A maioria das proteínas organelares são codificadas pelo núcleo, sintetizadas no citosol e direcionadas especificamente ao seu destino final. O direcionamento aos diferentes subcompartimentos subcelulares é feito por uma complexa e ampla maquinaria que envolve sequências de direcionamento, proteínas citossólicas e receptores organelares específicos. Entretanto, relativamente pouco se conhece sobre o processo na qual uma proteína recém sintetizada é transportada ao seu destino final. Parte significativa das proteínas destinadas às organelas possui a informação necessária ao seu transporte localizada na extremidade N-terminal. Vários estudos têm buscado caracterizar as etapas que envolvem a localização de uma proteína, desde os estágios iniciais após a sua síntese até os fatores que regulam o seu correto endereçamento. Modificações pós-traducionais, regiões 5-UTR, região C-terminal, transporte por vias alternativas e interações proteína-proteína podem agir na localização subcelular de proteínas. O estudo de redes biomoleculares se tornou um dos focos de estudo da biologia de sistemas e demonstra um enorme potencial na descoberta de diversos processos biológicos, como as interações proteína-proteína. As proteínas que possuem duplo direcionamento (DD) em Arabidopsis thaliana foram analisadas em redes de interação proteína-proteína (PPI) e proteínas que interagem com proteínas de DD foram escolhidas quanto a função e localização para a verificação de um eventual papel dessas proteínas na localização subcelular de outras proteínas. Para tanto, foram realizadas varreduras em ensaios de duplo-híbrido em levedura para os genes de GRF9 (14-3-3) e ATH7 (tiorredoxina tipo h). Os resultados para GRF9 incluem as proteínas peroxidase PRXR1 e dihidrolipoamida acetiltransferase. Já os resultados para ATH7 mostram a interação com glutamina sintetase (AT5G35630.3). A combinação dos estudos in silico com a varredura via duplo-híbrido de levedura abrem novas perspectivas no entendimento do controle da localização subcelular de proteínas. / Most organellar proteins are nuclear encoded, synthesized in the cytosol and then targeted to their destination. Specific subcellular targeting is conducted by a complex machinery for the specific localization of the proteins, which includes targeting sequences, cytosolic proteins and specific organelar receptors. However, little is known about the process that happens from the synthesis of a protein and the transport to its final destination. Most organellar proteins contain the information for their localization in the N-terminal sequence. Many studies have searched to characterize the steps involved in protein targeting, from the early stages after its synthesis to the cytosolic factors regulating its correct localization. Post-translational modifications, 5-UTR regions, the C-terminal extension on the protein, alternative transport pathways and proteinprotein interactions may influence the subcellular location of some proteins. The use of biomolecular netwoks has become one of the main focus of systems biology studies and possess a major potential in the discovery of several biological processes, such as protein-protein interactions (PPI). Dual-targeted (DT) proteins in Arabidopsis thaliana were analyzed through a PPI network, and the proteins displaying interactions with DT proteins were selected by their funtion and location. The selected proteins were analyzed for their eventual role in the subcellular targeting of other proteins. Screenings in yeast two-hibrid assays were performed for the genes GRF9 (14-3-3) and ATH7 (h-type thioredoxin). The results for GRF9 include a peroxidase (PRXR1) and dihydrolipamide acetyltransferase. The results for ATH7 include glutamine synthetase (AT5G35630.3). Combination of in silico analysis with yeast two-hibrid screenings provide new perspectives for understanding the control of subcellular localization.

O complexo FtsH em Arabidopsis, presente nos tilacóides, é formado pelas subunidades FtsH1/FtsH5 (tipo A) e FtsH2/FtsH8 (tipo B). Os tipos A e B apresentam grande identidade em seus domínios maduros, porém nenhuma similaridade é observada na região amino-terminal do peptídeo de trânsito. Em um experimento de importação em cloroplastos isolados, FtsH2 e FtsH5 foram importadas e subsequentemente integradas aos tilacóides através de um mecanismo de processamento em duas etapas que resultou em um domínio lumenal amino-proximal, uma única âncora transmembrânica e um domínio carboxi-proximal estromal. A integração da FtsH2 em tilacóides isolados foi totalmente dependente do gradiente de prótons, enquanto que a integração da FtsH5 foi dependente de NTPs, sugerindo que a inserção na membrana ocorre pelas vias TAT e Sec, respectivamente. Tal observação foi corroborada por experimentos de competição in-organello e inibição por anticorpos específicos. Os domínios amino-proximais até as âncoras transmembrânicas foram suficientes para a correta integração aos tilacóides. A região madura da FtsH2 apresentou incompatibilidade com a maquinaria Sec, como demonstrado pela troca de peptídeos de trânsito. A incompatibilidade não parece ser determinada por qualquer elemento específico da FtsH2, uma vez que nenhum domínio isolado apresentou incompatibilidade com a via Sec de transporte. Tal fato sugere uma incompatibilidade estrutural que requer a FtsH2 intacta. A descoberta que as subunidades FtsH do tipo A e B, que apresentam grande identidade e usam diferentes vias de integração para formar o mesmo complexo multimérico é uma observação nova e interessante para o estudo da biogênese de proteínas de membranas. O mecanismo de regulação que governa a atividade do complexo FtsH em Arabidopsis é ainda desconhecido, entretanto é proposta a existência de fatores adicionais. Dessa forma, a proteína plastidial FtsH de Arabidopsis foi usada como isca em um rastreamento por duplohíbrido de levedura. O rastreamento resultou em 48 colônias que ativaram os genes repórteres histidina e adenina. Entre todos os cDNAs sequenciados, foi encontrado um candidato em potencial denominado FIP (FtsH5 Interacting Protein). Experimentos GST Pull-Down também indicam uma interação entre FtsH5 e FIP. O precursor FIP radioativo foi incubado com cloroplastos de ervilha. Após a incubação, os cloroplastos foram lisados e separados em estroma e tilacóides. FIP permaneceu associada exclusivamente à fração membranosa dos tilacóides. A inserção na membrana foi verificada através da resistência ao tratamento com álcali e o tratamento dos tilacóides com protease resultou em um fragmento protegido, característico de proteínas inseridas na membrana. A construção FtsH5::GFP transformada em Nicotiana tabacum resultou no direcionamento do gene quimérico aos cloroplastos. Dessa forma, assim como FtsH5, FIP é uma proteína plastidial que está localizada na membrana dos tilacóides. Géis nativos utilizando FIP radioativa mostram que ela está associada a um complexo de aproximadamente 450 kDa, que é o tamanho esperado para o complexo tilacoidal FtsH em Arabidopsis. Como as proteínas FtsH apresentam tanto o domínio ATPase quanto protease, acreditamos que FIP pode de alguma forma modular a atividade do complexo FtsH nos tilacóides. / The Arabidopsis thylakoid FtsH protease complex is composed of FtsH1/FtsH5 (type A) and FtsH2/FtsH8 (type B) subunits. Type A and type B subunits display a high degree of sequence identity throughout their mature domains, but no similarity in their amino-terminal targeting peptide regions. In chloroplast import assays, FtsH2 and FtsH5 were imported and subsequently integrated into thylakoids by a two-step processing mechanism that resulted in an amino-proximal lumenal domain, a single transmembrane anchor, and a carboxyl proximal stromal domain. FtsH2 integration into washed thylakoids was entirely dependent on the proton gradient, whereas FtsH5 integration was dependent on NTPs, suggesting their integration by Tat and Sec pathways, respectively. This finding was corroborated by in organello competition and by antibody inhibition experiments. The amino proximal domains through the transmembrane anchors were sufficient for proper integration. The mature FtsH2 protein was found to be incompatible with the Sec machinery as determined with targeting peptide-swapping experiments. Incompatibility does not appear to be determined by any specific element in the FtsH2 domain as no single domain was incompatible with Sec transport. This suggests an incompatible structure that requires the intact FtsH2. That the highly homologous type A and type B subunits of the same multimeric complex use different integration pathways is a striking example of the notion that membrane insertion pathways have evolved to accommodate structural features of their respective substrates. The regulation mechanism which governs the Arabidopsis FtsH complexs activity is still unknown, but it is proposed the presence of additional factors. For this reason, the plastidial Arabidopsis FtsH5 was used as bait in a yeast two hybrid screening. The screening resulted in 48 colonies that activated the histidine and adenine reporter genes. Among all the sequenced cDNAs we have found a potential candidate named FIP (FtsH5 Interacting Protein). GST Pull-Down experiments also indicate an interaction between FtsH5 and FIP. Radiolabeled FIP was incubated with intact isolated chloroplasts. After incubation, intact chloroplasts were lysated and separated into stroma and thylakoids. FIP remained associated exclusively with the thylakoid membrane fraction. The insertion into membrane was verified throughout resistance to alkali treatment and the thylakoid protease treated fraction resulted in a protected fragment, characteristic of membrane-inserted proteins. Agroinfiltrated Nicotiana tabacum leaves with a FtsH5::GFP construct resulted that the chimeric gene was targeted to chloroplasts. Thus, as FtsH5, FIP is a plastidial protein which is located into thylakoid membrane. Blue native gels using radiolabeled FIP protein show that it runs associated with a complex around 450 kDa, which is the expected size for the Arabidopsis FtsH thylakoidal complex. As FtsH proteins present both ATPase and protease domains, we believe that FIP can somehow modulates the activity of the thylakoidal FtsH complex.

Proteínas que se ligam a RNAs geralmente regulam estabilidade, localização e tradução de mensageiros por interação com seqüências específicas na região 3 não traduzida. A proteína PUMILIO foi descrita pela primeira vez em Drosophila, apresentando a função de controlar a tradução de mensageiros alvo durante o desenvolvimento. Homólogos podem ser encontrados em outras espécies filogeneticamente distantes com funções similares. O seqüenciamento do genoma de Arabidopsis thaliana indicou a presença de quatro genes que codificam homólogos ao PUMILIO de Drosophila. Três deles estão no cromossomo II (APUM-1, 2 e 3), com alto grau de identidade e situados muito próximos uns dos outros. A terceira cópia está no cromossomo IV (APUM-4) e sua seqüência tem menor similaridade em relação aos três anteriores. Neste trabalho, foi clonado e caracterizado APUM-2. Ensaios de northern blot e RT-PCR indicaram que o mensageiro de Apum-2 pode ser encontrado em amostras de raiz, caule, folha, flores e frutos. Entretanto, sistemas repórteres, APUM-2::GUS e APUM-2::GFP, foram introduzidos no vegetal e a expressão foi observada nos ápices do caule e raízes, especificamente nas regiões meristemáticas. Também foram feitas construção para ensaios de genética reversa e as plantas contendo construções constitutivas (35S::APUM-2) se desenvolveram sem dominância apical e com grande quantidade de ramos, folhas e raízes secundárias, mostrando perturbações nos meristemas. Os resultados obtidos sugerem que APUM-2 possui papel relevante durante o desenvolvimento meristemático, possivelmente através de interações com mRNAs. / RNA-binding proteins often regulate the stability, localization, and translation of mRNAs by interaction with specific motifs in the 3-UTR. The PUMILIO protein from Drosophila was shown to control translation of specific mRNAs during development. PUMILIO homologs were found in several species and constitute the PUF family. The Arabidopsis thaliana sequencing project revealed four genes homolougs to PUMILIO. Three are situated in chromosome II (APUM-1, 2, and 3) that are nearly identical among themselves, and that contain a region that is 52% homologous to the PUMILIO. RNA-binding domain. The fourth is in chromosome IV (APUM-4), and shows low similarity to the other three. In this work, we characterized APUM-2 in further detail. Northern blots indicated that Apum-2 is expressed in shoot and root apices. Reporter transgenic plants were made that contained either APUM-2::GUS or Apum2::GFP constructs. Reporter gene expression confirmed that APUM-2 is active in shoot and root apices, specifically in the meristematic regions. Finally transgenic plants containing the 35S::APUM-2 construct were created. Constitutive APUM-2 expression resulted in plants with no apical dominance and a high number of leaves, stems and lateral roots. These results suggest that APUM-2 plays an important role during meristem development, possibly through a mRNA interaction.

CoordenaÃÃo de AperfeiÃoamento de NÃvel Superior / O feijÃo-de-corda (Vigna unguiculata) Ã uma leguminosa bastante utilizada na alimentaÃÃo de famÃlias de baixa renda da regiÃo nordeste do Brasil. Embora suas sementes sejam ricas em proteÃnas, estas sÃo deficientes em aminoÃcidos sulfurados na sua composiÃÃo. Dessa forma, um aumento na qualidade nutricional dessas sementes tem sido um dos principais objetivos dos programas de melhoramento genÃtico para essa espÃcie. AlÃm de determinar o padrÃo de deposiÃÃo de proteÃnas durante o desenvolvimento de embriÃes zigÃticos e somÃticos, nÃs pretendemos identificar genes com padrÃes especÃficos de expressÃo durante a embriogÃnese com a finalidade de utilizÃ-los em programas de melhoramento genÃtico. Utilizando a tÃcnica de eletroforese bidimensional (2D-PAGE) foi comparado o padrÃo protÃico de sementes em desenvolvimento com 10 dias apÃs a antese (DAA), sementes maduras e de suspensÃes celulares embriogÃnicas (SCE) obtidas de calos embriogÃnicos friÃveis (CEF) de feijÃo-de-corda. Para cada estÃgio de desenvolvimento da semente e para as SCE foram obtidos mapas de referÃncia proteÃmicos altamente reprodutÃveis numa faixa de pH de 3-10 e 4-7. VÃrios âspotsâ foram selecionados baseando-se na quantidade ou volume relativo de cada âspotâ e na taxa de expressÃo. Cerca de 800 (para sementes em desenvolvimento) e 130 (para SCE) âspotsâ protÃicos regulados para cima, para baixo, que se mantÃm constantes ou que sÃo especÃficos durante o desenvolvimento foram retirados dos gÃis 2D para anÃlise por espectrometria de massa. Algumas estratÃgias foram utilizadas para a identificaÃÃo das proteÃnas, como: PMF (peptide mass fingerprinting) e busca atravÃs de dados nÃo processados (MS/MS ion search). Dessa forma, cerca de 400 proteÃnas foram identificadas em sementes e cerca de 70 proteÃnas foram identificadas para SCE. A maioria das proteÃnas fram classificadas como proteÃnas do metabolismo primÃrio, energÃtico, proteÃnas de destinaÃÃo/proteÃnas de reserva e proteÃnas de defesa ou relacionadas a algum tipo de estresse. / Cowpea (Vigna unguiculata) is a leguminous plant highly utilized by low-income earners of Northeastern Brazil. However, proteins found in the crop are highly deficient in sulfur-containing amino acids. In line with this, improvement of nutritional quality of cowpea seeds has been one of the major goals of breeding programs of the crop. A starting point in this respect is a concerted effort to study the basic biochemical and physiological aspects of the development of cowpea seed. Besides determining the protein deposition pattern during the development of zygotic embryos and somatic embryos of the species, we set out to identify genes with specific pattern of expression during the two processes which will be of immense importance in cowpea breeding. Using the technique of two-dimensional electrophoresis (2D-PAGE), we compared the protein deposition pattern of developing cowpea seeds with 10 days after anthesis (DAA) and mature seeds and embryogenic cell suspension (ECS). From each developmental stage and for ECS, we obtained proteomic reference maps that were highly reproducible within the pH of 3-10 and 4-7. Various spots were selected based on quantity or relative volume and rate of expression. About 800 (for seeds development) and 130 (for ECS) proteins spots up- or down-regulated, remained constantly expressed and were specific for each developmental stage. The spots were removed from the 2-D gels, processed, and subjected to mass spectrometry. Some strategies like peptide mass fingerprinting (PMF) and non-processed data search (MS/MS ion search) were employed for protein identification. Over 400 proteins in seeds and over 70 proteins in ECS were identified. A large number of these proteins were found to be primary metabolism proteins, energy, protein destination and storage and defense proteins and others related to stress.

Spodoptera frugiperda (J. E. Smith) e Pseudoplusia includens (Walker) são importantes insetos-praga no algodoeiro (Gossypium hirsutum L.) devido às injúrias de desfolha e destruição de estruturas reprodutivas no caso de S. frugiperda. Os eventos de algodão Bt que expressam as proteínas Cry1Ac/Cry2Ab2 (Bollgard® II) e Cry1Ac/Cry1F (WideStrike(TM)) de Bacillus thuringiensis Berliner são ferramentas disponíveis para o controle dessas espécies-praga. A fim de subsidiar o Manejo da Resistência de Insetos (MRI) foram conduzidos estudos em laboratório para avaliar a sobrevivência e desenvolvimento de S. frugiperda e P. includens nos eventos de algodão Cry1Ac/Cry2Ab2 e Cry1Ac/Cry1F. Em bioensaios com discos de folhas, a eficácia de controle de neonatas nos dois eventos de algodão Bt foi superior a 80% para S. frugiperda e de 100% para P. includens. Em bioensaios com brácteas com neonatas de S. frugiperda, a eficácia de controle de ambos os eventos de algodão Bt também foi superior a 80%. As lagartas de S. frugiperda sobreviventes em algodão Bt apresentaram severa inibição de desenvolvimento larval em folhas (> 75%) e brácteas (> 44%). Em bioensaios com simulações de alimentação larval, as quais consistiam em grupos de lagartas alimentadas com o algodão Bt aos 0, 3, 6, 9, 12, 15 e 18 dias após a inoculação (DAI), S. frugiperda e P. includens demonstraram que a suscetibilidade dessas espécies diminuiu com o avançar do desenvolvimento larval. Para S. frugiperda, em todas as simulações de alimentação com o algodão Cry1Ac/Cry2Ab2 houve lagartas que atingiram as fases de pupa e adulto. Por outro lado, quando expostas ao algodão Cry1Ac/Cry1F, somente algumas das lagartas de 5º e 6º ínstares atingiram as fases de pupa e adulto. Para P. includens, somente lagartas no 6º ínstar atingiram as fases de pupa e adulto quando alimentadas com os dois eventos de algodão Bt. Os parâmetros biológicos de S. frugiperda sobreviventes em algodão Cry1Ac/Cry2Ab2 foram afetados negativamente com aumento da duração da fase larval (? 9 dias), baixa viabilidade larval (1,4%) e de insetos que completaram o ciclo biológico (0,9%), aumento no intervalo entre gerações (? 9 dias) e redução da taxa intrínseca de crescimento populacional (? 83%). Os eventos de algodão Cry1Ac/Cry2Ab2 e Cry1Ac/Cry1F são promissores no controle de S. frugiperda e P. includens. No entanto, a atividade inseticida dos dois eventos de algodão Bt em lagartas de S. frugiperda e P. includens diminui com o desenvolvimento larval e essa constatação deve ser considerada em programas de MRI, especialmente na disposição espacial do refúgio. / Spodoptera frugiperda (J. E. Smith) and Pseudoplusia includens (Walker) are important insect pests in cotton (Gossypium hirsutum L.) due to damage on leaves and reproductive structures in the case of S. frugiperda. The events of Bt cotton expressing proteins Cry1Ac/Cry2Ab2 (Bollgard® II) and Cry1Ac/Cry1F (WideStrike(TM)) from Bacillus thuringiensis Berliner are tools available to control these pest species. To develop an Insect Resistance Management (IRM), we performed laboratory studies to evaluate the survival and development of S. frugiperda and P. includens. In fresh leaf discs bioassays, the control efficacy of neonates in both Bt cotton events was greater than 80% mortality for S. frugiperda and 100% for P. includens. In fresh bracts bioassays to neonates of S. frugiperda, the control efficacy of both Bt cotton events was over 80%. The surviving larvae of S. frugiperda in Bt cotton showed severe growth inhibition (weight and instar) in leaves (> 75%) and bracts (> 44%). In simulations feed bioassays with larvae, which consisted of groups of larvae fed on Bt cotton at 0, 3, 6, 9, 12, 15 and 18 days after inoculation (DAI), S. frugiperda and P. includens showed that the susceptibility of species decreases with advancing larval development. For S. frugiperda, in all feed simulations with cotton Cry1Ac/Cry2Ab2 had caterpillars that reached pupae and adult stages. Moreover, when exposed to cotton Cry1Ac/Cry1F, only some caterpillars of 5th and 6th instars reached pupae and adult stages. For P. includens, only some caterpillars of 6th instar reached pupae and adult stages when fed with two events of Bt cotton. The biological parameters of S. frugiperda fed on cotton Cry1Ac/Cry2Ab2 were negatively affected with increasing duration of the larval stage (? 9 days), reduced larval viability (1,4%) and insects that completed the life cycle (0,9%), increased the generation time (? 9 days) and decreased the intrinsic rate of increase (? 83%). The events of cotton Cry1Ac/Cry2Ab2 and Cry1Ac/Cry1F are promising for the control of S. frugiperda and P. includens. However, the insecticidal activity of both events of Bt cotton in larvae of S. frugiperda and P. includens decreases through larval development and this finding should be considered in programs of MRI, especially in the spatial arrangement of the refuge.

A number of methods to isolate and quantify protein in Pinot noir wines were evaluated. The combination of precipitation by acetone containing 10% w/v trichloroacetic acid followed by quantification with the Bradford assay, reported in yeast invertase equivalents yielded the most accurate results when compared to micro-Kjeldahl analysis. The technique was validated by dialysis and proteolysis experiments and was used to assay protein in 57 Pinot noir wines. These wines were found to contain protein concentrations ranging from 49 to 102 mg/L. The mannan and tannin content of the wines was also measured, but no correlation between protein content and concentrations of these components was found. The presence of protein in red wines greater than 30 years old at concentrations typically found in white wines contradicts the notion that interactions with tannin severely reduce protein levels. / Graduation date: 2011

Title from first page of PDF document. Includes bibliographical references (p. 26-29).

The purpose of this study was to explore the influence of external product attributes on consumers’ perceived acceptance of pea protein as a protein substitute in food products. The interest in pea protein as a possible protein source was investigated and the various advantages of consuming pea protein were highlighted. In previous studies, pea protein was identified as a possible substitute for red meat (Davies & Lightowler, 1998), but also as protein containing the amino acid profile which corresponds with the profile recommended for adults by the Food and Agriculture Organisation (FAO) (Swendseid, 1981). Additionally the nutritional and health benefits, the environmental benefits of growing pea protein as well as the non-allergen declaration accompanying pea protein were discussed. The various factors that will influence the consumer’s decision making process when introduced to a new and unfamiliar protein source such as pea protein were investigated in terms of the external product attributes as well as the factors indicated in the Theory of Reasoned Action (TRA) (Fishbein & Ajzen, 1975). The external product attributes were identified as the influence of price, marketing of the product, packaging attributes of new food products, branding importance of a product and new product education and knowledge perceived necessary for acceptability of a new food product. The additional factors which were investigated, as indicated by the TRA, included the influence of the subjective norms or significant others, as well as the consumers’ personal attitude. attitude. Due to the limited insight and understanding of the influences that external product attributes have on a consumer’s perceived acceptance of pea protein as a protein substitute in food products, a qualitative exploratory research design was initiated. An exploratory research design was incorporated because this afforded the researcher the opportunity to gather thorough and comprehensive data or information and it also increased the richness and depth of explorations that could be researched (Key, 2002). The fact that an exploratory research design was incorporated, a non-probability sampling strategy was implemented. Additionally a convenient sampling strategy was also implemented because this type of sampling strategy represents sites or individuals from which data can easily be collected and where access is convenient (Cresswell, 2007). The unit of analysis for this research was female shoppers, being the main grocery and food shoppers in the household, who were living in suburban areas. The participants of the research, therefore, resided mainly in the Johannesburg and Pretoria suburban areas, within the Gauteng province and were chosen because these are two of the major suburban cities in South Africa. The data were collected through means of a multi-method approach whereby different data collection instruments were applied. In this regard focus group interviews, sentence completion exercise and a vignette were used which addressed the objectives set out in the study. From the findings of the study on the influence of external product attributes on consumers’ perceived acceptance of new products in general and new products containing pea protein, it was found that there were not any noticeable differences between a new food product and a new food product containing pea protein. The consumer did not indicate a greater willingness to accept general new products in comparison to a pea protein product. On the contrary, the fact that a new product contained pea protein did not make any difference. For consumers external product attributes in general played a major role in their purchasing decision making process. The most important requirement that the consumers had was that additional information regarding the pea protein should be provided. It was indicated by the participants that they would be interested in the pea protein product and consider purchasing it and incorporating it into their daily diet, providing they knew more about the product and had sufficient background information about the new product. Furthermore, the importance of marketing of a new product was highlighted and the fact that the packaging of the new product should be attractive and eye-catching. Price competiveness was mentioned, however, should a new and unfamiliar product be introduced by a well-known brand, this would lower the initial resistance to the new product. One of the main limitations of this study is the relatively small sample size from a particular area in South Africa and, therefore, resulting in a very selective sample. The sample, therefore, remains one of convenience which limits the ability to generalize or transfer findings to a bigger group of consumers. Furthermore the study location was within a limited area, namely Gauteng. For this reason no assumptions were made nor were findings considered to be transferable to other population groups or consumer groups. Another limitation is that the topic of this study is very new and unfamiliar; there are, therefore, no previous studies with which to compare the findings. This study was exploratory in nature and the instruments were chosen specifically to explore a new and unfamiliar topic. The transferability of this study might be limited, but care was taken to describe the research context in as much detail as possible. In doing so, an attempt was made to enhance the transferability, should another researcher wish to duplicate the study. From the findings of this study certain recommendation can be made as most participants were of the opinion that they would be interested in the pea protein product and consider purchasing it and incorporating it into their daily diet provided they knew more about the product and sufficient background information had been supplied. Furthermore, it was noted from the findings that these external attributes would influence the new product acceptability and, therefore, giving the research and developers as well as the marketing team of a manufacturing company the information necessary to understand the requirements from the consumers regarding the product containing pea protein better and how this product should be marketed as a healthy alternative to other vegetable protein sources. Further recommendations are that should a food manufacturing company wish to develop a pea protein product, they would need to promote the product beforehand and create an awareness of the advantages and benefits of pea proteins. This can be done by editorials in magazines as well as promotions in printed media. Additionally, the competition between products and brands is fierce and, therefore, manufacturing companies need to differentiate their products to keep consumers interested in their product or brand. Pea protein is a new and innovative product and can, therefore, add that differentiation and benefit to food products. Future research that can be considered is that of a sensory study, to determine the actual viability of the pea protein products based on sensory evaluation. One of the main criteria indicated for the acceptability of a food product containing pea protein has been the taste. The present research was theoretical and exploratory in nature and, therefore, there were no actual products which the participants could taste. Additionally, the inclusion criteria for this study was identified as female shoppers who are in charge of household grocery and food purchases and were, therefore, specifically recruited to participate in the research. However, based on a study done by Jensen (2011), it is recommended that further studies specifically include males because men are doing more of the household grocery shopping and they are not as brand loyal as females and would, therefore, be more willing to try and experiment with new food products. A study based on the gender differences of the acceptability of new products, could highlight different extrinsic attributes that would influence the purchasing decision. / Business Management / M. Consumer Sc.

Spodoptera frugiperda (J. E. Smith) e Pseudoplusia includens (Walker) são importantes insetos-praga no algodoeiro (Gossypium hirsutum L.) devido às injúrias de desfolha e destruição de estruturas reprodutivas no caso de S. frugiperda. Os eventos de algodão Bt que expressam as proteínas Cry1Ac/Cry2Ab2 (Bollgard® II) e Cry1Ac/Cry1F (WideStrike(TM)) de Bacillus thuringiensis Berliner são ferramentas disponíveis para o controle dessas espécies-praga. A fim de subsidiar o Manejo da Resistência de Insetos (MRI) foram conduzidos estudos em laboratório para avaliar a sobrevivência e desenvolvimento de S. frugiperda e P. includens nos eventos de algodão Cry1Ac/Cry2Ab2 e Cry1Ac/Cry1F. Em bioensaios com discos de folhas, a eficácia de controle de neonatas nos dois eventos de algodão Bt foi superior a 80% para S. frugiperda e de 100% para P. includens. Em bioensaios com brácteas com neonatas de S. frugiperda, a eficácia de controle de ambos os eventos de algodão Bt também foi superior a 80%. As lagartas de S. frugiperda sobreviventes em algodão Bt apresentaram severa inibição de desenvolvimento larval em folhas (> 75%) e brácteas (> 44%). Em bioensaios com simulações de alimentação larval, as quais consistiam em grupos de lagartas alimentadas com o algodão Bt aos 0, 3, 6, 9, 12, 15 e 18 dias após a inoculação (DAI), S. frugiperda e P. includens demonstraram que a suscetibilidade dessas espécies diminuiu com o avançar do desenvolvimento larval. Para S. frugiperda, em todas as simulações de alimentação com o algodão Cry1Ac/Cry2Ab2 houve lagartas que atingiram as fases de pupa e adulto. Por outro lado, quando expostas ao algodão Cry1Ac/Cry1F, somente algumas das lagartas de 5º e 6º ínstares atingiram as fases de pupa e adulto. Para P. includens, somente lagartas no 6º ínstar atingiram as fases de pupa e adulto quando alimentadas com os dois eventos de algodão Bt. Os parâmetros biológicos de S. frugiperda sobreviventes em algodão Cry1Ac/Cry2Ab2 foram afetados negativamente com aumento da duração da fase larval (? 9 dias), baixa viabilidade larval (1,4%) e de insetos que completaram o ciclo biológico (0,9%), aumento no intervalo entre gerações (? 9 dias) e redução da taxa intrínseca de crescimento populacional (? 83%). Os eventos de algodão Cry1Ac/Cry2Ab2 e Cry1Ac/Cry1F são promissores no controle de S. frugiperda e P. includens. No entanto, a atividade inseticida dos dois eventos de algodão Bt em lagartas de S. frugiperda e P. includens diminui com o desenvolvimento larval e essa constatação deve ser considerada em programas de MRI, especialmente na disposição espacial do refúgio. / Spodoptera frugiperda (J. E. Smith) and Pseudoplusia includens (Walker) are important insect pests in cotton (Gossypium hirsutum L.) due to damage on leaves and reproductive structures in the case of S. frugiperda. The events of Bt cotton expressing proteins Cry1Ac/Cry2Ab2 (Bollgard® II) and Cry1Ac/Cry1F (WideStrike(TM)) from Bacillus thuringiensis Berliner are tools available to control these pest species. To develop an Insect Resistance Management (IRM), we performed laboratory studies to evaluate the survival and development of S. frugiperda and P. includens. In fresh leaf discs bioassays, the control efficacy of neonates in both Bt cotton events was greater than 80% mortality for S. frugiperda and 100% for P. includens. In fresh bracts bioassays to neonates of S. frugiperda, the control efficacy of both Bt cotton events was over 80%. The surviving larvae of S. frugiperda in Bt cotton showed severe growth inhibition (weight and instar) in leaves (> 75%) and bracts (> 44%). In simulations feed bioassays with larvae, which consisted of groups of larvae fed on Bt cotton at 0, 3, 6, 9, 12, 15 and 18 days after inoculation (DAI), S. frugiperda and P. includens showed that the susceptibility of species decreases with advancing larval development. For S. frugiperda, in all feed simulations with cotton Cry1Ac/Cry2Ab2 had caterpillars that reached pupae and adult stages. Moreover, when exposed to cotton Cry1Ac/Cry1F, only some caterpillars of 5th and 6th instars reached pupae and adult stages. For P. includens, only some caterpillars of 6th instar reached pupae and adult stages when fed with two events of Bt cotton. The biological parameters of S. frugiperda fed on cotton Cry1Ac/Cry2Ab2 were negatively affected with increasing duration of the larval stage (? 9 days), reduced larval viability (1,4%) and insects that completed the life cycle (0,9%), increased the generation time (? 9 days) and decreased the intrinsic rate of increase (? 83%). The events of cotton Cry1Ac/Cry2Ab2 and Cry1Ac/Cry1F are promising for the control of S. frugiperda and P. includens. However, the insecticidal activity of both events of Bt cotton in larvae of S. frugiperda and P. includens decreases through larval development and this finding should be considered in programs of MRI, especially in the spatial arrangement of the refuge.

A soja geneticamente modificada MON 87701 × MON 89788, Glycine max (L.) Merrill, que expressa genes que codificam a proteína Cry1Ac de Bacillus thuringiensis var. kurstaki Berliner e a proteína 5-enolpiruvilchiquimato-3-fosfato sintase de Agrobacterium sp., foi liberada para uso comercial no Brasil em 2010. Para subsidiar um programa de Manejo da Resistência de Insetos (MRI) foi realizada a avaliação de risco de evolução de resistência a Cry1Ac para as pragas-alvo primárias, Anticarsia gemmatalis Hübner e Pseudoplusia includens (Walker), a praga-alvo secundária Heliothis virescens (Fabricius) e as pragas nãoalvo Spodoptera cosmioides (Walker), Spodoptera eridania (Stoll) e Spodoptera frugiperda (J. E. Smith). Em bioensaios com a proteína Cry1Ac purificada incorporada em dieta artificial, verificou-se que Cry1Ac foi extremamente ativa para A. gemmatalis [CL50 (IC 95%) = 0,23 (0,15 - 0,34) g Cry1Ac/mL de dieta], P. includens [CL50 (IC 95%) = 3,72 (2,65 - 4,86) g Cry1Ac/mL de dieta] e H. virescens [CL50 (IC 95%) = 0,026 (0,021 - 0,033) g de Cry1Ac/mL de dieta]. Em contraste, na máxima concentração testada de 100 g Cry1Ac/mL de dieta, S. cosmioides e S. eridania apresentaram mortalidade < 13%, e S. frugiperda de » 50%. Em bioensaios com tecido liofilizado de soja MON 87701 × MON 89788, diluído 25 vezes em dieta artificial, houve 100% de mortalidade para A. gemmatalis e H. virescens e até 96% para P. includens. Entretanto, as lagartas sobreviventes de P. includens apresentaram enfezamento larval > 95%. Em bioensaios com discos de folha em laboratório (para A. gemmatalis, P. includens e H. virescens), vagens (para H. virescens) e elevada infestação em condições de casa de vegetação e de infestação natural em campo (para A. gemmatalis e P. includens) foram obtidas alta eficácia da soja MON 87701 × MON 89788 no controle das pragas-alvo primárias e secundária. Por outro lado, a soja MON 87701 × MON 89788 apresentou baixa eficácia para S. cosmioides e S. eridania (mortalidade < 7%) e atividade intermediária para S. frugiperda (32 a 47% mortalidade). A soja MON 87701 × MON 89788 não afetou os parâmetros biológicos de S. cosmoides e S. eridania. Para S. frugiperda houve prolongamento da fase larval (» 5 dias), menor viabilidade larval e total, aumento no intervalo entre gerações (» 8 dias) e redução na taxa intrínseca de crescimento (» 41%). No contexto do MRI, a soja MON 87701 × MON 89788 expressa a proteína Cry1Ac em níveis que constituem alta dose para A. gemmatalis e H. virescens, e muito próximo a alta dose para P. includens. Por outro lado, para as espécies de Spodoptera, a soja MON 87701 × MON 89788 é um evento de baixa dose, pois permite que indivíduos suscetíveis completem o ciclo biológico. Em termos de MIP, a soja MON 87701 × MON 89788 apresenta um elevado nível de controle de A. gemmatalis, P. includens e H. virescens. No entanto, a soja MON 87701 × MON 89788 não ocasiona controle efetivo de espécies de Spodoptera. As informações obtidas no presente trabalho contribuirão para subsidiar programas de MRI e preservar a durabilidade dessa tecnologia para o MIP-Soja no Brasil. / Genetically modified MON 87701 × MON 89788 soybean, Glycine max (L.) Merrill, expressing genes that code for the Cry1Ac protein of Bacillus thuringiensis var. kurstaki Berliner and the 5-enolpyruvylshikimate-3-phosphate synthase protein of Agrobacterium sp., has been registered for commercial use in Brazil in 2010. To develop a program of Insect Resistance Management (IRM) for this event, we conducted resistance risk assessment to the primary target pests, Anticarsia gemmatalis Hübner and Pseudoplusia includens (Walker), the secondary target pest Heliothis virescens (Fabricius) and the non-target pests Spodoptera cosmioides (Walker), Spodoptera eridania (Stoll) and Spodoptera frugiperda (J. E. Smith) to the Cry1Ac protein. In bioassays with purified Cry1Ac protein incorporated into artificial diet, Cry1Ac was highly active for A. gemmatalis [LC50 (95% FL) = 0.23 (0.15 - 0.34) g Cry1Ac/mL of diet], P. includens [LC50 (95% FL) = 3.72 (2.65 - 4.86) g Cry1Ac/mL of diet] and H. virescens [LC50 (95% FL) = 0.026 (0.021 - 0.033) g Cry1Ac/mL of diet]. In contrast, even at the highest concentration tested of 100 g Cry1Ac/mL of diet, the mortality observed to S. cosmioides and S. eridania was < 13% and to S. frugiperda was » 50%. In bioassays with freeze-dried MON 87701 × MON 89788 soybean tissue, diluted 25 times in artificial diet, there was 100% mortality to A. gemmatalis and H.virescens and up to 96% for P. includens. However, the surviving P. includens larvae showed > 95% larval stunting. In bioassays with leaf discs in the laboratory to (A. gemmatalis, P. includens and H. virescens), pods (H. virescens), high pest infestation studies under greenhouse conditions and in natural infestation in the field (to A. gemmatalis and to P. includens) showed a very high level of efficacy against these primary and secondary target pests. On the other hand, soybean MON 87701 × MON 89788 showed low efficacy to S. cosmioides and S.eridania (mortality < 7%), and intermediate activity to S. frugiperda (32 to 47% of mortality). MON 87701 × MON 89788 soybean did not affect the biological parameters of S. cosmoides and S. eridania. For S. frugiperda, the larval stage was prolonged (» 5 days), with reduction on larval and total viability, increase in generation time (» 8 days) and reduction in the intrinsic rate of increase (» 41%). In the context of IRM, MON 87701 × MON 89788 soybean expresses the Cry1Ac protein at levels that are high-dose for A. gemmatalis and H. virescens, and near to the highdose for P. includens. On the other hand, for the Spodoptera species, MON 87701 × MON 89788 soybean is a low-dose event, because it allows susceptible individuals to complete larval developement. In terms of IPM, MON 87701 × MON 89788 soybean provided a high level of control of A. gemmatalis, P. includens and H. virescens. However, MON 87701 × MON 89788 soybean was not effective to control Spodoptera species. The information obtained in this research will contribute to develop IRM programs and to preserve the durability of this technology for soybean IPM in Brazil.