial of conflict among international norms applicable to the transboundary trade of GMOs, taking into account the tensions between the Cartagena Protocol on Biosafety and the relevant WTO agreements (SPS, TBT, GATT-94 and TRIPs). An analysis of the conflict clauses in these agreements, as well as in the Vienna Convention on the Law of Treaties is also made. In order to demonstrate the complexity of the international GMO legal framework, this work examines the determinations of the WTO panel on the biotech case involving the delay of European regulatory authorities to approve new biotechnological products. In this analysis special attention is given to the following points: (a) applicability of the Convention on Biological Diversity, of the Cartagena Protocol and of the precautionary principle in the interpretation of the covered agreements; (b) simultaneous application of the SPS and the TBT to the dispute; (c) application of provisions of the SPS which are mutually exclusive. From the analysis of these determinations one can infer that the potential of conflict among international norms applicable to GMO products is not limited to existing tensions between the Protocol and WTO agreements, but is also present among the latter agreements. In order to demonstrate the coincidence of challenges involved in the regulation of GMOs at the multilateral as well as at the domestic level, part of this work was dedicated to the analysis of a set of claims before the Brazilian courts involving conflicts of norms of different hierarchical status applicable to GMOs. These claims comprised divergent opinions regarding which constitutional provision should be applicable in order to define Federal and State Government´s respective competence to legislate on different aspects of GMOs regulation. These claims also involved direct attacks on the constitutionality and legality of some infra-constitutional norms. The growing judicialization of conflicts of norms that govern the release of genetically modified products illustrates some of the shortcomings of Brazil´s domestic legal framework - that also exist at the multilateral level - that doesn´t allow a mutually satisfactory accomodation between the goals of protection of biodiversity and human health on the hand and the free trade of GMOs on the other hand.

A liberação comercial de plantas geneticamente modificadas (PGMs) é assunto controverso, devido ao desconhecimento quanto aos potenciais impactos ambientais e sócioeconômicos que pode suscitar a curto, médio e longo prazo. De modo a regular as deliberações sobre o uso de organismos geneticamente modificados (OGMs) mecanismos regulatórios que se propõem a prever tais impactos estão sendo criados em diversas nações, sendo a análise de risco (AR) o instrumento de estudo ambiental normalmente empregado nos processos decisórios. No entanto, há contestações sobre seu uso como única ferramenta de análise ambiental de PGMs, já que não possibilita a avaliação dos impactos cumulativos, indiretos, de longo prazo e dos interesses das nações delineados em suas políticas, planos e programas (PPPs). Um instrumento proposto por alguns autores passível de abarcar tais considerações é a Avaliação Ambiental Estratégica, que busca inserir a variável ambiental durante as fases de planejamento que resultam na elaboração de PPPs. Este trabalho teve como objetivo avaliar o sistema regulatório envolvendo a liberação em escala comercial de PGMs no Brasil e as potenciais contribuições da AAE para o processo decisório, contemplando os princípios de uma boa governaça ambiental nos processos decisórios. Para tanto, aplicou-se um questionário eletrônico a agentes sociais interessados pelo tema visando identificar suas visões sobre o assunto, analisou-se os procedimentos utilizados na deliberação sobre o algodão MON1445 resistente ao herbicida glifosato e, por fim, efetuou-se a avaliação dos instrumentos análise de risco, estudo de impacto ambiental (EIA) e AAE quanto à inserção da variável ambiental nas tomadas de decisão. Os resultados obtidos foram: com relação ao sistema regulatório, cujos pressupostos encontram-se na Lei de Biossegurança, evidenciou-se a falta de legitimidade nas tomadas de decisão, as quais são realizadas pela CTNBio, enquanto que a Constituição brasileira atribui aos órgãos ambientais a deliberação sobre atividades potencialmente poluidoras, dentre estas aquelas que envolvam OGMs; a deficiência dos mecanismos de participação social, pois as audiências públicas ocorrem mediante decisão da CTNBio; a falta de acesso às informações, devido a não implementação do Sistema de Informações sobre Biossegurança (SIB), além do descumprimento de diversas legislações, que configuram no desrespeito à justiça ambiental. Em razão destes fatores, conclui-se que o sistema regulatório brasileiro sobre PGMs não se baseia em uma boa governança ambiental. Quanto aos instrumentos de avaliação de impactos ambientais, a análise de risco, em função dos aspectos acima levantados, não se adequa para a avaliação prévia de PGMs, podendo ser utilizada como uma metodologia que subsidie o EIA ou a AAE. O EIA, por sua vez, não tem a atribuição de avaliar ações que envolvam territórios abrangentes, mas sim de atividades pontuais que se dão em etapas posteriores de planejamento. Já a AAE contribuiria para a tomada de decisões no tocante à liberação comercial de PGMs, em função de seus princípios de transparência, envolvimento social, planejamento ambiental, abrangência de extensos recortes territoriais, avaliação de impactos cumulativos e de longo prazo e monitoramento ambiental contínuo. Deste modo, recomendou-se a utilização da AAE no planejamento ambiental envolvendo liberações comerciais de PGMs no Brasil. / The commercial release of genetically modified crops (GMCs) is controversial, due to the lack of knowledge about the potential environmental and socio-economic impacts that can lead to short, medium and long term. In order to regulate the deliberations on the use of genetically modified organisms (GMOs), regulatory mechanisms that are proposed to predict such impacts are being created in many nations, with risk analysis (RA) being the instrument normally used in environmental studies for decision-making. However, there are some doubts about its use as the only tool of environmental analysis of PGMs, since it does not allow the assessment of cumulative, indirect and long-term impacts and the interests of the nation outlined in their policies, plans and programs (PPPs). An instrument proposed by some authors likely to embrace such considerations is the Strategic Environmental Assessment, which seeks to insert the environmental variable during the planning stages that result in the development of PPPs. This thesis aimed to evaluate the regulatory system involving the commercial release of GMCs in Brazil and the potential contribution of SEA to decisionmaking process, incorporating the principles of good governance. To this end, we applied an electronic questionnaire to some social actors to identify their views on the subject, reviewed the procedures used in the deliberation of the glyphosate-resistant cotton MON1445 and, finally, we performed the evaluation of risk analysis, environmental impact statement (EIS) and SEA as tools of environmental parameter insertion in decision-making. The results obtained were: with respect to the regulatory system, whose assumptions are in the Biosafety law, there was a lack of legitimacy in decision-making, which are held by CTNBio, while the brazilian Constitution assigns the decision on potentially polluting activitities (among which is those related to the GMOs) to the environmental agencies; the deficiency of the mechanisms of social participation, since decisions for public hearings are taking by CTNBio; lack of access to information due to non-implementation of the Information Biosafety System (IBS), and the disrespect of various laws, whith disregard for environmental justice. Because of these factors, the brazilian regulatory system on PGMs is not based on good environmental governance. With regard to instruments for environmental studies, risk analysis, according to the aspects mentioned above, is not suitable for prior assessment of GMCs and can be used as a methodology that assists EIS or SEA. The EIS, in turn, does not have the assignment to assess actions that involve extensive territories, but the pontual activities that take place in later stages of planning. SEA, in turn, would contribute to the decision-making regarding the commercial release of GMPs, according to its principles of transparency, social involvement, environmental planning, coverage of extensive territorial areas, assessment of cumulative and long-term impacts and continuous environmental monitoring. Thus, recommendations were made for the use of SEA in environmental planning involving commercial releases of GMPs in Brazil.

Os efeitos do treinamento físico aeróbio sobre o sistema renina-angiotensina cardíaco (SRA) foram avaliados em camundongos com insuficiência cardíaca (IC) e em seus respectivos controles (WT). O treinamento físico foi conduzido em esteira rolante (8 semanas, 5 x/sem, 60 min por dia). A tolerância à realização de esforço físico, e análises estruturais e funcionais cardíacas foram avaliadas. Os camundongos apresentaram disfunção cardíaca e fibrose associadas ao aumento na expressão de angiotensina II cardíaca e ao aumento na atividade da enzima conversora de angiotensina cardíaca (ECA). O treinamento físico aeróbio reduziu os níveis de angiotensina II e de ECA cardíaca para os mesmos valores apresentados pelo grupo controle. Além disso, elevou a expressão da ECA2 cardíaca, preveniu a intolerância à realização de esforço físico e a disfunção cardíaca, com pouco impacto sobre o remodelamento cardíaco / The effects of aerobic exercise training on the cardiac renin-angiotensin system (RAS) was evaluated in mice with heart failure (HF) and control (WT). The exercise training was realized in a motor treadmill (8 weeks, 5d/wk, 60 min/day). The exercise tolerance, structural and function analysis were evaluated. Mice displayed cardiac dysfunction and fibrosis associated to increased in cardiac angiotensin II expression and angiotensin converting enzyme activity (ACE). The exercise training reduced cardiac angiotensin II and ACE levels to age-matched WT. In addition, increased the ACE2 expression, prevent the exercise intolerance and cardiac dysfunction, with little impact on cardiac remodeling

O algodão transgênico Bollgard® (algodão Bt) contém o gene cry1Ac da bactéria Bacillus thuringiensis, que confere a planta resistência a Lepidopteros. A expressão deste gene na planta pode acarretar efeitos ecológicos adversos à microbiota do solo e da rizosfera. Em casa-de-vegetação, a comunidade bacteriana associada ao algodão Bt foi comparada a do algodão convencional, em dois tipos de solos e quatro estádios fenológicos. Amostras de rizosfera foram avaliadas por técnicas dependentes e independentes de cultivo. As técnicas de contagem de bactérias e DGGE permitiram observar os efeitos do algodão Bt na densidade e diversidade de Pseudomonas e bactérias totais, durante os estádios iniciais de desenvolvimento da planta. A toxina Cry foi detectada na rizosfera de algodão Bt, em todo ciclo da cultura. Nas fases de formação do botão floral e abertura das maçãs, a atividade microbiana foi maior na rizosfera do algodão Bt. Esses resultados indicam o potencial do ambiente rizosférico em reestabelecer à estrutura da comunidade bacterina após um impacto temporal. / The transgenic cotton Bollgard® (Bt cotton) contains the cry1Ac gene from the Bacillus thuringiensis bacterium, which confers the plant resistance against some insects. The expression of this gene in the plant can cause adverse ecological effects on soil and rhizosphere microbiota. In a greenhouse experiment, the bacterial community associate to Bt cotton was compared to non-transgenic parental cultivar plants, in two types of soil at different plant development stages. Rhizosphere communities were evaluated by culture-dependent and independent approaches. Results reveal the effect of the Bt cotton in the density and diversity of Pseudomonas and total bacteria, during initial plant development stages. The Cry toxin was detected in the rhizosphere of Bt cotton, during all plant cycle. In the phases of flower formation and fruit opening, the microbial activity was greater in the rhizosphere of Bt cotton. These results show the potential of the rhizosphere to reestablish the original structure of the bacterial community after a temporary impact.

A presente pesquisa teve por objetivo estudar a biologia de Spodoptera frugiperda (J. E. Smith, 1797) no milho MON810, que expressa a proteína Cry1Ab de Bacillus thuringiensis Berliner, bem como avaliar a interação tritrófica: milho MON810 vs S. frugiperda vs Trichogramma spp.. A biologia de S. frugiperda foi avaliada, por três gerações sucessivas em laboratório, utilizando-se o milho convencional e MON810. Os insetos mantidos em folhas de milho MON810 apresentaram maior duração do período larval e pré-pupaL, com menor viabilidade do que aqueles alimentados em milho convencional. A fase de pupa (fêmea e macho) foi similar entre os tratamentos, apesar da menor viabilidade e menor peso de pupas no milho MON810. Apesar da ação deletéria da proteína Bt, presente no milho geneticamente modificado para as fases imaturas de S. frugiperda, a longevidade dos adultos, o período de oviposição, o número de posturas, o número médio de ovos por postura e a duração da fase de ovo foram semelhantes entre os tratamentos. O número total de ovos por fêmea, no entanto, foi menor no milho geneticamente modificado. A duração média do ciclo biológico (ovo - adulto) foi maior em insetos mantidos em milho MON810, sendo a viabilidade total menor neste substrato. Foi observado que a taxa líquida de reprodução (Ro) e a razão finita de aumento (ë) foram menores nos insetos alimentados em milho MON810. As lagartas de S. frugiperda apresentaram comportamento canibal independente do substrato alimentar utilizado; porém, tal canibalismo foi mais acentuado no milho MON810, provavelmente devido a presença da proteína Bt. O consumo de área foliar por lagartas de S. frugiperda foi menor no milho MON810, sendo a eficiência do alimento digerido (ECD) menor e o custo metabólico maior neste substrato, em relação ao milho convencional. Em nível de campo, através de experimentos com infestações artificiais e naturais de S. frugiperda, observou-se que o milho MON810 determinou a redução populacional deste lepidóptero, nos diferentes estádios fenológicos estudados, protegendo a cultura do dano da praga. Os estudos de interação milho MON810 vs S. frugiperda vs Trichogramma atopovirilia (Oatman & Platner, 1983) foram efetuados por cinco gerações consecutivas do parasitóide e da praga em laboratório. Observou-se que não houve diferenças quanto a capacidade de parasitismo, a porcentagem de emergência, o número de parasitóides emergidos, a razão sexual e a longevidade do parasitóide quando T. atopovirilia foi criado em ovos de fêmeas que alimentaram-se de milho MON810 ou milho convencional. A qualidade nutricional dos ovos de S. frugiperda foi semelhante entre os tratamentos, não ocorrendo prejuízos ao parasitismo por T. atopovirilia. Em campo, a distribuição de posturas de S. frugiperda, em relação ao estádio fenológico da planta e superfície da folhas, foi semelhante entre os milhos MON810 e convencional. A porcentagem de ovos naturalmente parasitados por Trichogramma spp. também foi similar entre estes tratamentos, ocorrendo a predominância da espécie T. pretiosum (Riley, 1879) em relação a T. Atopovirilia condições de campo. / The objectives of this research were to study the biology of Spodoptera frugiperda (J. E. Smith, 1797) on MON810, that expresses the protein Cry1Ab from Bacillus thuringiensis Berliner, and to evaluate the tritrophic interactions: MON810 vs S. frugiperda vs Trichogramma spp.. The biology of S. frugiperda was evaluated under laboratory conditions for three consecutive generations on MON810 and conventional corn leaves. Insects that fed on MON810 showed longer larval and pre pupae stages duration and lower viability, compared to the larvae that fed on conventional corn. The duration of pupae stage (for both female and male) was similar for both treatments, however the viability of this stage was lower on MON810 and the pupae were smaller on this food source. Despite of the deleterious effect of the Bt protein expressed by MON810 to the larvae of S. frugiperda, the adult longevity, oviposition stage duration, number of egg laying, number of eggs per egg laying and egg stage duration were also similar for both treatments. However the total number of eggs was lower on genetically modified corn. The total biological life cycle (egg - adult) was longer for insects that fed on MON810, and the total viability was lower on this food source. It was observed that the net reproduction rate (Ro) and finite increase rate (ë) were also lower for insects that fed on MON810. The cannibalistic behavior of S. frugiperda occurred regardless of the food source (conventional corn or MON810); but this behavior was more pronounced when MON810 leaves were used as food source. The leaf area consumption by S. frugiperda was lower on MON810 and the insects had lower efficiency of conversion of digested (ECD) and higher metabolic cost than on conventional corn. Field experiments carried out with artificial and natural infestations of S. frugiperda showed that MON810 was very effective to reduce the population of this pest in different corn phenological stages. The studies on the interaction among MON810 vs S. frugiperda vs Trichogramma atopovirilia (Oatman & Platner, 1983) were carried out under laboratory conditions for five consecutives generations of both parasitoid and pest. It was not observed difference between both treatments for the parasitism, emergence, number of parasitoids, sexual ratio, and parasitoid longevity when T. atopovirilia was reared on eggs from females that fed on either MON810 or conventional corn. These data suggest that the nutritional quality of S. frugiperda eggs was similar between MON810 and conventional corn as food source, with no effect in the parasitism by T. atopovirilia. Field experiments showed that the distribution of S. frugiperda egg laying, according to the phenological stage and surface of the leaf, was similar between MON810 and conventional corn. The natural parasitism of S. frugiperda eggs by Trichogramma spp. was similar between MON810 and conventional corn, with the predominance of Trichogramma pretiosum (Riley, 1879), followed by T. atopovirilia in field conditions.

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.