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<p>Availability of genome sequences for numerous bacterial species comprising of different bacterial strains allows elucidation of species and strain specific adaptations that facilitate their survival in widely fluctuating micro-environments and enhance their pathogenic potential. Different bacterial species use different strategies in their pathogenesis and the pathogenic potential of a bacterial species is dependent on its genomic complement of virulence factors. A bacterial virulence factor, within the context of this study, is defined as any endogenous protein product encoded by a gene that aids in the adhesion, invasion, colonization, persistence and pathogenesis of a bacterium within a host. Anecdotal evidence suggests that bacterial virulence genes are undergoing diversifying evolution to counteract the rapid adaptability of its host&rsquo / s immune defences. Genome sequences of pathogenic bacterial species and strains provide unique opportunities to study the action of diversifying selection operating on different classes of bacterial genes.</p>

<p>Availability of genome sequences for numerous bacterial species comprising of different bacterial strains allows elucidation of species and strain specific adaptations that facilitate their survival in widely fluctuating micro-environments and enhance their pathogenic potential. Different bacterial species use different strategies in their pathogenesis and the pathogenic potential of a bacterial species is dependent on its genomic complement of virulence factors. A bacterial virulence factor, within the context of this study, is defined as any endogenous protein product encoded by a gene that aids in the adhesion, invasion, colonization, persistence and pathogenesis of a bacterium within a host. Anecdotal evidence suggests that bacterial virulence genes are undergoing diversifying evolution to counteract the rapid adaptability of its host&rsquo / s immune defences. Genome sequences of pathogenic bacterial species and strains provide unique opportunities to study the action of diversifying selection operating on different classes of bacterial genes.</p>

Philosophiae Doctor - PhD (Biotechnology) / Availability of genome sequences for numerous bacterial species comprising of different bacterial strains allows elucidation of species and strain specific adaptations that facilitate their survival in widely fluctuating micro-environments and enhance their pathogenic potential. Different bacterial species use different strategies in their pathogenesis and the pathogenic potential of a bacterial species is dependent on its genomic complement of virulence factors. A bacterial virulence factor, within the context of this study, is defined as any endogenous protein product encoded by a gene that aids in the adhesion, invasion, colonization, persistence and pathogenesis of a bacterium within a host. Anecdotal evidence suggests that bacterial virulence genes are undergoing diversifying evolution to counteract the rapid adaptability of its host&rsquo;s immune defences. Genome sequences of pathogenic bacterial species and strains provide unique opportunities to study the action of diversifying selection operating on different classes of bacterial genes. / South Africa

Les avancées dans l'analyse de l'expression différentielle de gènes ont suscité un vif intérêt pour l'étude d'ensembles de gènes présentant une similarité d'expression au cours d'une même condition expérimentale. Les approches classiques pour interpréter l'information biologique reposent sur l'utilisation de méthodes statistiques. Cependant, ces méthodes se focalisent sur les gènes les plus connus tout en générant des informations redondantes qui peuvent être éliminées en prenant en compte la structure des ressources de connaissances qui fournissent l'annotation. Au cours de cette thèse, nous avons exploré différentes méthodes permettant l'annotation d'ensembles de gènes.Premièrement, nous présentons les solutions visuelles développées pour faciliter l'interprétation des résultats d'annota-tion d'un ou plusieurs ensembles de gènes. Dans ce travail, nous avons développé un prototype de visualisation, appelé MOTVIS, qui explore l'annotation d'une collection d'ensembles des gènes. MOTVIS utilise ainsi une combinaison de deux vues inter-connectées : une arborescence qui fournit un aperçu global des données mais aussi des informations détaillées sur les ensembles de gènes, et une visualisation qui permet de se concentrer sur les termes d'annotation d'intérêt. La combinaison de ces deux visualisations a l'avantage de faciliter la compréhension des résultats biologiques lorsque des données complexes sont représentées.Deuxièmement, nous abordons les limitations des approches d'enrichissement statistique en proposant une méthode originale qui analyse l'impact d'utiliser différentes mesures de similarité sémantique pour annoter les ensembles de gènes. Pour évaluer l'impact de chaque mesure, nous avons considéré deux critères comme étant pertinents pour évaluer une annotation synthétique de qualité d'un ensemble de gènes : (i) le nombre de termes d'annotation doit être réduit considérablement tout en gardant un niveau suffisant de détail, et (ii) le nombre de gènes décrits par les termes sélectionnés doit être maximisé. Ainsi, neuf mesures de similarité sémantique ont été analysées pour trouver le meilleur compromis possible entre réduire le nombre de termes et maintenir un niveau suffisant de détails fournis par les termes choisis. Tout en utilisant la Gene Ontology (GO) pour annoter les ensembles de gènes, nous avons obtenu de meilleurs résultats pour les mesures de similarité sémantique basées sur les nœuds qui utilisent les attributs des termes, par rapport aux mesures basées sur les arêtes qui utilisent les relations qui connectent les termes. Enfin, nous avons développé GSAn, un serveur web basé sur les développements précédents et dédié à l'annotation d'un ensemble de gènes a priori. GSAn intègre MOTVIS comme outil de visualisation pour présenter conjointement les termes représentatifs et les gènes de l'ensemble étudié. Nous avons comparé GSAn avec des outils d'enrichissement et avons montré que les résultats de GSAn constituent un bon compromis pour maximiser la couverture de gènes tout en minimisant le nombre de termes.Le dernier point exploré est une étape visant à étudier la faisabilité d'intégrer d'autres ressources dans GSAn. Nous avons ainsi intégré deux ressources, l'une décrivant les maladies humaines avec Disease Ontology (DO) et l'autre les voies métaboliques avec Reactome. Le but était de fournir de l'information supplémentaire aux utilisateurs finaux de GSAn. Nous avons évalué l'impact de l'ajout de ces ressources dans GSAn lors de l'analyse d’ensembles de gènes. L'intégration a amélioré les résultats en couvrant d'avantage de gènes sans pour autant affecter de manière significative le nombre de termes impliqués. Ensuite, les termes GO ont été mis en correspondance avec les termes DO et Reactome, a priori et a posteriori des calculs effectués par GSAn. Nous avons montré qu'un processus de mise en correspondance appliqué a priori permettait d'obtenir un plus grand nombre d'inter-relations entre les deux ressources. / The revolution in new sequencing technologies, by strongly improving the production of omics data, is greatly leading to new understandings of the relations between genotype and phenotype. To interpret and analyze data grouped according to a phenotype of interest, methods based on statistical enrichment became a standard in biology. However, these methods synthesize the biological information by a priori selecting the over-represented terms and focus on the most studied genes that may represent a limited coverage of annotated genes within a gene set. During this thesis, we explored different methods for annotating gene sets. In this frame, we developed three studies allowing the annotation of gene sets and thus improving the understanding of their biological context.First, visualization approaches were applied to represent annotation results provided by enrichment analysis for a gene set or a repertoire of gene sets. In this work, a visualization prototype called MOTVIS (MOdular Term VISualization) has been developed to provide an interactive representation of a repertoire of gene sets combining two visual metaphors: a treemap view that provides an overview and also displays detailed information about gene sets, and an indented tree view that can be used to focus on the annotation terms of interest. MOTVIS has the advantage to solve the limitations of each visual metaphor when used individually. This illustrates the interest of using different visual metaphors to facilitate the comprehension of biological results by representing complex data.Secondly, to address the issues of enrichment analysis, a new method for analyzing the impact of using different semantic similarity measures on gene set annotation was proposed. To evaluate the impact of each measure, two relevant criteria were considered for characterizing a "good" synthetic gene set annotation: (i) the number of annotation terms has to be drastically reduced while maintaining a sufficient level of details, and (ii) the number of genes described by the selected terms should be as large as possible. Thus, nine semantic similarity measures were analyzed to identify the best possible compromise between both criteria while maintaining a sufficient level of details. Using GO to annotate the gene sets, we observed better results with node-based measures that use the terms’ characteristics than with edge-based measures that use the relations terms. The annotation of the gene sets achieved with the node-based measures did not exhibit major differences regardless of the characteristics of the terms used. Then, we developed GSAn (Gene Set Annotation), a novel gene set annotation web server that uses semantic similarity measures to synthesize a priori GO annotation terms. GSAn contains the interactive visualization MOTVIS, dedicated to visualize the representative terms of gene set annotations. Compared to enrichment analysis tools, GSAn has shown excellent results in terms of maximizing the gene coverage while minimizing the number of terms.At last, the third work consisted in enriching the annotation results provided by GSAn. Since the knowledge described in GO may not be sufficient for interpreting gene sets, other biological information, such as pathways and diseases, may be useful to provide a wider biological context. Thus, two additional knowledge resources, being Reactome and Disease Ontology (DO), were integrated within GSAn. In practice, GO terms were mapped to terms of Reactome and DO, before and after applying the GSAn method. The integration of these resources improved the results in terms of gene coverage without affecting significantly the number of involved terms. Two strategies were applied to find mappings (generated or extracted from the web) between each new resource and GO. We have shown that a mapping process before computing the GSAn method allowed to obtain a larger number of inter-relations between the two knowledge resources.

A identificação de genes alvos, vias de sinalização e vias metabólicas para melhoramento de cana-de-açúcar associados a características de interesse, ainda são pouco conhecidos e estudados. Alguns estudos do transcriptoma através de plataformas de microarranjo têm buscado identificar listas de genes, para experimentos tecido- específico ou submetidos a condições de estresse bióticos e abióticos. Estudos pontuais destes dados tem sido associados a vias metabólicas ou vias de sinalização já descritas na literatura, de forma a identificar alterações relacionadas a padrões de expressão gênica. Porém, estas relações em cana-de-açúcar são pouco conhecidas e estudadas. O estudo e entendimento de cana-de-açúcar por meio da diversidade genética e de sua adaptação ao ambiente é um grande desafio, principalmente pela ausência de um genoma sequenciado e por possuir um genoma complexo. Apresentamos nossos resultados para tentar superar tais limitações e desafios para estudos de expressão gênica. Foram desenvolvidas metodologias para anotação funcional do transcriptoma, centradas na transferência de anotação, identificação de vias metabólicas e enzimas pelo método de similaridade bi-direcional, predição de genes full-length, análises de ortologia e desenho de oligonucleotídeos para microarranjos customizados, resultando no ORFeoma de cana-de-açúcar, na identificação e classificação de famílias de fatores de transcrição e identificação de genes ortólogos entre gramíneas. Além disso, desenvolvemos uma plataforma para processamento e análise automatizada de experimentos por microarranjo, para armazenamento, recuperação e integração com a anotação funcional. Adicionalmente desenvolvemos e implementamos métodos para seleção de genes diferencialmente e significativamente expressos, e abordagens para análise de enriquecimento de categorias, e escores de atividade de vias metabólicas. De forma a integrar a anotação funcional do transcriptoma aos estudos por expressão gênica, desenvolvemos a plataforma CaneRegNet e uma interface para integração desta rede de dados biológicos e conhecimentos, composta por aplicativos para consulta e prospecção de dados por análises de agrupamento e correlação entre experimentos de microarranjo, possibilitando a geração de novas hipóteses e predições dentro da organização da regulação celular. / The identification of target genes, metabolic and signaling pathways associated with characteristics of interest to the sugarcane improvement are still poorly known and studied. Some transcritptome studies through microarray platforms has tried to identify lists of genes, for tissue-specific experiments or subjected to conditions of biotic and abiotic stress. In the literature specific studies of these data has already been associated with metabolic or signaling pathway, in order to identify changes in these tracks related to patterns of gene expression. However, these relations are still little know and generally defined slightly. The study and understanding of sugarcane by means of genetic diversity and its adaptation to the environment is a major challenge, mainly due to the absence of a sequenced genome and by your complex genome. We present our results to surpass this barrier e challenges for the study of gene expression. Methodologies were developed for the transcriptome functional annotation, focused on the annotation transfer, identification of metabolic pathways and enzymes by the bi- directional method; prediction of full-length genes; ortology analysis and probe design for customized microarrays, resulting in the sugarcane ORFeome, the identification and classification of transcription factor families and identification of ortholog genes between grasses. Besides that, we have developed a plataform for automated processing and analysis for microarray experiments, to store, retrieve and integration with the functional annotation. Additionally, we have developed and implemented methods for identification of differentially and significantly expressed genes, and approaches for over-represented analysis and functional class scoring (FCS). To integrate the functional annotation and the studies by gene expression profile, we have developed the CaneRegNet platform and an interface to integrate this network of biological data and knowledge, composed by searching and data mining tools for clustering and correlations between microarray experiments, enabling the generation of new hypothesis and predictions around the organization of cellular regulation.

O feijão-comum (Phaseolus vulgaris) é atacado por uma gama de patógenos que afetam a produtividade das lavouras e a qualidade dos grãos. Dentre os patógenos de importância econômica para a cultura no Brasil, destaca-se o nematoide das galhas (Meloidogyne incognita). Embora haja relatos sobre a avaliação de cultivares na presença de M. incognita, as fontes de resistência tem se mostrado pouco efetivas. Por isso, pesquisas que possibilitem um melhor entendimento sobre a interação planta-nematoide são de extrema valia e devem nortear novas estratégias para o melhoramento do feijoeiro. Assim, no presente estudo, 18 cultivares de P. vulgaris foram avaliadas quanto à resistência a M. incognita raça 3, sendo que quatro comportaram-se como pouco suscetíveis, 11 como moderadamente suscetíveis e três altamente suscetíveis. A cultivar IPR Saracura mostrou menor grau de suscetibilidade e foi, então, usada na construção de 12 bibliotecas de RNAseq, visando à identificação dos genes envolvidos na reposta à infecção pelo nematoide. Foram adotados dois tratamentos, 4 e 10 DAI (dias após inoculação), compostos de plantas inoculadas e controles. Primeiramente, realizou-se o mapeamento dos transcritos de cada biblioteca, tomando como referência o genoma de P. vulgaris (G19833), o que resultou na identificação de 27.195 unigenes. Em seguida, foi realizada a quantificação da expressão dos transcritos mapeados e genes diferencialmente expressos foram identificados. No total, 191 genes do hospedeiro apresentaram expressão diferencial, considerando-se: i) o tratamento inoculado em relação ao controle; ii) a razão de expressão (Fold Change - FC) mínima absoluta igual a 4; iii) o nível de significância ? = 0,05. Do total, 120 genes foram identificados aos 4 DAI e 71 aos 10 DAI. As sequências mapeadas foram contrastadas àquelas dos bancos de dados NCBI e TAIR, usando a ferramenta BLASTx e, posteriormente, anotadas usando os softwares Blast2GO e MapMan. Detectou-se similaridade com genes codificadores de proteínas conhecidas para 90% (24.604/27.195) dos unigenes, sendo que 69% (16.991/24.604) deles foram anotados. Quanto à expressão diferencial, 98% (188/191) dos transcritos mostraram similaridade com proteínas conhecidas e 67% (127/188) puderam ser anotados. Os transcritos foram atribuídos a diferentes categorias funcionais putativas, predominando o termo ontológico \'processos metabólicos\', em ambas as plataformas. A anotação dos genes na plataforma MapMan mostrou abundância das categorias da via de resposta a estresse, com predominância de genes de defesa superexpressos aos 4 DAI e reprimidos aos 10 DAI. Por fim, 10 genes mostraram expressão diferencial tanto aos 4 como aos 10 DAI: sete deles foram estáveis, sendo superexpressos nas plantas inoculadas, e três apresentaram comportamentos opostos nos momentos avaliados. Ênfase foi dada a um gene que codifica uma \'probable inactive ADP-ribosyltransferase\' e a quatro genes de resposta a ferimento. / The common bean (Phaseolus vulgaris) is attacked by a range of pathogens, which affect crop yield and the quality of grains. Among the pathogens of economic significance to the crop in Brazil, the root-knot nematodes (Meloidogyne incognita) deserve attention. Though there are some reports on cultivar evaluation in presence of M. incognita, the resistance sources have not being effective. Therefore, it is of valuable importance research projects that could lead to a better understanding of plant-nematode interaction and to indicate new strategies for common bean breeding. In the present study, 18 cultivars of P. vulgaris were evaluated in regard to their resistance to M. incognita race 3; four were less susceptible, 11 moderately susceptible, and three were highly susceptible. \'IPR Saracura\' behaved as the less susceptible cultivar and then was selected for the construction of 12 RNAseq libraries, aiming at the identification of genes differentially expressed in response to nematode infection. Two treatments were adopted, 4 and 10 days after inoculation (DAI), each comprised of inoculated and control plants. Firstly, the transcripts were mapped to the reference genome of P. vulgaris (G19833), resulting in the identification of 27,195 unigenes. Then, the mapped transcript\'s expression was quantified and differentially expressed genes were identified. In total, 191 genes of the host plant showed differential expression taking into consideration: i) the inoculated treatments in relation to their control; ii) an absolute fold change (FC) >= 4; iii) a level of significance ? = 0,05. Of the total, 120 genes were detected at 4 DAI and 71 at 10 DAI. The mapped sequences were compared against those deposited in NCBI and TAIR databanks using BLASTx and subsequently annotated using Blast2GO and MapMan softwares. Similarity to known proteins was detected for 90% of the unigenes (24,604/27,195) and 69% (16,991/24,604) of them were annotated. Regarding assessing differential expression, 98% (188/191) of the transcripts showed similarity to known proteins and 67% (127/188) were annotated. Transcripts were attributed to different putative functional categories and the ontological term \'metabolic process\' was predominant within both platforms. Gene annotation within MapMan platform showed predominance of stress-related pathway categories, with prevalence of defense genes overexpressed at 4 DAI and repressed at 10 DAI. Finally, 10 genes showed differential expression at both 4 and 10 DAI: seven were stably overexpressed in the inoculated plants, and three showed an opposite behavior regarding the evaluation periods. Attention was given to a gene encoding a probable inactive ADP-ribosyltransferase and four genes related to wound response.

Organohalide-respiring Bacteria (OHRB) play key roles in the reductive dehalogenation of natural organohalides and anthropogenic chlorinated contaminants. Reductive dehalogenases (RDases) catalyze the cleavage of carbon-halogen bonds, enabling respiratory energy conservation and growth. Large numbers of RDase genes, a majority lacking experimental characterization of function, are found on the genomes of OHRB. In silico genomics tools were employed to identify shared sequence features among RDase genes and proteins, predict RDase functionality, and elucidate RDase evolutionary history. These analyses showed that the RDase superfamily could be divided into proteins exported to the membrane and cytoplasmic proteins, indicating that not all RDases function in respiration. Further, Hidden Markov models (HMMs) and multiple sequence alignments (MSAs) based upon biochemically characterized RDases identified previously uncharacterized members of an RDase superfamily, delineated protein domains and amino acid motifs serving to distinguish RDases from unrelated iron-sulfur proteins. Such conserved and discriminatory features among RDases may facilitate monitoring of organohalide-degrading microbial communities or improve accuracy of genome annotation. Phylogenetic analyses of RDase superfamily sequences provided evidence of convergent evolution and horizontal gene transfer (HGT) across distinct OHRB genera. Yet, the low frequency of RDase transfer outside the genus level and the absence of RDase transfer between phyla indicate that RDases evolve primarily by vertical evolution or HGT is restricted among related OHRB strains. Polyphyletic evolutionary lineages within the RDase superfamily comprise distantly-related RDases, some exhibiting activities towards the same substrates, suggesting a longstanding history of OHRB adaptation to natural organohalides. Similar functional and phylogenetic analyses provided evidence that nitrous oxide (N₂O, a potent greenhouse gas) reductase (nosZ) genes from versatile OHRB members of the Anaeromyxobacter and Desulfomonile genera comprised a nosZ sub-family evolutionarily distinct from nosZ found in non-OHRB denitrifiers. Hence, elucidation of RDase and NosZ sequence diversity may enhance the mitigation of anthropogenic organohalides and greenhouse gases (i.e., N₂O), respectively. The tetrachloroethene-respiring bacterium Geobacter lovleyi strain SZ exhibited genomic features distinguishing it from non-organohalide-respiring members of the Geobacter genus, including a conjugative pilus transfer gene cluster, a chromosomal genomic island harboring two RDase genes, and a diminished set of c-type cytochrome genes. The G. lovleyi strain SZ genome also harbored a 77 kbp plasmid carrying 15 out of the 24 genes involved in biosynthesis of corrinoid, likely related to this strains ability to degrade PCE to cis-DCE in the absence of supplied corrinoid (i.e., vitamin B₁₂). Although corrinoids are essential cofactors to RDases, the strictly organohalide-respiring Dehalococcoides mccartyi strains are corrinoid auxotrophs and depend upon uptake of extracellular corrinoids via Archaeal and Bacterial salvage pathways. A key corrinoid salvage gene in D. mccartyi, cbiZ, occurs at duplicated loci adjacent to RDase genes and appears to have been horizontally-acquired from Archaea. These comparative genome analyses highlight RDase dependencies upon corrinoids and also suggest mobile genomic elements (e.g., plasmids) are associated with organohalide respiration and corrinoid acquisition among OHRB. In summary, analyses of OHRB genomes promise to enable more complete modeling of metabolic and evolutionary processes associated with the turnover of organohalides in anoxic environments. These efforts also expand knowledge of biomarkers for monitoring OHRB activity in anoxic environments, and will improve our understanding of the fate of chlorinated contaminants.

Upon ageing, a progressive disruption of protein homeostasis often leads to extensive protein aggregation and neurodegeneration. It is therefore important to study at the proteome level the origins and consequences of such disruption, which so far have remained elusive. Addressing this problem has recently become possible by major advances in mass spectrometry-based (MS) proteomics, which allows the identifications and quantification of thousands of proteins in a variety of biological samples. In the first part of this thesis, I analyse proteome-wide MS data for the nematode worm C. elegans upon ageing, in wild type (WT), long-lived and short-lived mutant strains. By comparing the total abundance and the soluble abundance for nearly 4000 proteins, I provide extensive evidence that proteins are expressed in adult worms at levels close to their solubility limits. With the use of sequence-based prediction tools, I then identify specific physico-chemical properties associated with this age-related protein homeostasis impairment. The results that I obtained reveal that the total intracellular protein content remains constant, in spite of the fact that the proteome undergoes wide remodeling upon ageing, resulting into severe protein homeostasis disruption and widespread protein aggregation. These results suggest a protein-dependent decrease in solubility associated with the protein homeostasis failure. In the second part of the thesis, I determine and classify potential interactions of misfolded protein oligomers with other proteins. This phenomenon is widely believed to give rise to cytotoxicity, although the mechanisms by which this happens are not fully understood. To address this question, I process and analyse MS data from structurally different oligomers (toxic type A and nontoxic type B) of the protein HypF-N, incubated in vitro with proteins extracted from murine cell cultures. I find that more than 2500 proteins are pulled down with the misfolded oligomers. These results indicate that the two types of oligomers interact with the same pool of proteins and differ only in the degree of binding. Functional annotation analysis on the groups reveals a preference of the oligomers to bind proteins in specific biological pathways and categories, including in particular mitochondrial membrane proteins, RNA-binding proteins and molecular chaperones. Overall, in this study I complement the powerful and high-throughput experimental approach of MS proteomics with bioinformatics analyses and prediction algorithms to define the physical, chemical and biological features of protein homeostasis disruption upon ageing and the interactome of misfolded oligomers.

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No. of bitstreams: 2 Tese - Ricardo Diogenes Dias Silveira - 2014.pdf: 2417341 bytes, checksum: dd7932e4849f3c8cf7a4ff97d173eb77 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2014-03-31 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The upland rice is sensitive to drought especially during the reproductive phase, when even moderate stress can result in drastic reduction in yield. Upon the occurrence of the drought a variety of genes is induced in plants, triggering a complex network of responses that extends from the perception and recognition of sign of stress, through activation of adaptive response genes. The objective of this thesis was to study the transcriptome of two Brazilian cultivars of upland rice (Douradão and Primavera) contrasting in relation to drought tolerance, and subjected to two experiments under water deficit in two consecutive years. In the first year, corresponding to Article 1, the transcripts from leaf samples were sequenced by RNA-seq, and in the second year, corresponding to Article 2, the transcripts from leaf and root samples were sequenced, for both cultivars, under normal and limited irrigation during the reproductive phase. In Article 1, the sequencing showed in Douradão 27,618 transcripts, from which 24,090 (87.2 %) showed homology to rice genes and 27,221 transcripts in Primavera, from which 23,663 (86.9 %) showed homology to rice genes. Douradão had 493 diferentially expressed genes (DEGs) and Primavera 1,154 DGEs. In Article 2 it were identified 44,978 and 37,898 transcripts in leaf and root tissues, respectively. Douradão showed 3,554 and 840 diferentially expressed genes (DEGs), in leaf and root tissues, respectively, while Primavera showed 1,141 and 1,975 DEGs on those tissues, respectively.It were identified genes from different metabolic routes related to distinct drought tolerance mechanisms in leaf and root tissues, such as cell signaling -related genes, transcription factors, functional protective proteins and cell cellular detoxification enzymes. A set of expressed genes from both tissues were validated by RT–qPCR and most of them were similar to the results of RNA-seq results. The rice transcripts not annotated were submitted to an orthology analysis in relation to the Arabidopsis thaliana databank, which revealed one gene related to the root cell growth Douradão. The 16 genes validated by RT-qPCR will be used as molecular markers for marker assisted selection in the upland rice breeding program and are candidate genes for use in transformation of rice cultivars susceptible to drought. / O arroz de terras altas é sensível à seca principalmente durante a fase reprodutiva, quando até mesmo o estresse moderado pode resultar na redução drástica de produtividade. Diante da seca uma variedade de genes é induzida nas plantas, desencadeando uma complexa rede de respostas que se estende desde a percepção e reconhecimento do sinal de estresse até a ativação de genes de resposta adaptativa. O objetivo desta tese foi estudar o transcriptoma de duas cultivares brasileiras de arroz de terras altas (Douradão e Primavera), constrastantes em relação à tolerância à seca, e submetidas a dois experimentos sob déficit hídrico em dois anos consecutivos. No primeiro ano, correspondente ao Artigo 1, foram sequenciados os transcritos provenientes de amostras de folhas, e no segundo ano, correspondente ao Artigo 2, foram sequenciados os transcritos provenientes de amostras de folhas e raízes, para as duas cultivares, sob condições normais e restritas de irrigação durante a fase reprodutiva das plantas. No Artigo 1, o sequenciamento revelou 27.618 transcritos em Douradão, com 24.090 (87,2%) de homologia à genes de arroz e 27.221 transcritos na cultivar Primavera, dos quais 23.663 (86,9%) apresentaram homologia aos genes de arroz. Douradão apresentou 493 genes diferencialmente expressos (GDEs) e Primavera 1.154 GDEs. No Artigo 2 foram identificados 44.978 transcritos do tecido foliar e 37.898 transcritos do tecido radicular, considerando o número total de transcritos de ambas as cultivares. Douradão apresentou 3.554 e 840 GDEs, respectivamente, no tecido foliar e radicular, enquanto que Primavera apresentou, 1.141 e 1.975 GDEs. Foram identificados vários genes atuantes em rotas metabólicas envolvidas em diferentes mecanismos de tolerância a seca nos tecidos foliar e radicular, como por exemplo, genes relacionados à sinalização celular, fatores de transcrição, proteínas protetoras funcionais da célula e enzimas de detoxificação celular. Um conjunto de genes expressos em ambos os tecidos foram validados via RT-qPCR e a maioria deles tiveram resultados similares aos resultados de RNA-seq. Foi realizada uma análise de ortologia envolvendo os transcritos não anotados em arroz contra o banco de dados de Arabidopsis thaliana, a qual revelou um gene relacionado ao crescimento celular de raízes na cultivar Douradão. Os 16 genes validados via RT-qPCR relacionados a tolerância à seca serão utilizados como marcadores moleculares em seleção assistida no programa de melhoramento de arroz de terras altas e são genes candidatos a serem utilizados na transformação de genótipos sensíveis à seca.