Seed development, germination and dormancy, considered as the most important phenomena in seeds, are regulated by several plant hormones; gibberellin (GA) and abscisic acid (ABA) being the major players acting antagonistically. The regulation of these seed related processes by GA and ABA is dependent partly on the endogenous levels of the two hormones, which in turn are determined by the balance between their biosynthesis and catabolism. This thesis investigated the spatial and temporal expression patterns of several members of the GA and ABA biosynthetic and catabolic gene families during grain development using a non-dormant cultivar and during imbibition using grains collected from dormant and non-dormant cultivars of barley. In addition, the thesis examined the effect of exogenous ABA treatment, and after-ripening of seeds collected from dormant cultivars on the expression patterns GA and ABA metabolism genes during grain development and imbibition, respectively. The results suggest that specific members of the gene families related to the metabolic pathways of the two hormones exhibit distinct spatial and temporal roles in the regulation of barley grain development, dormancy and germination. / October 2015

Infestation of cattle by Lone Star ticks, Amblyomma americanum, leads to damage of hides intended for leather, weight loss, infertility, and potentially death of cattle, which contribute to production losses for farmers. Public concerns regarding chemical residues in food and the environment necessitate development of chemical-free alternative tick controls, such as breeding for tick-resistant phenotypes and developing anti-tick vaccines. Thus, the goal of this study was elucidation of mechanisms that mediate immune responses in cattle infested with A. americanum using gene expression techniques. Methods for isolation of total RNA from bovine tick bite-site biopsies and blood leukocytes were optimized to provide RNA suitable for gene expression studies. Tick bite-site biopsies (6 mm) and blood leukocytes were collected from a total of 13 calves (N=6, Group 4 and N=7, Group 5 calves) during experimental tick infestations to determine A. americanum tick-susceptible and -resistant phenotypes. Microarray experiments compared gene expression in tick bite-sites from tick-susceptible, moderately tick-resistant, and highly tick-resistant calves. A total of 35 genes were profiled in tick bite-site biopsies and 12 genes were evaluated in blood leukocytes via gene-specific qRT-PCR assays, and analyzed for each phenotype and for each group of calves as a whole. Analysis of microarray data revealed differential expression of IL-1R-mediators among the three cattle phenotypes. Expression profiles generated by qRT-PCR for TLR-mediating genes such as TLR2, TLR4, CD14, and MyD88 suggest that a MyD88-dependent signaling pathway may mediate the development of acquired immunity in cattle infested with Lone Star ticks. Additionally, increased expression for IL12, IFNgamma, and TNFalpha suggests that a Th1-type cell-mediated reaction may be activated, whereas increased expression of IL6, IL10, and IGHG1 supports the involvement of a Th2-type humoral-mediated response at tick bite-sites in cattle infested with at A. americanum. Regression analyses identified strong correlations between factors involved in pattern recognition in tick bite-site biopsies, including associations between TLR4 and IL1alpha, and between IL1alpha and IL1RN. In conclusion, this dissertation reports optimal methodology for gene expression studies in tick-infested cattle and provides preliminary data concerning the underlying mechanisms associated with the immune response in Lone Star tick-infested cattle.

The main focus of this thesis has been to study of domestication traits. Identification of genes of domestication traits has a huge potencial for plant breeding. The experimental part has been devoted to the study of pod dehiscence as a domestication trait in pea (Pisum sativum L.). The goal of this work has been to evaluate the expression of candidate genes of pod dehiscence in pea (Pisum sativum L.). For this experiment was used JI64 line pea (Pisum sativum ssp. elatius L.) and JI92 (Afghan cultural type of pea Pisum sativum ssp. sativum L.) and recombinant inbred lines (RILs) arising from reciprocal crossing. Using the method of qRT-PCR (Quantitative Reverse Transcription PCR) have been analyzed candidate genes IDEH, SHAT, bZIP, SPT, NAC and compared with the expression of the reporter gene tubulin.

Aspergillus flavus exemplifies a pathogenic fungus that remains a significant contributor to the loss of corn (Zea mays) crops. The production of carcinogenic aflatoxins renders the crop hazardous for consumption and causes significant loss to farmers. Therefore, the prevention of A. flavus contamination continues to persist as a topic for research intervention. Host resistance to this pathogen provides a promising source of defense for the corn plant. Corn inbred line Mp313E was previously identified to exhibit significant resistance against the A. flavus fungal infection and aflatoxin contamination. Quantitative trait loci (QTL) mapping has previously established four major QTL locations associated with aflatoxin resistance in the corn inbred line Mp313E. Near-isogenic lines were developed containing these previously identified QTLs from backcrossing of inbred lines Mp313E (resistant donor parent) and Va35 (susceptible recurrent parent). Quantitative RT-PCR (qRT-PCR) was used to study gene expression patterns of 17 genes selected from plant-pathogen interaction pathways. Furthermore, genomic primer analysis was used for establishment of 15 candidate genes for marker- assisted breeding.

O carcinoma epidermoide bucal (CEB) é uma malignidade epitelial que causa grande mortalidade. As metalotioneínas (MTs) são proteínas envolvidas na homeostasia de metais e eventos oxidativos. Estudos de expressão proteica a apontaram como marcadora de prognóstico e comportamento metastático para o CEB. A análise da expressão gênica das mesmas tem auxiliado no entendimento deste papel para outros tumores, mas ainda não foi estudada no CEB. O objetivo deste trabalho foi avaliar o perfil de expressão gênica das MTs no CEB e em fragmentos de mucosa oral (MOC), além de sua relação com dados clínicopatológicos, comportamento metastático e prognóstico. Para tal, foram utilizadas amostras armazenadas a -80º C no Banco Nacional de Tumores do Instituto Nacional de Câncer, constando de 35 casos de CEB de língua e/ou assoalho bucal e 35 MOC. Todos os fragmentos foram submetidos ao qRT-PCR com uso de TaqMan® para os genes: MT1A, MT1B, MT1E, MT1F, MT1G, MT1H, MT1X, MT2A, MT3 e MT4. A expressão dos genes MT1B e MT1H foi raramente detectada. Houve queda significativa de expressão dos genes MT1A, MT1X, MT3 e MT4 e aumento significativo de expressão de MT1F no CEB com relação à MOC. Casos de baixa expressão de MT1G tiveram pior prognóstico. A alta expressão de MT1X indicou casos não metastáticos e a alta expressão de MT3 indicou casos metastáticos. Em suma, foi demonstrado pela primeira vez o perfil gênico das MTs no CEB, indicando que a mesma pode fornecer informações sobre o prognóstico e comportamento metastático do CEB. / The oral squamous cell carcinoma (OSCC) is a malignancy that causes high mortality. Metallothioneins (MTs) are proteins involved in metal homeostasis and antioxidant events. Studies regarding its protein expression indicated its potential as marker of prognosis and metastatic behavior, also for OSCC. The analysis of its specific gene expression can clarify its importance in these aspects and no study has been done for OSCC. The aim of this work was to evaluate the profile of gene expression of MTs in OSCC and samples of non-neoplastic oral mucosa (OM), evaluating its relationship with clinic-pathologic characteristic, metastatic behavior and prognosis for OSCC. For this, tissue samples archived at -80º C at the National Bank of Tumors of the Brazilian National Institute of Cancer were collected, in a total of 35 cases of OSCC and 35 fragments of OM. All tissues were submitted to qRTPCR with TaqMan® for the genes: MT1A, MT1B, MT1E, MT1F, MT1G, MT1H, MT1X, MT2A, MT3 e MT4, besides the constitutive gene GAPDH. Expressions of MT1B and MT1H were rarely detected. There was significant loss of expression for MT1A, MT1X, MT3 and MT4 and gain of expression for MT1F comparing OSCC with OM. Cases with down-regulation of MT1G had the worst prognosis. Up-regulation of MT1X indicated non-metastatic cases whereas up-regulation of MT3 indicated metastatic ones. In conclusion, this study shows for the first time the profile of gene expression of MTs on OSCC indicating distinctive patterns of regulation for each, and giving associations with prognosis and metastatic behavior of cases.

O carcinoma epidermoide bucal (CEB) é uma malignidade epitelial que causa grande mortalidade. As metalotioneínas (MTs) são proteínas envolvidas na homeostasia de metais e eventos oxidativos. Estudos de expressão proteica a apontaram como marcadora de prognóstico e comportamento metastático para o CEB. A análise da expressão gênica das mesmas tem auxiliado no entendimento deste papel para outros tumores, mas ainda não foi estudada no CEB. O objetivo deste trabalho foi avaliar o perfil de expressão gênica das MTs no CEB e em fragmentos de mucosa oral (MOC), além de sua relação com dados clínicopatológicos, comportamento metastático e prognóstico. Para tal, foram utilizadas amostras armazenadas a -80º C no Banco Nacional de Tumores do Instituto Nacional de Câncer, constando de 35 casos de CEB de língua e/ou assoalho bucal e 35 MOC. Todos os fragmentos foram submetidos ao qRT-PCR com uso de TaqMan® para os genes: MT1A, MT1B, MT1E, MT1F, MT1G, MT1H, MT1X, MT2A, MT3 e MT4. A expressão dos genes MT1B e MT1H foi raramente detectada. Houve queda significativa de expressão dos genes MT1A, MT1X, MT3 e MT4 e aumento significativo de expressão de MT1F no CEB com relação à MOC. Casos de baixa expressão de MT1G tiveram pior prognóstico. A alta expressão de MT1X indicou casos não metastáticos e a alta expressão de MT3 indicou casos metastáticos. Em suma, foi demonstrado pela primeira vez o perfil gênico das MTs no CEB, indicando que a mesma pode fornecer informações sobre o prognóstico e comportamento metastático do CEB. / The oral squamous cell carcinoma (OSCC) is a malignancy that causes high mortality. Metallothioneins (MTs) are proteins involved in metal homeostasis and antioxidant events. Studies regarding its protein expression indicated its potential as marker of prognosis and metastatic behavior, also for OSCC. The analysis of its specific gene expression can clarify its importance in these aspects and no study has been done for OSCC. The aim of this work was to evaluate the profile of gene expression of MTs in OSCC and samples of non-neoplastic oral mucosa (OM), evaluating its relationship with clinic-pathologic characteristic, metastatic behavior and prognosis for OSCC. For this, tissue samples archived at -80º C at the National Bank of Tumors of the Brazilian National Institute of Cancer were collected, in a total of 35 cases of OSCC and 35 fragments of OM. All tissues were submitted to qRTPCR with TaqMan® for the genes: MT1A, MT1B, MT1E, MT1F, MT1G, MT1H, MT1X, MT2A, MT3 e MT4, besides the constitutive gene GAPDH. Expressions of MT1B and MT1H were rarely detected. There was significant loss of expression for MT1A, MT1X, MT3 and MT4 and gain of expression for MT1F comparing OSCC with OM. Cases with down-regulation of MT1G had the worst prognosis. Up-regulation of MT1X indicated non-metastatic cases whereas up-regulation of MT3 indicated metastatic ones. In conclusion, this study shows for the first time the profile of gene expression of MTs on OSCC indicating distinctive patterns of regulation for each, and giving associations with prognosis and metastatic behavior of cases.

Made available in DSpace on 2015-03-26T13:47:15Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1676673 bytes, checksum: 5c833a936878e6ed4a6d88b1073a8552 (MD5) Previous issue date: 2013-04-24 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Escherichia coli is a microorganism of environmental origin that is highly adaptive; its ability to form biofilms under certain conditions can be critical to its resistance to antimicrobial treatments. The aim of this study was to evaluate the expression profiles of the genes luxS (autoinducer synthase-2), fliC (flagellin), csgA (large subunit of the fimbriae curli ) and fimA (largest subunit type I fimbriae), which are involved in biofilm formation, in E. coli obtained from mastitic milk in the presence of antimicrobials on abiotic and biotic environments. The genetic profile of 27 isolates was evaluated by pulsed field gel electrophoresis (PFGE) with XbaI enzyme digestion. Four isolates of E. coli, two strong biofilm producers (GBP) and two weak producers (WBP) which possessed the genes luxS, fliC, csgA and fimA, were selected by conventional PCR. The minimum inhibitory concentration (MIC) of antimicrobials was assessed for performing other tests at the concentration of ½ MIC. The biofilm production was detected in the presence of antimicrobials and visualized through scanning electron microscopy (SEM). Assay of adhesion/invasion of bacterial cells in bovine mammary alveolar cell (MAC-T) culture in the presence of antimicrobials was quantified in CFU/mL and visualization was achieved by confocal microscopy. Subsequently, the expression profiles of the genes luxS, fliC, csgA and fimA were analyzed at different times after inoculation on abiotic and biotic surfaces. Nine (I to IX) different phylogenetic groups with similarity > 90% were identified by PFGE. The results revealed the presence of the genes luxS, fimA and csgA in all 27 isolates, whereas the fliC gene was only detected in 16. The MICs ranged from 0.05 to 8 μg/mL, with the highest values obtained for gentamicin (8 μg/mL) and ampicillin (6 μg/mL). Enrofloxacin and cotrimoxazole induced biofilm formation in at least one isolated WBP, and ampicillin and gentamicin in one isolated GBP. Also, the reducing effect of enrofloxacin and cotrimoxazole was observed in at least one isolated GBP. There was no increase in the ability of adxiv hesion/invasion of the isolates of E. coli in the presence of antibiotics, but an increase in the internalization of the WBP isolate when treated with antimicrobials was observed through confocal microscopy. The four genes were differentially expressed when exposed to ½ MIC concentrations of antimicrobials with changes in the order of 1.5 to 22 times. The growth on the biotic surface favored the expression of luxS and fimA genes in relation to abiotic surfaces. The sub-inhibitory concentrations of the antimicrobials studied were able to induce the formation of biofilms by some isolates, as well as inducing invasion into host cells, which is a situation that might be occurring in vivo. This could result in a reservoir of bacteria in the udder of the animal, which could explain the increase in cases of persistent mastitis in herds. / Escherichia coli é um micro-organismo de origem ambiental altamente adaptativo e sua habilidade em formar biofilmes em determinadas condições pode ser fundamental para sua resistência a tratamentos com antimicrobianos. O objetivo desse estudo foi avaliar os perfis de expressões dos genes luxS, fliC, csgA e fimA envolvidos com a formação de biofilmes em E. coli obtidos de leite mastítico na presença de antimicrobianos em meio biótico e abiótico. Foi avaliado o perfil genético de 27 isolados por meio de gel de eletroforese em campo pulsado (PFGE) com a digestão enzimática XbaI. Quatro isolados de E. coli , dois fortes produtores de biofilmes (GPB) e dois fracos produtores (FPB) que possuíam os genes da sintase do autoindutor-2 (luxS), flagelina (fliC), subunidade maior da fímbria curli (csgA) e subunidade maior da fímbria do tipo I (fimA) foram selecionados por PCR convencional. Foi avaliada a concentração inibitória mínima (MIC) dos antimicrobianos para realização dos demais testes com a concentração de ½ MIC. A produção de biofilme foi detectada na presença de antimicrobianos e visualizada em microscopio eletrônico de varredura (MEV). Foram realizados ensaios de adesão/invasão de células bacterianas em cultivo de células alveolares mamárias bovinas (MAC-T) na presença de antimicrobianos, quantificados em UFC/mL, e visualizadas por meio de microscopia confocal. Posteriormente, foram analisados os perfis de expressão dos genes luxS, fliC, csgA e fimA em diferentes tempos e em superfície de inoculação abiótica e biótica. Nove (I a IX) grupos filogenéticos diferentes com similaridade >90 % foram identificados pelo PFGE. Os resultados revelaram a presença de luxS, fimA e csgA em todos os 27 isolados, enquanto o gene fliC foi detectado em 16. As MICs variaram entre 0,05 a 8 μg/mL, sendo os maiores valores obtidos para gentamicina (8 μg/mL) e para ampicilina (6 μg/mL). A enrofloxacina e o cotrimoxazol induziram a formação de biofilme em pelo menos um isolado FPB, ampicilina e gentamicina em um GPB. Também foi observado efeito reduxii tor da enrofloxacina e cotrimoxazol em pelo menos um isolado GPB. Não foi observado aumento na capacidade de adesão/invasão dos isolados de E. coli na presença de antimicrobianos, no entanto observou-se aumento na internalização do isolado FPB quando tratado com os antimicrobianos através da microscopia confocal. Os quatro genes foram diferencialmente expressos quando submetidos à concentração de ½ MIC dos antimicrobianos com alterações da ordem de 1,5-22 vezes. O crescimento em superfície biótica favoreceu a expressão dos genes luxS e fimA em relação à abiótica. A concentração subinibitória dos antimicrobianos estudados foi capaz de induzir a formação de biofilmes por alguns isolados, além de induzir a invasão às células hospedeiras, situação que poderia estar ocorrendo in vivo, resultando em um reservatório de bactéria no úbere do animal, o que poderia explicar também, o crescente aumento de casos de mastites persistentes nos rebanhos.

Made available in DSpace on 2014-08-20T13:44:24Z (GMT). No. of bitstreams: 1 dissertacao_daisy_leticia-ramirez_monzon.pdf: 495372 bytes, checksum: 4109f5d12e44f58ba6cd6d0affc3d675 (MD5) Previous issue date: 2012-02-07 / The soybean (Glycine max L. Max) is one of the main crops grown in Brazil, the second largest world producer. Its evolution has been strongly supported by the development of technologies that allowed the acreage increase. Currently, the use of biotechnology tools aims to contribute to the development of new genotypes with characteristics that can continue increase the production. The identification of genes responsible for characters that can assist in developing varieties with improved seed quality may constitute something innovative. The seed tegument has an important function as for the seeds resistance to deterioration. In this sense, the partial characterization of the protein expression FLA 14 (Arabinogalactan), directly related to the cell wall synthesis in the seed tegument may contribute to the advancement of these studies. The aim of this work was to generate and validate primers capable of quantifying the gene expression of the FLA 14 protein in four soybean genotypes, two with black tegument (TP and IAC 222) and higher lignin content and two with yellow tegument (CD 202 and Potência) and lower lignin content. The plant tissue (tegument) was collected 50 days after the anthesis. In order to obtain the gene expression among genotypes, it was used the qRT-PCR technique, where it was tested four combinations of primers, constructed from sequence models of soybeans ESTs. With the validation results, the primers that were found within the standards were the FLA 14p1, FLA 14p2 and β-Actina. They were all used at the expression studies. The results of the relative expression showed that soybean genotypes with black tegument and higher lignin content express the FLA 14 protein quantitatively superior to the genotypes with yellow tegument and lower lignin content. / A soja (Glycine max L. Merr.) é uma das principais culturas produzidas no Brasil, sendo considerado o segundo maior produtor mundial. Sua evolução foi fortemente amparada pelo desenvolvimento de tecnologias que possibilitaram o aumento da área cultivada. Atualmente o emprego de ferramentas da biotecnologia busca contribuir para o desenvolvimento de novos genótipos com características que possam continuar incrementando a produção. A identificação de genes responsáveis por caracteres que possam auxiliar no desenvolvimento de variedades com melhor qualidade da semente, pode se constituir em algo inovador. O tegumento apresenta uma função importante quanto à resistência das sementes à deterioração. Nesse sentido, a caracterização parcial da expressão do gene da proteína FLA 14 (Arabinogalactano), relacionadas diretamente com a síntese da parede celular no tegumento da semente pode contribuir para o avanço dos estudos. O objetivo do trabalho foi gerar e validar primers capazes de quantificar a expressão gênica da proteína FLA 14 em quatro genótipos de soja, dois desses com tegumento preto (TP e IAC 222) e maior conteúdo de lignina, e outros dois com tegumento amarelo (CD 202 e Potência) e menor conteúdo de lignina. O tecido vegetal (tegumentos) foi coletado aos 50 dias após a antese. Para a análise da expressão gênica entre os genótipos, utilizou-se a técnica de qRT-PCR, onde foram testadas quatro combinações de primers construídos a partir de modelos de sequência de ESTs da soja. Com os resultados da validação, os primers que se encontraram dentro dos padrões foram os FLA 14p1, FLA 14p2 e β-Actina utilizando-se no estudo de expressão. Os resultados da expressão relativa permitiram concluir a expressão do gene da proteína FLA 14 é quantitativamente superior nos genótipos de soja com tegumento preto e conteúdo superior de lignina em relação aos genótipos com tegumento amarelo e menos teor de lignina.

La graine de soja contient de grandes quantités d'isoflavones (génistéine, daidzéine et glycitéine). En raison de leurs propriétés phytoestrogéniques, ces composés peuvent avoir des effets bénéfiques sur la santé humaine, mais ils peuvent aussi être perçus comme perturbateurs endocriniens, en particulier dans les laits pour nourrissons. La teneur et la composition en isoflavones de la graine diffèrent selon la fraction considérée. Les cotylédons contiennent de la génistéine et de la daidzéine, tandis que les germes, avec une teneur quatre à dix fois supérieure, contiennent majoritairement de la daidzéine et de la glycitéine. Le génotype influence fortement la teneur en isoflavones totales. Le déterminisme du pourcentage des isoflavones est essentiellement génétique. Ce travail porte sur l'étude du déterminisme génétique à l'origine des variations de teneurs et de compositions en isoflavones dans les germes et les cotylédons de la graine, en tenant compte également du net décalage de l'accumulation entre ces deux compartiments, au cours du développement de la graine. Dans un premier temps, les gènes des isoflavone synthases (IFS) de variétés très différenciées pour leurs teneurs et profils d'isoflavones ont été séquencés, puis les expressions des gènes clefs de la biosynthèse (neuf chalcone synthases (CHS), une chalcone réductase (CHR), quatre chalcone isomérases (CHI) et les deux isoflavone synthases (IFS) ont été suivies par RT-PCR quantitative dans les cotylédons et dans les germes, à trois stades critiques du développement de la graine (25, 40 et 60 jours après floraison). La seconde partie de ce travail a été consacrée à l'étude de l'expression de différents gènes candidats de la flavonoïde 6-hydroxylase (F6H) catalysant la première étape de la synthèse de la glycitéine. Le polymorphisme des séquences génomiques IFS1 et IFS2 des isoflavone synthases n'a pas montré de lien avec les différences de teneurs en isoflavones entre les variétés. L'activité transcriptionnelle des gènes de biosynthèse des isoflavones souligne l'existence d'une régulation bien distincte de cette synthèse dans ces deux compartiments. Les taux d'expression des gènes cibles ne sont pas toujours reliés avec les différences de teneurs ou de profils dans les germes et les cotylédons, suggérant ainsi l'effet prépondérant des régulations post-traductionnelles, notamment dans la formation du complexe multienzymatique de biosynthèse de ces composés. Nous avons aussi mis en évidence une forte expression du gène CHS9 codant pour la chalcone synthase 9, avec un profil correspondant plus à l'accumulation des isoflavones dans le germe que dans les cotylédons. Les gènes CHS7 et CHS8 codant pour les chalcone synthases 7 et 8, déjà signalés comme fortement corrélés à la synthèse des isoflavones, sont plus liés à l'accumulation dans les cotylédons que dans les germes. Ces travaux montrent aussi que le gène F6H signalé dans la littérature ne s'exprime pas dans les germes. En revanche, deux candidats dont la séquence est similaire à 79% ont été étudiés. Le gène F6H3 est le seul à s'exprimer dans la graine, uniquement dans le germe. Son expression n'a pas été détectée dans les germes d'une lignée mutante qui ne produit pas de glycitéine. Ce gène est donc un candidat potentiel clef pour la synthèse de la glycitéine dans le germe. La structure particulière de l'enzyme correspondante pourrait indiquer une forte implication de l'architecture du complexe enzymatique et des contraintes qui en découlent dans l'utilisation préférentielle d'une voie ou d'une autre dans ce schéma de biosynthèse. / The soybean seed contains large amounts of isoflavones (genistein, daidzein, and glycitein). Owing to their phytoestrogenic properties, these compounds can have beneficial effects on human health, but they can also be considered as endocrine disruptors, for example in infant formulas. The isoflavone content and composition in the seed depend on the considered fraction. The cotyledons contain only genistein and daidzein, while the hypocotyls are four to ten times more concentrated and contain three isoflavones, mostly daidzein and glycitein. The genotype has a strong influence on total isoflavone content, and even more on the percentage of individual isoflavones in cotyledons and hypocotyls. The objective of this work is to investigate the genetic determinism that underlies such contrasted contents and compositions between the two seed fractions, and the relation between main biosynthetic steps and genotypic differences. First, the genes of isoflavone synthases (IFS) were sequenced in varieties with highly contrasted content and composition. The expression of different keys genes of the biosynthesis (nine chalcone synthases (CHS), a chalcone reductase (CHR), four chalcone isomerases (CHI) and the two isoflavone synthases (IFS) have then been followed by quantitative RT - PCR in the cotyledons and hypocotyls, at three critical stages of seed development (25, 40 and 60 days after flowering). Second, the expression of different candidate genes for the flavonoid 6-hydroxylase (F6H) which catalyzes the first step in the synthesis of the glycitein has been investigated. The polymorphism of the genomic sequences IFS1 and IFS2 of isoflavone synthases was not correlated with differences in isoflavone contents. The transcriptional activity of key genes of the biosynthesis of isoflavones pointed out the existence of a distinct regulation of isoflavone biosynthesis between the two seed fractions. The expression levels of target genes were not always related to differences in isoflavone content or compositions in the hypocotyls and cotyledons. This suggests the overriding effect of post-translational regulation, especially in the formation of multienzyme complex of biosynthesis of these compounds. The chalcone synthase gene CHS9 was highly expressed, with a profile similar to the accumulation of isoflavones in hypocotyls. The chalcone synthase genes CHS7 and CHS8 expressions, already reported as highly correlated to the biosynthesis of isoflavones were more related to accumulation in the cotyledons than in hypocotyls. This work has also shown that the F6H gene, reported in the literature was not expressed in the hypocotyls. However, two candidates with as highly similar coding sequence (79%) have been studied. The F6H3 gene is the only one expressed in the seed, more precisely in the hypocotyls but it was not expressed in the cotyledons. Moreover, it was not expressed in a mutant line which did not accumulate glycitein. This gene is therefore a key potential candidate for the synthesis of the glycitein in hypocotyls. The particular structure of the corresponding enzyme may indicate a strong involvement of the architecture of the multienzyme complex of isoflavones biosynthesis and the constraints arising in the preferential use of a track or another in this scheme of biosynthesis.

Barley (Hordeum vulgare L.) is the fourth largest produced cereal worldwide. About two thirds of barley production is used to animal feed. When used to feed monogastric animals, the main shortcoming of barley grains is the deficiency of essential amino acids, especially lysine, threonine and methionine. The unbalanced amino acid composition is due to the main storage protein, the hordeins, which account for about 50% of total grain protein content. The nitrogen fertilization promotes C-hordein expression and accumulation, the hordein subgroup with the lowest content of essential amino acids, and the highest content of non-essential amino acids. Due to the importance of grain protein content and composition in the end use grain quality the key objective of the present study was to obtain a detailed insight into synthesis and accumulation of barley grain proteins and their relation to improved nutritional quality. An integrated proteomic and transcriptomic analysis have been undertaken in a set of transgenic antisense barley lines with the grain protein profile altered in comparison to the non-transgenic line cv. Golden Promise. The results were presented in three manuscripts in the thesis (chapters 2, 3 and 4). The first manuscript (chapter 2) reported a new grain protein extraction method combined with multi-method protein evaluation, including biochemical quantification, amino acid composition, sodium dodecyl-polyacrylamide gel electrophoresis (SDS-PAGE) couple with mass spectrometry (MS) identification and a gel free shotgun MS identification and relative quantification. The results showed the changeability of proteins between protein groups and the importance of choosing an adequate proteomic-based method for protein identification according to the complexity of protein mixtures. In the second manuscript (chapter 3) a differential protein profile of non-transgenic barley cv. Golden Promise and the transgenic antisense C-hordein barley lines was achieved by two-dimensional gel electrophoresis (2-DE) for salt soluble proteins and the differentially expressed proteins were identified by MS. The key results showed that the suppression of C-hordeins, the poor nutritional hordein subgroup, does not exclusively affects hordein synthesis and accumulation, and that the more balanced amino acid composition of these lines may be a consequence of distinct protein sources among different transgenic events, though a stable lysine-rich proteins upregulation occurs in all lines. In the third manuscript (chapter 4) the effects of nitrogen fertilization on hordein family at transcriptional and proteome level were assessed. The main results showed differential responses to N nutrition between non-transgenic and transgenic lines. In relation to C-hordein, specific C-hordein downregulation effect and in particular different responses to N were verified among subgroups of C-hordein multigene family in the transgenic line at transcriptional and proteomic level. In summary, the multi-method strategy used in the present work was successfully applied to obtain comprehensive information about barley grain proteins synthesis and accumulation and explain, at least in part, their relation to improved nutritional quality. These results can be useful in barley breeding programs aiming selective alterations of specific alleles/homologues to change amino acid composition by changing the relative proportions of the grain proteins in order to improve the barley grains nutritional quality. / A cevada (Hordeum vulgare L.) é o quarto cereal mais produzido no mundo. Cerca de dois terços desta produção é utilizada na alimentação animal. A principal desvantagem dos grãos de cevada na alimentação de animais monogástricos é a deficiência de aminoácidos essenciais, principalmente lisina, treonina e metionina. Esta composição desfavorável ocorre devido à principal proteína de reserva dos grãos, as hordeínas, que representam cerca de 50% do teor total de proteína no grão. O nitrogênio promove a expressão e o acúmulo de C-hordeínas, o subgrupo com o menor teor de aminoácidos essenciais e o maior teor de aminoácidos não essenciais. Devido à importância do teor e composição de proteínas nos grãos na determinação de sua qualidade no uso final, o principal objetivo do presente trabalho foi obter uma visão detalhada sobre a síntese e acúmulo de proteínas de grãos de cevada e sua relação com a melhoria da qualidade nutricional. Análises proteômicas e transcriptômicas integradas foram realizadas em um conjunto de linhagens transgênicas de cevada com o perfil de proteínas de reserva alterados em comparação à linhagem não transgênica cv. Golden Promise. Os resultados foram apresentados na forma de três manuscritos (capítulos 2, 3 e 4). O primeiro (capítulo 2) descreveu um novo método de extração de proteínas dos grãos em combinação com métodos de estudo de proteínas diversos, incluindo a quantificação bioquímica, composição de aminoácidos, eletroforese em gel de poliacrilamida-dodecil sulfato de sódio (SDS-PAGE) seguido de identificação por espectrometria de massas (MS) e estratégia shotgun para identificação e quantificação relativa das proteínas. Os resultados mostram a mutabilidade das proteínas entre os diferentes grupos e a importância da escolha de um método adequado para a sua identificação de acordo com a complexidade das misturas proteicas. No segundo manuscrito (capítulo 3) o perfil proteico diferencial da linhagem não transgênica e transgênica foi obtido por eletroforese bidimensional (2-DE) para proteínas solúveis, e aquelas expressas diferencialmente foram identificadas por MS. Os resultados demonstram que a supressão das C-hordeínas não afeta exclusivamente a síntese e o acúmulo de hordeínas, e que a composição de aminoácidos mais equilibrada destas linhagens pode ser uma consequência de fontes de proteína distintas entre os diferentes eventos de transgenia, embora a regulação positiva de proteínas ricas em lisina foi estável. No terceiro manuscrito (capítulo 4) foram avaliados os efeitos da adubação nitrogenada sobre a família das hordeínas. Os resultados mostraram que as respostas foram diferentes entre as linhagens não transgênica e transgênica. Um efeito específico de supressão e respostas particulares foi verificado entre os subgrupos da família multigênica das C-hordeínas na linhagem transgênica. Em resumo, a estratégia de pesquisa multimétodo foi aplicada com sucesso na obtenção de informações abrangentes sobre a síntese e o acúmulo de proteínas nos grãos de cevada, e pelo menos em parte, explicou sua relação com a melhoria da qualidade nutricional. Esses resultados podem ser úteis em programas de melhoramento de cevada que visam alterações seletivas de alelos/homólogos específicos para alterar a composição de aminoácidos, através de mudanças nas proporções relativas das proteínas dos grãos.