Algodão geneticamente modificado que expressa a toxina Cry1Ac de Bacillus thuringiensis Berliner tem sido plantado no Brasil desde 2006. Entre as pragas-alvo da tecnologia, Alabama argillacea (Hüeb.) é uma espécie monófoga e apresenta alto potencial de risco de evolução da resistência. Para a implantação de um programa de manejo da resistência de A. argillacea à toxina Cry1Ac no Brasil, os principais objetivos do trabalho foram: a) estabelecer a linhas-básicas de suscetibilidade à toxina Cry1Ac em populações de A. argillacea e definir concentrações diagnósticas para o monitoramento da resistência e b) isolar e caracterizar locos microssatélites para avaliar a variabilidade e estruturação genética de populações de A. argillacea no Brasil. As linhas-básicas de suscetibilidade foram estimadas por meio de bioensaio de imersão de discos de folhas em soluções contendo a toxina Cry1Ac para populações de A. argillacea coletadas nos estados da Bahia, Goiás, Mato Grosso e Mato Grosso do Sul, durante as safras agrícolas de 2008 e 2009. Foram isolados e caracterizados dez locos microssatélites. Para avaliar a variabilidade genética foram estimadas as heterozigosidades observadas e esperadas. Para o estudo da estruturação genética foram estimadas as estatísticas F e feita a análise de agrupamento (distância de Nei) e análise Bayesiana. Baseado na estimativa da CL50 foram encontradas variações naturais de até seis vezes na suscetibilidade à toxina Cry1Ac entre as populações testadas. A partir da análise conjunta dos dados de concentração-mortalidade das populações testadas, foram definidas as concentrações diagnósticas de 10 e 32 µg de Cry1Ac/ml de água para futuros programas de monitoramento da resistência. O número médio de alelos por loco foi de 7,1 (variando de dois a 23 alelos). As heterozigosidades observada e esperada médias foram de 0,532 e 0,329. O índice de fixação intrapopulacional médio (f FIS) foi de 0,268, com variação entre os locos de -0,008 a 0,736. O índice de fixação da espécie (FIS) estimado através da análise de variância foi de 0,244 (IC 95% de 0,093 a 0,418). O valor de FST estimado foi de 0,036 (IC 95% de 0,007 a 0,080). Esse valor de FST não diferiu significativamente de zero, indicando a ausência de estruturação genética. Contudo foi detectado certo grau de endogamia intrapopulacional. A estruturação espacial da variabilidade genética não foi detectada, pois as populações avaliadas apresentaram uma coesão que é mantida pela alta taxa de migração (6,7 migrantes por geração). Entretanto, foi identificada indícios de estruturação genética determinada pelo tempo, uma vez que tanto o agrupamento baseado em distâncias genéticas quanto à análise Bayesiana identificaram grupos que são formados por populações coletadas em safras agrícolas diferentes. As causas ligadas a essa mudança na variabilidade genética não puderam ser identificadas, entretanto pode se inferir que possivelmente causas naturais ou práticas de manejo estejam determinando eventos de gargalo genético. Devido ao intenso fluxo gênico entre populações de A. argillacea no Brasil, estratégias de manejo da resistência devem ser implantadas no âmbito nacional. / Genetically modified cotton expressing Cry1Ac toxin of Bacillus thuringiensis Berliner has been planted in Brazil since 2006. Among target pests of this technology, Alabama argillacea (Hüeb.) is a monophagous species and offers a high potential risk of resistance evolution. In order to implement a resistance management program of A. argillacea to Cry1Ac toxin in Brazil, the objectives of this research were: a) to establish baseline susceptibility to Cry1Ac toxin in A. argillacea populations and define diagnostic concentrations for resistance monitoring and b) to isolate and characterize microsatellite loci to evaluate the variability and genetic structure of A. argillacea populations in Brazil. The baseline susceptibility data were estimated with leaf-disc bioassays by dipping into different concentration of Cry1Ac solution. Populations of A. argillacea were collected in Bahia, Goiás, Mato Grosso and Mato Grosso do Sul States, during 2008 and 2009 cotton-growing seasons. Ten microsatellite loci were isolated and characterized. The genetic variability was evaluated estimating observed and expected heterozygosities. For the studied of genetic structure, the F statistics was estimated, and Cluster analysis (Nei´s distance) and Bayesian analysis were performed. Based on estimation of LC50, natural variation up to 6-fold was detected in the susceptibility to Cry1Ac among tested populations. Based on analysis of concentration-mortality data by combining all populations, diagnostic concentrations of 10 and 32 µg of Cry1Ac/ml of water were defined for monitoring resistance. The mean number of alleles per loci was 7.1 (varying from 2 to 23 alleles). The observed and expected heterozigosities was 0,523 e 0, 395. The mean intrapopulation fixation index (f FIS) 0,268, varying from -0.008 to 0.736 between loci. The species fixation index (FIS) estimated by analysis of variance was 0.244(95% CI of 0.093 to 0.418). The estimated value of FST was 0.036 (95% CI of 0.007 to 0.080). The FST value was not significantly different from zero, indicating absence of genetic structure However, some degree of intrapopulational inbreeding was detected. Spatial structure of genetic variability was not detected because tested populations showed cohesion kept by high migration rate (6.7 migrants per generation). However, evidence of genetic structure across time was detected by Cluster analysis of genetic distance as well as by Bayesian analysis with group formation by population collection seasons. Factors affecting changes in genetic variability were not identified; however, natural factors or management practices may be determining some genetic bottleneck events. Due to intense gene flow among A. argillacea populations in Brazil, resistance management strategies must be implemented in a national basis.

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

Made available in DSpace on 2014-08-20T13:32:54Z (GMT). No. of bitstreams: 1 tese_janaina_guimaraes_venzke.pdf: 370814 bytes, checksum: ca6312b2d70800b452ae33ccea99bab6 (MD5) Previous issue date: 2009-02-26 / The most cultivated genetically modified plant in Brazil is the soybean [Glycine max (L.) Merr.] resistant to the systemic herbicide glyphosate. There are 12.3 millions of cultivated hectares. This soybean was obtained through the introduction of the gene that codes for the 5-enolpyruyilshikimic-acid3-phosphate synthase enzyme, (EPSPS, E.C 2.5.1.19, CP4), of the shikimic pathway, resistant to glyphosate that keeps active the aromatic amino acids biosynthetic pathway. To the soybean genetical modification the parameters in law were reviewed and the maximum glyphosate limit permitted in beans that was 0.2 mg/kg was increased to 10 mg/kg to GMRR soybean. Although Brazil is the 3rd biggest worldwide producer, cultivates the GMRR soybean for more than ten years, and represents 50% of the overall production of soybean in Brazil, just a few studies have been conducted in the country to evaluate in biological assays, the impact of this technology on the food quality and safety. Thus, the present work seeks for to evaluate the influence of the genetic modification of the soybean on the nutritional quality, on the possible effects of chronical exposition, as well as the effects on the fertility and the development of Wistar rats and the on the endocrine system. The nutritional quality was evaluated through the protein value, in 30 male rats, just weaned, distributed in five groups, fed along 28 days with the following diets, 10% protein ration GMRR soybean no isogenic, GMRR soybean isogenic, conventional soybean, milk (casein) or for 10 days with a non-protein diet. The weight gain and the food intake of diets did not present statistical relevance. The same behavior was observed in the variables of growth and the nitrogen balance study. In the chronical exposition of 40 male and 39 female rats distributed in four groups that consumed genetically modified soybean no isogenic and isogenic, conventional soybean and the standard group without soybean it signs of systemic toxicity on males have been evident in the neutrophilia, lymphocytopenia in the group conventional soybean compared with the standard group, lymphoid hyperplasia of the lungs in the groups without soybean; the signs of reproductive toxicity through of 8 the increase on relative weight of the tests and epididymis and also through the decrease of the sperm concentration. On the females the effect on the reproductive system became evident through the fertility pregnancy and weaning index. These results point out that the protein quality of the genetically modified soybean is preserved, but there is an association of factors that trigger a probable endocrine disruption. Studies are necessary to elucidate the mechanism of interaction that cause toxicity on males and females fed with conventional soybean and genetically modified soybean that presented detectable glyphosate levels. / A espécie vegetal geneticamente modificada mais cultivada no Brasil é a soja [Glycine max (L.) Merr.] resistente ao herbicida sistêmico pós-emergente glifosato, com 12,3 milhões de hectares cultivados. Essa soja foi obtida pela introdução do gene correspondente à enzima 5-enolpiruvilchiquimato-3-fosfato sintase (EPSPS, E.C 2.5.1.19, CP4), enzima da via de chiquimato, resistente ao glifosato, mantendo ativa a via biossintética de aminoácidos aromáticos. A modificação genética fez com que parâmetros preconizados pela legislação brasileira fossem revistos e o limite máximo permitido de glifosato nos grãos que era de 0,2 mg/kg aumentasse para 10 mg/kg. Embora o Brasil cultive há mais de dez anos a soja resistente ao glifosato e seja o terceiro maior produtor mundial dessa leguminosa, com 50% da produção nacional com genótipos transgênicos, poucos estudos têm sido conduzidos no país com o intuito de avaliar, em ensaios biológicos, o impacto dessa tecnologia sobre o meio ambiente, a qualidade e a segurança do alimento. Sendo assim, o presente trabalho objetivou estudar a influência do consumo de grãos de soja geneticamente modificada resistente ao glifosato (GMRR) isogênica à convencional e não isogênica, tratada com este herbicida, sobre a qualidade nutricional, possíveis efeitos à exposição crônica, bem como efeitos sobre a fertilidade e o desenvolvimento de ratos Wistar e a possibilidade deste, desregular o sistema endócrino. A qualidade nutricional foi avaliada através do valor protéico mediante índices biológicos, em 30 machos recém desmamados, distribuídos em cinco grupos, alimentados por 28 dias com ração contendo 10% de proteína de soja GMRR não isogênica, soja GMRR da isogênica, soja convencional, leite (caseína) ou por 10 dias com ração aproteica. O ganho de massa corporal e o consumo alimentar das dietas não diferiu entre os tratamentos. Esse mesmo comportamento foi observado no que concerne às variáveis de crescimento e índices de balanço nitrogenado. Na exposição crônica de 40 machos e 39 fêmeas distribuídos em 4 grupos que consumiram dietas contendo sojas GMRR (isogênica e não isogênica), soja convencional e grupo padrão sem soja, 6 sinais de toxicidade sistêmica nos machos foram evidenciados na neutrofilia, linfopenia do grupo que consumiu soja convencional em relação ao grupo padrão, e na hiperplasia linfóide dos pulmões nos animais que consumiram soja GMRR e convencional; sinais de toxicidade reprodutiva foram observados através do aumento da massa relativa dos testículos e epidídimos e redução da concentração espermática. Nas fêmeas, os efeitos no sistema reprodutivo foram evidenciados através dos índices de fertilidade, e desmame. Estes resultados indicam que a qualidade protéica da soja modificada geneticamente é preservada, porém há uma associação de fatores que desencadeiam em uma provável desregulação endócrina. Outros estudos são necessários para elucidar o mecanismo de interação que provoca efeitos de toxicidade em machos e fêmeas alimentados com soja convencional e GMRR que apresentam níveis detectáveis de glifosato.

Les canaux de type T Cav3.2 sont des canaux calciques activés pour de faibles dépolarisations membranaires. Ils ont un rôle important dans la régulation de l’excitabilité neuronale, particulièrement dans les neurones des ganglions rachidiens dorsaux (DRG) où ils sont impliqués dans la transmission de la douleur. Il est établi que les canaux Cav3.2, natifs et recombinants, sont inhibés par de faibles concentrations de métaux divalents tels que le zinc et le nickel et qu’ils sont modulés par des agents oxydo-réducteurs. In vitro, la mutation ponctuelle de l’histidine 191 en glutamine (H191Q) diminue fortement la sensibilité du canal Cav3.2 pour ces différents composés et il est proposé que cette régulation joue un rôle physiologique. L’objectif de ce travail de thèse a été d’étudier l’impact physiologique de cette modulation sur l’excitabilité neuronale et dans la perception de la douleur. Pour ce faire, nous avons généré une souris knock-in (KI) portant la mutation H191Q sur Cav3.2. L’étude électrophysiologique a été réalisée sur une population de neurones de DRG particulière : les cellules D-hair qui sont des mécanorécepteurs exprimant de grands courants Cav3.2. Nous avons validé que la sensibilité des canaux Cav3.2 neuronaux des souris KI est diminuée pour le zinc, le nickel et l’ascorbate. Nous montrons que cette régulation modifiée favorise une augmentation de l’excitabilité de ces neurones. Pour étudier l’impact de cette modulation in vivo, nous avons effectué des études comportementales. Les souris KI ne présentent pas de différence dans la perception de la douleur mécanique et thermique, ni dans l’hyperalgésie induite par l’inflammation et la neuropathie. Toutefois, dans le test à la formaline les souris KI montrent une réponse exacerbée dans la phase tardive. En résumé, nous décrivons ici un modèle animal original pour l’étude de la régulation metal/redox du canal Cav3.2 et identifions un rôle de cette modulation dans l’excitabilité des neurones D-Hair. Nos résultats obtenus in vivo indiquent cependant que cette modulation des canaux Cav3.2 aurait un impact limité dans les voies de la douleur. / Cav3.2 T-type channels are low-voltage activated calcium channels. They have an important role in the regulation of neuronal excitability, particularly in neurons of the dorsal root ganglia (DRG) where they are involved in pain transmission. It is established that Cav3.2 channels are inhibited by low concentrations of divalent metals such as zinc and nickel, and are modulated by redox agents. In vitro, the histidine191-to-glutamine mutation (H191Q) greatly reduces the Cav3.2 channel sensitivity to these compounds and it is proposed that this regulation plays a physiological role. The objective of this thesis was to study the physiological impact of this modulation on neuronal excitability and pain perception. To do this, we generated a knock-in (KI) mouse carrying the H191Q mutation on Cav3.2. Electrophysiological study was carried out on a particular population of DRG neurons, the D-hair cells, which are mechanoreceptors that express large Cav3.2 currents. We show that the sensitivity to zinc, nickel and ascorbate of the neuronal Cav3.2 channels is significantly reduced in the KI mouse. We also show that this modified regulation promotes an increase in the excitability of these neurons. To study the impact of this modulation in vivo, we performed behavioral studies. KI mice show no difference in the perception of mechanical and thermal pain, nor in hyperalgesia induced by inflammation and neuropathy. However, KI mice show an exaggerated response in the late phase in the formalin test. In summary, we describe here an original animal model to study the metal/redox regulation of Cav3.2 channel and identify a role of this modulation in the excitability of D-Hair neurons. Our results indicate, however, that this modulation of Cav3.2 channel may have a limited impact in the pain pathways.

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.

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 do trabalho foi avaliar a qualidade, a quantidade e a capacidade de amplificação por PCR de DNA extraído de grãos de soja e milho, seus derivados e produtos acabados contendo como ingredientes obtidos desses grãos, com vistas à detecção de resíduos de organismos geneticamente modificados em alimentos. Para a amplificação de DNA pela PCR convencional, não houve melhor adequação de um protocolo de extração. Ambos métodos, CTAB e coluna de sílica tiveram desempenho comparável para as 32 matrizes avaliadas. A técnica de PCR em tempo real se mostrou mais sensível à qualidade do DNA testado e nesse contexto, o método CTAB se mostrou mais eficiente do que o método de coluna de sílica. Independentemente do método de extração utilizado não foi possível detectar DNA em óleos de soja e milho e em alguns derivados de amido, sugerindo que a aplicabilidade da lei de rotulagem pode esbarrar num entrave técnico no caso de algumas matrizes alimentares altamente processadas. / The aim of the study was to evaluate the quality, the quantity and the amplifiability by PCR of DNA isolated from soybean and maize grains and their by-products targeting the detection of genetically modified organisms in food. PCR amplification of DNA samples isolated either from CTAB and silica-column extraction methods achieved comparable performances. Both extraction methods showed similar results for the 32 tested matrices. The DNA amplification by real time PCR appeared to be affected by the quality of the isolated DNA. In this context, the CTAB extraction method showed to be more suitable when compared to the silica-column method. No DNA was amplified from soy and maize oils, as well as from some starch by-products, regardless the DNA extraction method used. It suggests that, the labeling requirement may rely on technical issues considering some high processed foodstuffs.

Farmer-led seed systems (FSS) provide the backbone for small-scale farmers and many rural communities that use traditional methods of farming to produce seeds that grow and adapt to local conditions. FSS differ from one community and farmer to the next, depending on the methods and practices used to maintain seed varieties. Seed diversity can enhance FSS by improving livelihoods and strengthening farmers' networks, thus contributing to resilient communities. Although nuanced, the dualistic agricultural system in South Africa consists largely of subsistence (small-scale) and commercial (large-scale) farming and includes different crop management systems and post-harvest practices. In South Africa, maize (Zea mays) is a major staple grain crop with a significant role as animal and poultry feed. The North West region is one of the highest white-maize-producing provinces in South Africa. Maize seed systems include both traditional, openpollinated varieties (OPVs) and cultivars such as modern hybrids and genetically modified (GM) seed varieties, including those engineered for specific purposes. The dominant GM maize is that designated for pest resistance using Bacillus thuringienesis (Bt), a soil bacterium which produces a toxin that is fatal to a wide variety of insects such as moths and flies. Many small-scale farmers prefer their own traditional seeds for breeding, planting, selection, selling and consuming. However, FSS based on traditional varieties are threatened by modern cultivars which may be introduced in different ways including through seed exchange, purchasing at shops or by pollination from nearby commercial farms. This study was conducted in the Sespond community of the North West Province. The aim of the study was to understand how small-scale farmers in Sespond maintain traditional maize varieties through selection and storage in a complex agricultural landscape that incorporates both formal and informal seed systems. The formal system represents industrialised farms and companies that work with commercial seed. The informal system represents small-scale farmers who rely on their own seed. Qualitative methods included mapping software which was used to obtain visual agricultural data in and around Sespond. Semi-structured interviews were conducted with 30 small-scale farmers to collect information about their farming practices, including the maize varieties planted. Quantitative methods included collecting 20 maize samples from different farmers for genetic analysis. Agdia® immunostrip tests were used to detect for the presence of Crystal protein (Cry protein) produced by the Bt bacterium, engineered to improve the resistance of maize against insects. The results showed that 13 samples were negative for the protein and seven samples were positive for the protein. A key finding is that small-scale farmers are not able to detect the different maize varieties in their seed systems. This represents a threat for traditional seed varieties in the community as without this knowledge, farmers are not able to adequately manage their production and storage systems. Farmers made use of alternative storage methods such as the mill to reduce seed damage they experienced at home. However, the findings of this research showed that there was an increasing risk of farmers' traditional maize being mixed with GM maize at the mill. Farmers' rights to plant and consume traditional maize were therefore undermined. This study recommends that (a) efforts are made to increase awareness among farmers that help to distinguish transgenes from hybrids and traditional maize varieties; (b) measures are implemented at mills to both improve the transparency about the storage and processing of traditional maize and to separate traditional maize from hybrid and GM maize.

With many genetic traits discovered and many more in progress, it is imperative to the industry that firms (biotechnology companies) decide on the trait valuation and pricing. This includes more than one trait (also referred to as stacked traits) in a single variety of crop; the risk and uncertainty of expected returns associated with the development and release of a variety increases even more in case of stacked traits. The purpose of this thesis is to develop a model that can be used for the valuing and pricing of genetically modified (GM) traits that are random, sporadic, and non-persistent (e.g. drought tolerance, heat/cold stress) using the real option approach. The efficiency gain in case of occurrence of random event and expression of GM traits will be measured and used as a decision factor in determining the value of GM trait(s) at different phases of development. Risk premiums representing the value of GM trait to growers is calculated across risk averse attitudes. The return to labor and management (RTLM) provided by a GM trait is used to calculate the risk premiums when variation in parameters is allowed to be same as that reflected in historical data and gains from GM traits are realized. Monte Carlo simulation and stochastic efficiency with respect to a function (SERF) are used to estimate the certainty equivalents that decision makers would place on a risky alternative relative to a no risk investment. Certainty equivalents are estimated across a range of risk aversion coefficients and used to rank alternatives and determine where preferences among alternatives change while estimating risk premiums for the base case (no trait), drought tolerance, cold tolerance, NUB, and All traits (all traits combined into one as a stacked trait). Premiums provide perspective on the magnitude of differences in relative preferences among choices. The range of ARAC utilized was from 0.00 to 0.15 for all three crops. The risk premiums are treated as a potential source of revenue in the model as a technology fee charged by a biotech company. This thesis uses the Real Option methodology to evaluate GM traits as Option values at various stages of development. This approach helps managers decide the best possible option in making a certain decision today. It is also helpful in comparing different pathways (series of decisions) and thus better exploits the potential cash return in the future from investments made today (Figure D.1, Figure D.2). Three possible options to "continue", "wait", and "abandon" were modeled in this thesis. Such modeling determines the possible option values of GM traits at different stages of development depending on the kind of choices made at different points of time. This thesis shows that various GM traits that are out-of-money (OTM) at initial stages have increased probability of being in-the-money (ITM) at later stages of development. Sensitivities show that a share of potential technology fees and acreage of GM crops play a significant role in option values being ITM. Stacked traits provide a better chance of being ITM, thus the option to continue will be exercised by management. The option to wait causes reduction in option value. Among individual traits, drought tolerance has the greatest maximum option value in most cases. Therefore, if management has to choose the development of only one GM trait, it is most likely to choose to invest in the development of drought tolerance.

<p><strong>Apstrakt je obrađen tehnologijama za optičko prepoznavanje teksta (OCR).</strong></p><p>Imajući u vidu naučni i praktičan značaj i nedostatak podataka o aplikaciji ultrafiltracije (UF), alternativnih koaguli&scaron;ućih enzima, posebno genetskog himozina, kao i enzima za akceleraciju zrenja u tehnologiji tvrdih sireva, za predmetna istraživanja je odabran Kačkavalj, popularni sir mediteranskog porekla.<br />U okviru istraživanja je u industrijskim uslovima izvr&scaron;ena proizvodnja KačkavaIja iz retentata dobijenog nakon ultrafiltracije mleka po LCR (Low Concentrated Retentate) postupku (UF Kačkavalj). Ispitan je uticaj koagulanasa mikrobiolo&scaron;kog porekla (M. miehei proteaza, Rennilase) i genetski modifikovanog himozina (Maxiren) na kvalitet finalnog sira i upoređen sa klasičnim analozima (kontrolni Kačkavalj). Istražena je mogućnost skraćenja perioda zrenja obe varijante Kačkavalja (klasični i UF) dodatkom &bdquo;koktela&quot; enzima za ubrzanje zrenja proteolitičkog (Accelase) i lipolitičkog (Paletase) dejstva.<br />Na osnovu rezultata dobijenih ispitivanjem fizičko-hemijskog sastava, mikro-strukture i senzornog kvaliteta varijanti konvencionalnog Kačkavalja i UF analoga tokom jednogodi&scaron;njeg zrenja, utvrđeno je da se dejstvo rekombinovanog himozina (Maxiren-a) i standardnog himozina signifikantno ne razlikuje, potvrđujući dosada&scaron;nja saznanja da je genetski modifikovani himozin potpuno kompetentan proteolitički agens za koagulaciju mleka u procesu proizvodnje sira. Kod konvencionalnog i UF Kačkavalja proizvedenog primenom mikrobiolo&scaron;ke M. miehei proteaze (Rennilase) ustanovljena je najniža koncentracija mlečne masti u suvoj materiji tokom celokupnog ispitivanog perioda, &scaron;to se sa ekonomskog aspekta negativno odražava na kvalitet sira. Sve varijante UF Kačkavalja se tokom zrenja razlikuju od klasičnih analoga po hemijskom sastavu (sadržaj mlečne masti, ukupnog N, NPN, RN itd.), profilu proteolitičke degradacije as- i p-kazeina, modelu distribucije identifikovanih is- parljivih komponenata arome, reolo&scaron;kim i senzornim karakteristikama i mikrostrukturi usled<br />usporenog intenziteta promena proteinske i lipidne faze UF sireva tokom zrenja. Efekat ubrzanja zrenja Kačkavalja tretiranog &bdquo;koktelom&quot; enzima lipolitičkog i proteolitičkog dejstva je izraženiji kod konvencionalnog nego UF uzorka.</p> / <p><strong>Abstract was processed by technology for Optical character recognition (OCR).</strong></p><p>The scientific and practical significance and the lack of literature data on the<br />application of ultrafiltration (UF), as well as the alternative coagulating enzymes, particularly genetic chymosin, and enzymes for accelerated ripening in hard cheese technology were essential reasons for choosing this field for research. The object of presented investigations was Kashkaval, the popular mediterranean hard variety cheese.<br />In the scope of these investigations, Kashkaval was industrially produced using retentate obtained by UF of milk according to LCR (Low Concentrated Retentate) procedure (UF Kashkaval). Effects of clotting enzymes of microbiological origine (M. miehei protease, Rennilase) and of genetically modified chymosin (Maxiren) on the quality of final cheese product was also investigated and compared to conventional analogues (control Kashkaval). The possibility for reduced ripening period of both Kashkaval types (traditional and UF) was investigated by the addition of &bdquo;coctail&quot; of enzymes which accelerate the cheese ripening, involving the proteolytic (Accelase) and lypolytic (Palatase) activity.<br />According to the results obtained by the investigations of physico-chemical composition, microstructure and sensory quality of conventional Kashkaval and UF analogues in the course of one-year ripening period, it was possible to derive the conclusion that the effects of recombined chymosin (Maxiren) and the standard one are insignificantly different, confirming thus the former knowledge that the genetically modified chymosin is an adequate proteolytic agent for milk coagulation in cheese production. The lowest level of milk fat in dry matter over the whole investigated period was established both for conventional and UF Kashkaval samples produced by the application of M. miehei protease (Rennilase), whereas from the economic standpoint this indicated negative effects on cheese quality. All UF Kashkaval varieties differ during the period of ripening from conventional analogues concerning chemical composition (content of milk fat, total N, NPN, RN etc.), profile of proteolytic degradation of as- and p-casein, distribution of identified volatile aroma<br />components, rheological and sensory characteristics as well as the microstructure due to the slower intensity of changes in protein and lipid phases of UF cheese samples during ripening. The &bdquo;cocktail&quot; of proteolytic and lipolytic enzymes showed more pronounced effects on the Kashkaval ripening in the case of conventionally produced cheese sample.</p>