Made available in DSpace on 2016-06-02T20:21:27Z (GMT). No. of bitstreams: 1 3871.pdf: 3349931 bytes, checksum: 699bd6092d3da7218a1550f52bb10cdb (MD5) Previous issue date: 2011-05-27 / Financiadora de Estudos e Projetos / Litopenaeus vannamei is known as Pacific white shrimp and is the main species marketed worldwide. Its geographical distribution includes the Pacific coast, ranging from Mexico to Peru. Due to its outstanding economic importance, this species has been farmed and showing great adapting levels to captivity, having been introduced in several countries, including Brazil. However, despite all the concern to properly manage the farming populations, many countries have not had a renewal of their breeding herds because of the risk of introduction of exogenous pathogens, which has increased the degree of inbreeding of these stocks and decreased levels of genetic diversity. To monitor this loss, microsatellite markers or SSRs (Simple Sequence Repeats), from genome arbitrary and expressed regions have been used. In the specific case of SSRs present in expressed regions of the genome (ESTs, Expressed Sequence Tags) or SSRs-ESTs, they allow the access to the genetic variability of populations, and the ESTs markers devoid of SSRs may be related to genes of interest, subsidizing the development of breeding programs based on Marker Assisted Selection (MAS). Moreover, EST-SSRs loci may show an excellent rate of transferability in taxonomically related species, since they are in more conserved regions of the genome. Within this context, this study aimed (i) the validation population of EST-SSR loci isolated through dataming from L. vannamei ESTs database (www.shrimp.ufscar.br), (ii) the genomic annotation of ESTs and EST-SSRs markers (iii) the determination of EC numbers (Enzyme Codes), aiming respectively, (i) the characterization of polymorphic markers (ii) the description of genes and their protein products and (iii) the establishment of information to describe the possible pathways for the penaeid group. Therefore, we tested 32 EST-SSRs loci in PCR reactions. After establishing the best pattern of reaction and subsequent loci genotyping, nine SSRs-ESTs were polymorphic, with allele number ranging from two to 20, levels of observed heterozygosity from 0.32 to 0.86 and average PIC (Polymorphism Information Content) of 0.78. No pair of loci presented in linkage disequilibrium. However, after Bonferroni correction, we found that one of these showed a significant deficit of heterozygotes. The nine polymorphic loci from L.vannamei showed satisfactory amplification in at least one of the seven native species tested: the marine ones Xiphopenaeus kroyeri, Farfantepenaeus brasiliensis, Farfantepenaeus paulensis, Rimapenaeus constrictus and Litopenaeus schmitti and the freshwater ones Macrobrachium amazonicum and Macrobrachium jeskii, being useful also for genetic studies of these species. The genome annotation performed by access in ShEST website (Litopenaeus vannamei EST Genome Project) showed that only three of nine loci 4 have the gene and its protein product described. The other loci showed no matches with any other genomic database available for research. In this work, gene product could be elucidated for 99% of ESTs, being possible to establish 209 EC numbers, highlighting enzymes responsible for xenobiotics metabolism, immunity, energy production, reproduction, oxidative stress, among others. These codes were used for construction of some metabolic pathways present in the penaeid group, contributing for building a wide database of the genome of this important animal group. These data may be applied in genetic improvement programs as well as gene expression studies, such as realtime PCR and microarrays. / Litopenaeus vannamei é conhecido como camarão branco do Pacífico e é a principal espécie de peneídeo comercializada no mundo. Sua distribuição geográfica compreende a costa do Oceano Pacífico, indo desde o México até o Peru. Devido a sua relevante importância econômica, esta espécie passou a ser cultivada, adaptando-se bem às condições de cativeiro, tendo sido introduzida em diversos países, incluindo o Brasil. Entretanto, apesar de toda preocupação em manejar adequadamente as populações de cultivo, muitos países não têm tido renovação de seus estoques reprodutores ou plantéis, devido ao risco de introdução de patógenos exógenos, o que tem aumentado o grau de endogamia desses estoques e diminuição dos níveis de diversidade genética. Para monitorar esta perda de variabilidade genética, marcadores microssatélites ou SSRs (Simple Sequence Repeats), oriundos de regiões arbitrárias e expressas do genoma desta espécie, vêm sendo utilizados. No caso específico de SSRs presentes em regiões expressas do genoma (ESTs, Expressed Sequence Tags) ou SSRs-ESTs, além destes permitirem acessar a variabilidade genética das populações, as ESTs desprovidas de SSRs podem estar relacionadas a genes de interesse, podendo auxiliar o desenvolvimento de programas de melhoramento genético baseados na Seleção Assistida por Marcadores (MAS). Além disso, locos SSRs-ESTs podem apresentar uma excelente taxa de transferabilidade em espécies taxonomicamente relacionadas, uma vez que se encontram em regiões mais conservadas do genoma. Dentro deste contexto, este trabalho teve como objetivos (i) a validação populacional de locos SSRs-ESTs, isolados via dataming no banco de dados de ESTs de L. vannamei (www.shrimp.ufscar.br); (ii) a anotação genômica de marcas ESTs e locos SSRs-ESTs (iii) e a determinação dos EC numbers (códigos enzimáticos), visando, respectivamente, (i) a caracterização de marcadores polimórficos, (ii) a descrição de genes e seus respectivos produtos protéicos e (iii) o estabelecimento de informações para descrever as possíveis vias metabólicas para o grupo de peneídeos. Para tanto foram testados 32 locos SSRs-ESTs em reações de PCR. Após estabelecimento do melhor perfil de reação e posterior genotipagem dos locos, nove SSRs-ESTs mostraram-se polimórficos, com número de alelos variando de 4 a 20, níveis de heterozigozidade observada de 0,32 a 0,86 e valores médios de PIC (Polymorphism Information Content) de 0,78. Nenhum loco apresentou-se em desequilíbrio de ligação. Entretanto, após correção de Bonferroni, constatou-se que um deles apresentou déficit significativo de heterozigotos. Os nove locos polimórficos para L. vannamei apresentaram amplificação satisfatória em pelo menos uma das sete espécies nativas testadas: as marinhas Xiphopenaeus kroyeri, 2 Farfantepenaeus brasiliensis, Farfantepenaeus paulensis, Rimapenaeus constrictus e Litopenaeus schmitti e as de água doce Macrobrachium amazonicum e Macrobrachium jeskii podendo se constituir em marcas úteis para os estudos genéticos também dessas espécies. A anotação genômica realizada via acesso à página de anotação do Projeto ShEST (Projeto Genoma EST de Litopenaeus vannamei) demonstrou que apenas três dos nove locos polimórficos têm o seu gene e produto protéico já descritos. Os demais locos não apresentaram blasts positivos com nenhuma outra base de dados genômicos disponíveis para pesquisa. No caso das sequências anotadas, o produto gênico pôde ser elucidado para 99% destas, sendo possível estabelecer 209 EC numbers, destacando-se enzimas responsáveis pela degradação de xenobióticos, imunidade, produção de energia, reprodução, estresse oxidativo, dentre outras. Estes códigos enzimáticos foram utilizados para determinação de algumas vias metabólicas presentes no grupo dos peneídeos, contribuindo assim para a construção de uma ampla base de dados do genoma deste importante grupo animal, podendo ser utilizada em estudos de conservação, programas de melhoramento genético e em análises de expressão gênica, como PCR em tempo real e microarrays.

MiRNAs são pequenas moléculas de RNAs endógenos não codificantes com aproximadamente 23nt que atuam na regulação da expressão gênica. A sua função é inibir a tradução de genes transcritos através de um mecanismo que viabiliza a ligação do miRNA com o mRNA alvo levando à inibição da tradução ou a degradação do RNA mensageiro. Estudos evidenciam a relação dos miRNAs com diversos processos biológicos como proliferação celular, diferenciação, desenvolvimento e doenças. Uma vez que estão envolvidos na regulação gênica, também alteram as vias metabólicas. Atualmente, as ferramentas computacionais disponíveis para o estudo dos miRNAs são o miRBase, microCosm, o miRGen e o miRNAmap. Elas possuem informações sobre as sequências dos miRNAs, genes alvos e sobre elementos que estão próximos à região dos miRNAs. Embora o avanço até o momento, não existia que relacionasse os miRNAs com as vias metabólicas, para isso foi construída a plataforma miRNApath que auxilia no estudo da função dos miRNAs por meio da análise do seus alvos dentro vias metabólicas. De modo semelhante, também não existia uma abordagem que relacione dados de expressão miRNAs e seus alvos dentro de um mesmo experimento. Para tanto, neste trabalho foi feita uma abordagem utilizando bibliotecas de SAGE (Serial Analysis of Gene Expression) que será incorporada no miRNApath. O miRNApath encontra-se disponível em http://lgmb.fmrp.usp.br/mirnapath. / MiRNAs are small molecules of endogenous non-coding RNAs with approximately 23nt in length that acts over gene expression regulation. Its function is inhibit the translation of gene transcripts through a mechanism that links the miRNA with its mRNA target leading to a translational repression or degradation. Studies show the relation of RNAs in many biological processes like cell proliferation, dierentiation and development of diseases. Since they are involved in gene regulation, they also change the metabolic pathways. Currently, the available computational tools for the study of miRNAs are miRBase, microCosm, miRGen and miRNAmap. They have information about miRNAs sequences, targets and features. Despite the the advances, until now, there is no tool that correlates the miRNAs with metabolic pathways, therefore we developed the miRNApath platform that helps in the analysis of miRNAs function through the study of its targets that are into the metabolic pathway. In the same way, there is no approach that put together information of expression of miRNAs and its targets in the same experiment. In this work we develop an approach with SAGE (Serial Analysis of Gene Expression ) libraries that will be integrated to miRNApath. The plataform is avaible at http://lgmb.fmrp.usp.br/mirnapath.

Lors de cette thèse, nous avons présenté trois principaux types d'analyses du métabolisme, dont la plupart impliquaient la symbiose : dialogue métabolique entre un trypanosomatide et son symbiote, analyses comparatives de réseaux métaboliques et exploration de données métabolomiques. Tous ont été essentiellement basés sur des données de génomique où les capacités métaboliques ont été prédites à partir des gènes annotés de l'organisme cible, et ont été affinées avec d'autres types de données en fonction de l'objectif et de la portée de chaque analyse. Le dialogue métabolique entre un trypanosomatide et son symbiote a été explorée avec des objectifs fonctionnels et évolutifs qui comprenaient une analyse des voies de synthèse des acides aminés essentiels et des vitamines telles que ces voies sont classiquement définies, une exploration de réseaux complets métaboliques et une recherche de potentiels transferts horizontaux de gènes des bactéries vers les trypanosomatides. Les analyses comparatives effectuées ont mis l'accent sur les capacités métaboliques communes de bactéries appartenant à différents groupes de vie, et nous avons proposé une méthode pour établir automatiquement les activités métaboliques communes ou spécifiques à chaque groupe. Nous avons appliqué notre méthode d'énumération d'histoires métaboliques à la réponse de la levure à une exposition au cadmium comme une validation de cette approche sur une réaction au stress bien étudiée. Nous avons montré que la méthode a bien capté la connaissance que nous avons de cette réponse en plus de permettre de nouvelles interprétations des données métabolomiques mappées sur le réseau métabolique complet de la levure / In this thesis, we presented three main types of analyses of metabolism, most of which involved symbiosis: metabolic dialogue between a trypanosomatid and its symbiont, comparative analyses of metabolic networks and exploration of metabolomics data. All of them were essentially based on genomics data where metabolic capabilities were predicted from the annotated genes of the target organism, and were further refined with other types of data depending on the aim and scope of each investigation. The metabolic dialogue between a trypanosomatid and its symbiont was explored with functional and evolutionary goals which included analysing the classically defined pathways for the synthesis of essential amino acids and vitamins, exploring the genome-scale metabolic networks and searching for potential horizontal gene transfers from bacteria to the trypanosomatids. The comparative analyses performed focused on the common metabolic capabilities of different lifestyle groups of bacteria and we proposed a method to automatically establish the common and the group-specific activities. The application of our method on metabolic stories enumeration to the yeast response to cadmium exposure was a validation of this approach on a well-studied biological response to stress. We showed that the method captured well the underlying knowledge as it extracted stories allowing for further interpretations of the metabolomics data mapped into the genome-scale metabolic model of yeast

Genes can be associated in numerous ways, e.g. by co-expression in micro-arrays, co-regulation in operons and regulons or co-localization on the genome. Association of genes often indicates that they contribute to a common biological function, such as a pathway. The aim of this thesis is to predict metabolic pathways from associated enzyme-coding genes. The prediction approach developed in this work consists of two steps: First, the reactions are obtained that are carried out by the enzymes coded by the genes. Second, the gaps between these seed reactions are filled with intermediate compounds and reactions. In order to select these intermediates, metabolic data is needed. This work made use of metabolic data collected from the two major metabolic databases, KEGG and MetaCyc. The metabolic data is represented as a network (or graph) consisting of reaction nodes and compound nodes. Interme- diate compounds and reactions are then predicted by connecting the seed reactions obtained from the query genes in this metabolic network using a graph algorithm.<p>In large metabolic networks, there are numerous ways to connect the seed reactions. The main problem of the graph-based prediction approach is to differentiate biochemically valid connections from others. Metabolic networks contain hub compounds, which are involved in a large number of reactions, such as ATP, NADPH, H2O or CO2. When a graph algorithm traverses the metabolic network via these hub compounds, the resulting metabolic pathway is often biochemically invalid.<p>In the first step of the thesis, an already existing approach to predict pathways from two seeds was improved. In the previous approach, the metabolic network was weighted to penalize hub compounds and an extensive evaluation was performed, which showed that the weighted network yielded higher prediction accuracies than either a raw or filtered network (where hub compounds are removed). In the improved approach, hub compounds are avoided using reaction-specific side/main compound an- notations from KEGG RPAIR. As an evaluation showed, this approach in combination with weights increases prediction accuracy with respect to the weighted, filtered and raw network.<p>In the second step of the thesis, path finding between two seeds was extended to pathway prediction given multiple seeds. Several multiple-seed pathay prediction approaches were evaluated, namely three Steiner tree solving heuristics and a random-walk based algorithm called kWalks. The evaluation showed that a combination of kWalks with a Steiner tree heuristic applied to a weighted graph yielded the highest prediction accuracy.<p>Finally, the best perfoming algorithm was applied to a microarray data set, which measured gene expression in S. cerevisiae cells growing on 21 different compounds as sole nitrogen source. For 20 nitrogen sources, gene groups were obtained that were significantly over-expressed or suppressed with respect to urea as reference nitrogen source. For each of these 40 gene groups, a metabolic pathway was predicted that represents the part of metabolism up- or down-regulated in the presence of the investigated nitrogen source.<p>The graph-based prediction of pathways is not restricted to metabolic networks. It may be applied to any biological network and to any data set yielding groups of associated genes, enzymes or compounds. Thus, multiple-end pathway prediction can serve to interpret various high-throughput data sets. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Polyhydroxybutyrate (PHB) is a polyhydroxyalkanoate that has been extensively studied as a potential biodegradable replacement for petrochemically derived plastics. The synthesis pathway of PHB is native to Ralstonia eutropha, but the genes for the PHB pathway have successfully been introduced into Escherichia coli through plasmids such as the pBHR68 plasmid. However, the production of PHB needs to be more cost-effective before it can be commercially produced. A mathematical model for PHB synthesis was developed to identify target genes that could be genetically engineered to increase PHB production. The major metabolic pathways included in the model were glycolysis, acetyl coenzyme A (acetyl-CoA) synthesis, tricarboxylic acid (TCA) cycle, glyoxylate bypass, and PHB synthesis. Each reaction in the selected metabolic pathways was modeled using the kinetic mechanism identified for the associated enzyme. The promoters and transcription factors for each enzyme were incorporated into the model. The model was validated through comparison with other published models and experimental PHB production data. The predictive model identified 16 enzymes as having no effect on PHB production, 5 enzymes with a slight effect on PHB production, and 9 enzymes with large effects on PHB production. Decreasing the substrate affinity of the enzyme citrate synthase resulted in the largest increase in PHB synthesis. The second largest increase was observed from lowering the substrate affinity of glyceraldehyde-3-phosphate dehydrogenase. The predictive model also indicated that increasing the activity of the lac promoter in the pBHR68 plasmid resulted in the largest increase in the rate of PHB production. The predictive model successfully identified two genes and one promoter as targets for genetic engineering to create an optimized strain of E. coli for PHB production. The substrate-binding sites for the genes gltA (citrate synthase) and gapA (glyceraldehyde-3-phosphate dehydrogenase) should be genetically engineered to be less effective at binding the substrates. The lac promoter in the pBHR68 plasmid should be genetically engineered to more closely match the consensus sequence for binding to RNA polymerase. The model predicts that an optimized strain of E. coli for PHB production could be achieved by genetically altering gltA, gapA, and the lac promoter.

京都大学 / 新制・課程博士 / 博士(エネルギー科学) / 甲第24252号 / エネ博第450号 / 新制||エネ||84(附属図書館) / 京都大学大学院エネルギー科学研究科エネルギー基礎科学専攻 / (主査)教授 森井 孝, 教授 片平 正人, 教授 佐川 尚 / 学位規則第4条第1項該当 / Doctor of Energy Science / Kyoto University / DFAM

L’objectif de ce projet était de faire le lien entre gènes et métabolites afin d’éventuellement proposer des métabolites à mesurer en lien avec la fonction de gènes. Plus particulièrement, nous nous sommes intéressés aux gènes codant pour des protéines ayant un impact sur le métabolisme, soit les enzymes qui catalysent les réactions faisant partie intégrante des voies métaboliques. Afin de quantifier ce lien, nous avons développé une méthode bio-informatique permettant de calculer la distance qui est définie comme le nombre de réactions entre l’enzyme encodée par le gène et le métabolite dans la carte globale du métabolisme de la base de données Kyoto Encyclopedia of Genes and Genomes (KEGG). Notre hypothèse était que les métabolites d’intérêt sont des substrats/produits se trouvant à proximité des réactions catalysées par l’enzyme encodée par le gène. Afin de tester cette hypothèse et de valider la méthode, nous avons utilisé les études d’association pangénomique combinées à la métabolomique (mGWAS) car elles rapportent des associations entre variants génétiques, annotés en gènes, et métabolites mesurés. Plus précisément, la méthode a été appliquée à l’étude mGWAS par Shin et al. Bien que la couverture des associations de Shin et al. était limitée (24/299), nous avons pu valider de façon significative la proximité entre gènes et métabolites associés (P<0,01). En somme, cette méthode et ses développements futurs permettront d’interpréter de façon quantitative les associations mGWAS, de prédire quels métabolites mesurer en lien avec la fonction d’un gène et, plus généralement, de permettre une meilleure compréhension du contrôle génétique sur le métabolisme. / The objective of this project was to link genes and metabolites in order to ultimately predict which metabolites to measure in order to adequately reflect the function of a given gene. Specifically, we were interested in genes, which code for proteins that regulate substrate metabolism, hence enzymes that catalyze reactions that are part of metabolic pathways. In order to quantify this link, we have developed a bioinformatics method to calculate a distance, which is defined as the number of reactions separating a given selected gene-encoded enzyme and its metabolite of interest in Kyoto Encyclopedia of Genes and Genomes (KEGG) database’s metabolic overview map. Our hypothesis was that metabolites of interest are products/substrates found at proximity of the reactions catalyzed by the selected gene-encoded enzyme. In order to test our hypothesis and validate the method, we have used genome-wide association study of metabolites levels (mGWAS) because these studies report associations between genetic variants, annotated to genes, and measured metabolites. More specifically, we used the mGWAS conducted by Shin et al. Even though the coverage of the associations reported by Shin et al. was limited (24/299), we significantly validated the proximity between gene-metabolite associated pairs (P<0.01). Overall, the method and its future developments will allow the quantitative interpretation of mGWAS associations, predict which metabolite to measure with regards to the function of a gene and, in general, enable a better understanding of the genetic control of metabolism.

La comparaison inter-espèces de voies métaboliques est une problématique importante en biologie. Actuellement, les connaissances sont générées à partir d'expériences sur un nombre relativement limité d'espèces dites modèles. Mieux connaître une espèce permet de valider ou non une inférence faite à partir de ces données expérimentales et de déterminer si ou dans quelle mesure des résultats obtenus sur une espèce modèle peuvent être transposés à une autre espèce. Cette thèse propose une méthode de comparaison inter-espèces de voies métaboliques. Elle compare chaque étape d'une voie métabolique en exploitant les annotations dans Gene Ontology qui leur sont associées. Ce travail valide l'intérêt des mesures de similarités sémantiques pour interpréter ces annotations, propose d'utiliser conjointement une mesure de particularité sémantique et propose une méthode basée sur des motifs de similarité et de particularité pour interpréter chaque étape de voie métabolique. De nombreuses mesures sémantiques quantifient la similarité entre des produits de gènes en fonction des annotations qu'ils ont en commun. Nous en avons identifié et utilisé une adaptée à la problématique de comparaison inter-espèces. En se focalisant sur la part commune aux produits de gènes comparés, les mesures de similarité sémantiques ignorent les caractéristiques spécifiques d'un seul produit de gène. Or la comparaison inter-espèces de voies métaboliques se doit de quantifier non seulement la similarité des produits de gènes qui interviennent dans celles-ci, mais également leurs particularités. Nous avons développé une mesure de particularité sémantique répondant à cette problématique. Pour chaque étape de voie métabolique, nous calculons un profil composé de sa valeur de similarité et de ses deux valeurs de particularité sémantiques. Il n'est pas possible d'établir formellement que deux produits de gènes sont similaires ou que l'un d'eux a des particularités significatives sans disposer d'un seuil de similarité et d'un seuil de particularité. Jusqu'à présent, ces interprétations se faisaient sur la base d'un seuil implicite ou arbitraire. Pour combler ce manque, nous avons développé une méthode de définition de seuils pour les mesures de similarité et de particularité sémantiques. Nous avons enfin appliqué une mesure de similarité inter-espèces et notre mesure de particularité pour comparer le métabolisme des lipides entre l'Homme, la souris et la poule. Nous avons pu interpréter les résultats à l'aide des seuils que nous avions définis. Chez les trois espèces, des particularités ont pu être observées, y compris au niveau de produits de gènes similaires. Elles concernent notamment des processus biologiques et des composants cellulaires. Les fonctions moléculaires présentent une forte similarité et peu de particularités. Ces résultats sont biologiquement pertinents. / Cross-species comparison of metabolic pathways is an important task in biology. It is a major stake for both human health and agronomy. Currently, knowledge is acquired from some experiments on a relatively low number of species referred to as ``models''. A better understanding of a species determines whether to validate or not an inference made from these experimental data. It also determines whether or to what extent results obtained on model species can be transposed to another species. This thesis proposes a cross-species metabolic pathways comparison method. Our method compares each step of a metabolic pathway using the associated Gene Ontology annotations. This work validates the interest of the semantic similarity measures for interpreting these annotations, proposes to use jointly a semantic particularity measure and proposes a method based on similarity and particularity patterns to interpret each metabolic pathway step. Several gene products are involved throughout a metabolic pathway. They are associated to some annotations in order to describe their biological roles. Based on a shared ontology, these annotations allow to compare data from different species and to take into account several level of abstraction. Several semantic measures quantifying the similarity between gene products from their annotations have been developed previously. We have identified and used a semantic similarity measure appropriate for cross-species comparisons. Because they focus on the common part of the compared gene products, the semantic similarity measures ignore their specific characteristics. Therefore, cross-species metabolic pathways comparison has to quantify not only the similarity of the gene products involved, but also their particularity. We have developed a semantic particularity measure addressing this issue. For each pathway step, we proposed to create a profile combining its semantic similarity and its two semantic particularity values. Concerning the results interpretation, it is not possible to establish formally that two gene products are similar or that one of them have some significant particularities without having a similarity threshold and a particularity threshold. So far, these interpretations were based on an implicit or an arbitrary threshold. To address this gap, we developed a threshold definition method for the semantic similarity and particularity measures. We last applied a cross-species similarity measure and our particularity measure to compare the lipid metabolism between human, mice and chicken. We then interpreted the results using the previously defined thresholds. In all three species, we observed some particularities, including on similar genes. They concerned notably some biological processes and cellular components. The molecular functions present a strong similarity and few particularities. These results are biologically relevant.

Orientador: Fernando Sebastián Baldi Rey / Resumo: A compreensão de processos regulatórios e organização molecular dos organismos vivos progrediram consideravelmente na última década. As metodologias também evoluíram com o sequenciamento de DNA e RNA e de ferramentas genômicas permitindo a análise de centenas ou milhares de genes, proteínas ou metabólitos. O uso simultâneo dessas informações auxilia na obteção de informações relevantes sobre as variáveis que envolvem as variações fenotípicas de características de interesse. O objetivo do presente estudo foi integrar dados fenotípicos, genotípicos e transcriptômicos em busca de aprimoramento sobre os mecanismos genéticos e metabólicos que determinam o perfil de ácidos graxos na carne de bovinos Nelore, a fim de contribuir para o melhoramento da composição de ácidos graxos da carne. Foram utilizados machos da raça Nelore terminados em confinamento, abatidos com média de idade 24 meses. Amostras do músculo L. thoracis, entre a 12ª a 13ª costela foram coletadas para as análises de perfil de ácidos graxos, extração de RNA e de DNA. Os resultados foram apresentados nos capítulos 2 e 3. No capítulo 2, o objetivo foi identificar genes diferencialmente expressos pelo método RNA-seq e perfil de ácidos graxos no músculo L. thoracis com uso de componentes principais (principal components: PC). Foram selecionados dois grupos de 10 animais, os quais possuíam valores de PC1 e PC2 extremos (Alto x Baixo) para os grupos somatórios de ácidos graxos (AG): ácidos graxos saturados (AGS), ácidos g... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The understanding of regulatory processes and molecular organization of organisms has progressed considerably in the last 10 years.The methodologies also evolved with the sequencing of DNA, RNA and genomic tools allowing the analysis a lot of genes, proteins or metabolites. The simultaneous use of this information should help to obtain relevant information about the variables that result the phenotypic variations of traits of interest. The objective of the present study was to integrate phenotypic, genotypic and transcriptomic studies in order to clarify the genetic and metabolic mechanisms that determine the fatty acid profile in Nelore beef, in order to contribute to the improvement of the fatty acid composition of the meat. Nelore males were used in feedlot, coming from farms that integrate three breeding programs and slaughtered with an average of 24 months. Samples of the L. thoracis muscle between the 12th to 13th rib were collected for analysis of fatty acid profile, RNA and DNA extraction. The results were presented in chapters 2 and 3. In chapter 2, the objective was to identify genes differentially expressed by RNA-seq method and fatty acid profile in the L. thoracis muscle with the use of Principal Components (PC). Two groups of 10 animals were selected, which had PC1 and PC2 extreme values (High x Low) for the fatty acids (FA) groups: saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), polyunsaturated fatty acids (PUFA), omega 3 (n-3) and omega 6 (n-6... (Complete abstract click electronic access below) / Doutor

A levedura Saccharomyces cerevisiae, amplamente utilizada na conversão de glicose e frutose a etanol, não é capaz de fermentar a xilose presente na biomassa lignocelulósica de resíduos agroindustriais. Apesar da introdução da via metabólica dessa pentose em linhagens de S. cerevisiae, a fermentação da xilose simultaneamente com outros açúcares ainda é pouco eficiente. A proposta deste trabalho foi aumentar a eficiência do consumo da xilose por linhagens de S. cerevisiae introduzindo genes de transportadores exógenos identificados em leveduras selvagens que naturalmente fermentam pentoses. A via do metabolismo da xilose foi integrada no genoma de uma linhagem industrial brasileira de S. cerevisiae usada na produção de etanol. A partir desta, linhagens isogênicas foram criadas e mostraram ser mais eficientes no metabolismo da xilose em meio sintético e capazes de co-fermentar glicose e xilose na presença de altas concentrações de inibidores resultantes da hidrólise da biomassa lignocelulósica. Os tranportadores identificados foram testados nas linhagens industriais geneticamente modificadas criadas neste estudo e em linhagens laboratoriais. Não foi possível confirmar a eficiência dos transportadores nas linhagens, embora os resultados mostraram diferenças nas curvas de crescimento das linhagens industriais expressando os transportadores. Este trabalho foi o início de um estudo dos fatores envolvidos no metabolismo da xilose e servirá como base para que futuros trabalhos sejam realizados na obtenção de uma linhagem mais eficiente para produção de etanol de segunda geração. / The yeast Saccharomyces cerevisiae, which efficiently ferments glucose and fructose to ethanol, is unable to ferment xylose present in lignocellulosic biomass of agroindustrial residues. Although the introduction of xylose metabolic pathways in S. cerevisiae strains has been described in the literature, the simultaneous fermentation of xylose and glucose in these modified strains is still very inefficient. The aim of this study was to increase the xylose consumption efficiency of S. cerevisiae by introduction of exogenous genes identified in wild yeast that naturally ferment pentose. The xylose metabolism pathway was integrated into the genome of a Brazilian industrial strain of S. cerevisiae used for the production of ethanol, which was then used to obtain isogenic modified strains. The isogenic strains showed to be more effective in xylose metabolism in synthetic medium and able to co-ferment glucose and xylose in the presence of high concentrations of inhibitors resulting hydrolysis of lignocellulosic biomass. The transporters identified were inserted into genetically modified industrial strains of S. cerevisiae created in this study and also in laboratory strains. It was not possible to confirm the transporters efficiency in laboratory strains but the results showed differences in the growth curves of the industrial strains expressing the transporters. This work was the beginning of a study of the factors involved in xylose metabolism and it will help to prepare future work to obtain an efficient strain for lignocellulosic ethanol production.