Primordial germ cells (PGCs) are the embryonic precursors of the germ cell lineage. Segregation of the chicken germ line from somatic cells occurs very early in embryonic development. By day two of incubation chicken PGCs can be isolated from the circulating blood. The in vitro culture of chicken PGCs has significant potential as a tool for the investigation of germ cell development and as a cell-based system for the production of genetically modified chickens. The isolation, culture and manipulation of migratory chicken PGCs reported previously have not been independently validated. Initial attempts to isolate and culture chicken PGCs by reproducing a published protocol proved difficult. Key components of the published culture medium are by their nature variable, including the use of BRL-conditioned medium and animal sera. The protocol also stated that addition of SCF to the culture medium is essential but did not identify the source of SCF used. Several components of the culture conditions were tested including sources and batches of bovine and chicken sera and the growth factors FGF2 and SCF. Chicken PGCs from wild type and GFPexpressing chicken embryos were cultured and several cell lines established, proliferating for more than 100 days in culture. After seventy days in culture a single chicken PGC cell line was shown to retain the potential to develop into functional sperm. This was demonstrated by injection of the cultured chicken PGCs into early chick embryos, which were hatched and produced offspring derived from the injected chicken PGCs. To understand and produce a more robust system for the isolation and propagation of chicken PGCs three signalling pathways, AKT, MAPK and JAK/STAT, were investigated. When any of these signalling pathways were blocked, using chemical inhibitors, chicken PGC proliferation in vitro was significantly inhibited, showing the pathways to be essential for chicken PGC proliferation. Chicken PGCs were treated with individual components of the standard culture medium, FGF2, SCF, animal sera, BRL-conditioned medium, LIF and IGF, and the activation status of the key signalling pathways was assessed by western blot. Individual components of the culture medium induced activation of the AKT and MAPK pathways but not the JAK/STAT pathway. These data increase our understanding of PGC biology and are the first steps towards the development of a feeder- and serum-free medium for the growth of chicken PGCs. Published methods for the genetic manipulation of chicken PGCs are inefficient. To improve the efficiency of stable transgene integration, transposable element-derived gene transfer vectors were assessed for their ability to transpose into the genome of chicken PGCs. Comparison of Tol2 and piggyBac transposable elements, carrying reporter transgenes, demonstrated that both can be used to genetically-modify chicken cells. The incidence of stable transposition achieved was higher when using the Tol2 transposable element in comparison to the piggyBac element. The genetically-modified chicken PGCs formed functional gametes, demonstrated by injection of genetically modified chicken PGCs into host embryos which were hatched and produced transgenic offspring expressing the reporter gene construct.

This thesis develops biophysically-based data-driven mathematical models of intracellular calciumdynamics in ventricularmyocytes for both normal and genetically modified mouse hearts, based on species- and temperature-consistent experimental data. The models were subsequently applied to quantitatively examine the changes in calcium dynamics in mice with cardiomyocyte-specific knockout (KO) of the cardiac sarco/endoplasmic reticulum ATPase (SERCA2) gene, to determine the contributing mechanisms which underlie the ultimate development of heart failure in these animals. In Chapter 1, with emphasis on calcium dynamics and calcium regulation in heart failure, an overview of cardiac electrophysiology, excitation-contraction coupling and mathematical models of cardiac electrophysiology is provided. In Chapter 2, models of calcium dynamics in the ventricular myocytes from the C57BL/6 mouse heart at a physiological temperature is developed and validated based on species- and temperature-consistent measurements. In Chapter 3, the C57BL/6 model framework is re-parameterised to experimental data from the control and SERCA2 KO mice at 4 weeks after gene deletion. The models are then used to quantitatively characterise changes in calcium dynamics in the KO animals and the role of the compensatory mechanisms. In Chapter 4, the model framework is extended to include differential distributions of ion channels in the sarcolemma and the calcium dynamics in the sub-sarcolemmal space, with parameters in these sub-components fitted to experimentally measured calcium dynamics from the control and KO cardiomyocytes at 7-week after gene deletion. Finally in Chapter 5, conclusions are drawn, the limitations of this study are discussed, and the future extensions to this study are described.

This thesis describes the development of a framework for computational modelling of electromechanics in the mouse, with the purpose of being able to integrate cellular and tissue scale observations in the mouse and investigate physiological hypotheses. Specifically, the framework is applied to interpret electromechanical coupling mechanisms and the progression of heart failure in genetically modified mice. Chapter 1 introduces the field of computational biology and provides context for the topics to be investigated. Chapter 2 reviews the biological background and mathematical bases for electromechanical models, as well as their limitations. In Chapter 3, a set of efficient computational methods for coupled cardiac electromechanics was developed. Among these are a modified Newton method combined with a solution predictor which achieves a 98% reduction in computational time for mechanics problems. In Chapter 4, this computational framework is extended to a multiscale electromechanical model of the mouse. This electromechanical model includes our novel cardiac cellular contraction model for mice, which is able to reproduce murine contraction dynamics at body temperature and high pacing frequencies, and provides a novel explanation for the biphasic force-calcium relation seen in cardiac myocytes. Furthermore, our electromechanical model of the left ventricle of the mouse makes novel predictions on the importance of strong velocity-dependent coupling mechanisms in generating a plateau phase of ventricular pressure transients during ejection. In Chapter 5, the framework was applied to investigate the progression of heart failure in genetically modified 'Serca2 knockout' mice, which have a major disruption in mechanisms governing calcium regulation in cardiac myocytes. Our modelling framework was instrumental in showing for the first time the incompatibility between previously measured cellular calcium transients and ventricular ejection. We were then able to integrate new experimental data collected in response to these observations to show the importance of beta-adrenergic stimulation in the progression of heart failure in these knockout mice. Chapter 6 presents the conclusions and discusses possibilities for future work.

Master of Science / Department of Agricultural Economics / Timothy Dalton / Previous research in low-income countries reveals that genetically modified (GM) maize has the potential to increase yield and reduce labor use; however, other issues, especially regarding Roundup Ready (RR) maize, remain mostly unexplored. This research examines the impact of GM maize on yield, cost, and risk among 184 smallholders during the 2009-10 maize production season in two regions in KwaZulu-Natal, South Africa; Hlabisa and Simdlangetsha. Two hybrid maize varieties; Pannar and Carnia, and three GM varieties; Bt, RR, and BR (stacked with Bt and RR) are produced. In both regions, producers of RR and BR maize pay 47% more per kilogram of seed and use 44% less labor per hectare compared to other varieties. Due to low labor costs, net returns from RR and BR varieties are 25% and 40% higher than other varieties in Hlabisa and Simdlangetsha, respectively. Stochastic dominance analysis is used to compare net returns of all five varieties in both regions. RR maize is second-degree stochastic dominant to all other varieties in Simdlangetsha, while no variety is stochastically dominant in Hlabisa. Stochastic efficiency with respect to a function (SERF) analysis indicates that RR maize is the preferred variety for producers over the entire range of risk preferences in both regions. While average maize gross returns are $713 per hectare, risk premiums between $18 and $221 must be paid to RR maize producers, depending on region and farmer risk preference, to persuade them to switch to the second-most preferred variety. Econometric analysis indicates significant yield gains of at least 8% from RR maize, although the yield gain varies greatly when input endogeneity is taken into account. Elasticities of output with respect to labor are 0.41 and 0.82 for RR and non-RR maize respectively, and 0.61 and 0.33 with respect to land. A cost function analysis indicates that RR maize has 19% lower costs per maize plot, which increases to at least a 35% advantage when controlling for selectivity bias. Nonparametric kernel density estimation also reveals consistently lower total and average costs of RR maize at most levels of output, suggesting technological benefits to smallholder farmers from RR maize not available through conventionally-bred hybrids.

M. Tech. (Biotechnology, Department of Health Sciences), Vaal University of Technology / Amaranthus tricolor (A. tricolor) is a nutritious vegetable crop that is used as a subsistence and cash crop in the rural areas in Africa. Its yield and production is severely limited by abiotic stresses such as drought. Mutation technology, using gamma irradiation, was previously employed as a tool to create genetic variation in order to select for lines with improved drought-tolerance. During irradiation, 160 Gy (Gray) was selected as the optimal dosimetry that allowed subsequent seed germination. The resulting mutant lines were screened over several generations under field and greenhouse conditions and seven promising drought-tolerant lines were selected. Here we report on physiological and morphological studies of two of these Amaranthus mutant lines (#2 and #5) to confirm the enganced drought-tolerance. Plants were grown in the greenhouse in plastic pots containing germination mix with fertiliser. They were exposed to 21 days of well-watered condition, 19 days of drought-stress conditions and 7 days of re-watering. shoot height, leaf area, protein content and relative water content (RWC) of the fresh and dry material were determined colorimetrically under well-watered and drought-stress conditions, while anthocyanin was only measured during well-watered conditions. Shoot height, leaf area, number of leaves per plant and the protein content were significantly reduced under water-stress conditions. Under well-watered condition mutant #5 grew faster with the shoot length significantly higher than mutant #2 and the wild type. Even though drought adversely affected shoot lenght, mutant#5 still performed better than mutant #2 and the wild type under drought-stress conditions. While under both well-watered and drought-stress conditions, the wild type plants had bigger leaf area compared to the two mutant lines. After 16 days of drought-stress conditions, all the leaves of the wild type plants were dried out, as a result no wild type plants recovered after 8 days re-watering. Meanwhile, both mutant #2 and #5 plants recovered significantly after 8 days of re-watering. The wild type was tolerant compared to the two mutant lines. Protein content for mutant #2 plants was higher under both well-watered and drought-stress conditions but was not significantly different from mutant #5 plants compared to the wild type plants after 19 days of drought-stress conditions. Furthermore, genetic diversity was examined in all the Amaranthus lines using random amplified polymorphic DNA (RAPD) analysis. Nineteen arbitrary RAPD markers were used of which two detected polymorphisms (OPA) 07 and OPA 16).

A presente tese investiga como o Ambiente Institucional influencia o modo de governança de direitos de propriedade e a estratégia das firmas. O estudo divide-se em duas partes. A primeira parte empreende uma investigação teórica que se constitui em três etapas. Primeiro, examina-se o modelo de direitos de propriedade de Barzel (1994, 1997, 2003). Segundo, analisa-se uma abordagem de estratégia competitiva baseada em direitos de propriedade (Property Rights Perspective; Foss e Foss, 2001). Especificamente, argumenta-se que tal abordagem representa uma extensão do modelo de Barzel e demonstra-se que os conceitos introduzidos na etapa anterior possibilitam uma formulação mais geral da Property Rights Perspective, conduzindo a uma definição de estratégia competitiva que concilia as noções de strategizing e economizing (Williamson, 1991). A terceira etapa, por fim, examina particularmente o estabelecimento de estratégias de proteção de direitos de propriedade. Um modelo heurístico baseado em Williamson (1996) é proposto e com base nele três formas básicas de proteção são definidas em função da qualidade do Ambiente Institucional: estratégia centrada no sistema legal, no estabelecimento de mecanismos privados e no abandono de atributos valiosos. A segunda parte da pesquisa busca evidências empíricas que suportem o modelo teórico. O estudo examina três casos de proteção de direitos de propriedade sobre a tecnologia transgênica em sementes de soja: EUA, Brasil e Argentina. Cada um dos casos representa, respectivamente, uma forma de proteção de direitos como definido pelo modelo heurístico. A pesquisa examina também evidências econométricas que consolidam a análise empírica. De forma geral, o presente estudo desenvolve uma abordagem para o exame da apropriação de valor, colocando-se na interface entre a Economia de Direitos de Propriedade, o estudo da Estratégia e a análise do Ambiente Institucional. / The current thesis investigates how the Institutional Environment influences the mode of governance of property rights and the strategy of firms. The study is divided into two parts. The first part undertakes a theoretical investigation consisting of three steps. In the first step, the author examines the model of property rights developed by Barzel (1994, 1997, 2003). In the second step, an approach to competitive strategy based on property rights (Property Rights Perspective, Foss and Foss, 2001) is analyzed. Specifically, the author states that this approach represents an extension of Barzels model and demonstrates that the concepts introduced in the previous step allow a more general formulation of the Property Rights Perspective, leading to a definition of competitive strategy that reconciles the notions of strategizing and economizing (Williamson, 1991). The last step examines particularly the establishment of strategies for protection of property rights. A heuristic model based on Williamson (1996) is proposed and three strategies for protection of property rights are defined in terms of the quality of the Institutional Environment: strategy based on the legal system, on the establishment of private mechanisms, and on the abandon of valuable attributes. The second part of the research seeks empirical evidence to support the theoretical model. The study examines three cases of protection of property rights on genetically modified (GM) technology in soybean seeds: the US, Brazil, and Argentina. Each case represents, respectively, a strategy as defined by the heuristic model. The research also examines econometric evidence that consolidate the empirical analysis. Overall, the current study develops an approach for examining the appropriation of value, placing itself in the interface between the Property Rights Economics, the study of Strategy, and the assessment of the Institutional Environment.

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

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

O uso da associação entre glifosato e atrazina para a cultura do milho geneticamente modificado tolerante ao glifosato é uma das opções de controle de plantas daninhas nesta cultura. Portanto, o objetivo principal desse trabalho foi avaliar a influência do uso desta associação em um Latossolo vermelho-escuro proveniente do bioma Cerrado do Brasil através dos ensaios de degradação e mineralização desses herbicidas, carbono da biomassa microbiana e carbono mineralizado pelo solo. Os tratamentos para os ensaios de mineralização e degradação constaram da combinação entre 14C-glifosato na dose de campo (2,88Kg ha-1) a 0, 1/2, 1 e 2 vezes a dose de campo de atrazina (3,00Kg ha-1) e 14C-atrazina na dose de campo a 0, 1/2, 1 e 2 vezes a dose de campo de glifosato. A mineralização dos herbicidas foi medida aos 0, 3, 7, 14, 21, 28, 35, 42, 49, 56 e 63 dias e a degradação aos 0, 7, 28 e 63 dias após o início do experimento. A avaliação do carbono da biomassa microbiana foi realizada aos 21 e 63 dias após o início do ensaio e foram utilizados os mesmos tratamentos com a inclusão de uma prova em branco (solo sem herbicida). O ensaio de mineralização de carbono pelo solo foi feito através da quantificação do CO2 desprendido aos 3, 7, 14, 21, 28, 35, 42, 49, 56 e 63 dias após o início do ensaio, e também teve a inclusão de uma prova em branco. Os resultados demonstraram influência na degradação e mineralização da atrazina devido a presença do glifosato. A meia-vida de mineralização de atrazina teve uma variação de aproximadamente 100 dias quando foi comparada a aplicação individual de atrazina a associação com o dobro da dose de glifosato. A influência da atrazina na degradação e mineralização de glifosato não foi nítida. A presença de atrazina provocou queda no carbono da biomassa microbiana do solo e ocorreu um aumento na velocidade e quantidade de carbono mineralizado pelo solo. Não houve alteração no carbono da biomassa microbiana do solo e mineralização de carbono pelo solo devido a adição de glifosato. Nos tratamentos em associação, a presença do glifosato no sistema impediu a redução da biomassa microbiana devido ao efeito da atrazina. A associação entre glifosato e atrazina favoreceu a mineralização de carbono pelo solo comparada a aplicação individual de glifosato. Esses resultados demonstram a necessidade por parte da pesquisa em considerar a possibilidade de interação entre os diversos xenobióticos, o que pode alterar seus comportamentos individuais no solo. / The use of glyphosate and atrazine in association for transgenic corn tolerant to glyphosate is an option to weed control in this case. Therefore, the aim of this work was to assess the influence of this association in an Oxisoil from Brazil through the degradation, mineralization, microbial biomass and carbon mineralization of soil tests. The treatments of mineralization and degradation tests consisted of the combination between 14C-glyphosate in the field rate (2,88Kg ha-1) and 0, ½, 1 and 2 times the field rate of atrazine (3,00Kg ha-1). The mineralization of herbicides was measured at 0, 3, 7, 14, 21, 28, 35, 42, 49, 56, and 63 days and the degradation was measured at 0, 7, 28 and 63 days after the beginning of the tests. The evaluation of microbial biomass was performed at 21 and 63 days after the beginning of the test and was used the same treatments of the degradation and mineralization tests, but it was included a control (soil without application of herbicides). The test of carbon mineralization of soil was done by measuring the CO2 evolved at 0,7, 14, 21, 28, 35, 42, 49, 56 and 63 days after the beginning of the test and had the same control of the microbial biomass test. The results showed an influence on degradation and mineralization of atrazine due to the presence of glyphosate. The half-life of atrazine mineralization had a variation of about 100 days when it was compared the atrazine application alone to its association with glyphosate at double rate. The influence of atrazine in degradation and mineralization of glyphosate wasnt clear. The presence of atrazine caused decrease in the microbial biomass of soil and occurred an increase in speedy and amount of carbon mineralized by soil. No change was observed in microbial biomass and carbon mineralized by soil due to glyphosate application. In the treatments that was used the association, the presence of glyphosate in the system prevented decrease of microbial biomass due to the effect of atrazine. The association between glyphosate and atrazine favored the carbon mineralization by soil when compared to glyphosate applied alone. These results demonstrate a need to consider the possibility of interactions between several xenobiotics, wich can modify their behaviors in the soil.

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