Le Glypican-3 (GPC3) est surexprimé dans 72% des carcinomes hépatocellulaire (CHC). C’est un co-récepteur membranaire du récepteur WNT, qui appartient à la famille des protéoglycanes à sulfates d'héparane. L'objectif général de ma thèse vise à étudier les mécanismes de régulation post-transcriptionnelle de l’expression du GPC3 dans le CHC. Pour cela, j’ai développé un test fonctionnel qui m’a permis de cribler une bibliothèque de 876 microARNs humains. Ceci a conduit à l’identification de 5 microARNs régulateurs de l’expression de l’ARNm codant pour le GPC3 via sa région 3’ non traduite (NT). Mon travail de thèse porte plus particulièrement sur le miR-1271 et le miR-1291 car ils sont dérégulés dans le CHC et sont respectivement inhibiteur et inducteur de l’expression du GPC3. Dans un premier projet, j’ai démontré que le miR-1271 cible directement la région 3’NT du GPC3 et diminue la stabilité de son ARNm. Ce microARN est sous-exprimé dans le CHC et son expression corrèle négativement avec celle de l'ARNm du GPC3 dans les CHC associés à une infection par le virus de l’hépatite B. Dans un deuxième projet, j’ai démontré que le miR-1291 régule positivement l’expression du GPC3 en inhibant un facteur intermédiaire. Une analyse in silico a permis d’identifier IRE1α comme candidat. IRE1α est une protéine transmembranaire du réticulum endoplasmique (RE) qui participe à « l’Unfolded Protein Response », une réponse adaptative activée lors de l’accumulation de protéines mal conformées dans le RE. J’ai démontré qu’IRE1α clive l’ARNm codant pour le GPC3 grâce à son activité endoribonucléase. D’autre part, le miR-1291 cible directement l’ARNm codant pour IRE1α dans sa région 5’NT ce qui inhibe son expression et induit une surexpression du GPC3. Le miR-1291 est surexprimé dans le CHC et son expression corrèle positivement avec celle de l’ARNm du GPC3. En conclusion, mon travail de thèse m’a permis de mettre en évidence et de caractériser deux nouveaux microARNs (miR-1271 et miR-1291) contrôlant l’expression du GPC3 par des mécanismes directs ou indirects. La pertinence physiopathologique de ces régulations dans le CHC est en accord avec les niveaux d’expression respectifs de ces microARNs, qui pourraient contribuer à la surexpression du GPC3 dans ces tumeurs. / Glypican-3 (GPC3) is overexpressed in 72% of hepatocellular carcinoma (HCC). It is a co-receptor for WNT receptor and belongs to the heparan sulfate proteoglycans family. The general objective of my PhD thesis was to study the mechanisms by which GPC3 is post-transcriptionnally regulated in HCC. To this end, I developed a functional test that allowed me to screen a library of 876 human microRNAs. This led me to identify 5 microRNAs that regulate the expression of GPC3 mRNA through its 3’Untranslated Region (UTR). The work presented in this thesis particulary focuses on miR-1271 and miR-1291 as both microRNAs present a deregulated expression in HCC and are respectively inhibitor and activator of GPC3 mRNA expression. In a first project, I demonstrated that miR-1271 directly binds to GPC3 mRNA 3’UTR and affects its stability. This microRNA is underexpressed in HCC and its expression negatively correlates with that of GPC3 mRNA in a subgroup of HCC corresponding to those associated with hepatitis B virus infection. In a second project, I demonstrated that miR-1291 postively regulates the expression of GPC3 mRNA by targeting an intermediate factor. An in silico analysis led to the identification of the Inositol Requiring Enzyme 1 alpha (IRE1α) as a potential candidate. IRE1α is an endoplasmic reticulum (ER) resident type I transmembrane protein and contributes to the signaling of the Unfolded Protein Response (UPR). The UPR is an adaptive response activated upon accumulation of improperly folded proteins in the ER. I showed that IRE1α cleaves GPC3 mRNA through its endoribonuclease activity. Moreover I demonstrated that miR-1291 directly targets IRE1α mRNA through its 5’UTR, thereby decreasing its expression and contributing to GPC3 mRNA overexpression. MiR-1291 is overexpressed in HCC and its expression positively correlates with that of GPC3 mRNA. In summary, the work carried out during my PhD allowed the identification and the characterization of two new microRNAs (miR-1271 and miR-1291) that control the expression of GPC3 mRNA through direct or indirect mechanisms. The pathophysiological relevance of these regulatory mechanisms is in agreement with the respective expression levels of these microRNAs in HCC, which could therefore contribute to the overexpression of GPC3 in those tumors.

La protéine multifonctionnelle E4F1 fut initialement identifiée comme une cible de l'oncoprotéine virale E1A au cours de l'infection par l'adénovirus. Nos données, ainsi que celles d'autres laboratoires, montrent qu'E4F1 intervient à de multiples niveaux de régulation de la voie p53, une voie de signalisation très fréquemment inactivée au cours de la tumorigenèse. Au cours de ma thèse, j'ai mis en évidence et caractérisé un nouveau niveau de régulation impliquant la protéine E4F1 et un autre régulateur important de la voie p53 : l'oncoprotéine Mdm2. Mes travaux ont permis de montrer, pour la première fois, qu'E4F1 et Mdm2 sont présents dans un même complexe multiprotéique associé à la chromatine et dont le recrutement est indépendant de p53. La formation du complexe E4F1-Mdm2 est sous la dépendance de certains stress cellulaires, telle que la réponse aux stress oxydatifs. De plus, Mdm2 et E4F1 sont capables de s'ubiquitinyler mutuellement sans promouvoir leur protéolyse. Nos résultats préliminaires suggèrent que ce complexe chromatinien Mdm2-E4F1 est impliqué dans le contrôle d'un programme transcriptionnel apparenté à une réponse au stress oxydatif. L'ensemble de ces données suggère des fonctions de Mdm2 au niveau de la chromatine, indépendantes de sa fonction bien décrite de régulateur du suppresseur de tumeur p53. / E4F1 is a multifunctional protein that was first identified as a cellular target of the viral oncoprotein E1A during adenoviral infection. We, and others, have shown that E4F1 impinges at different level of the p53 pathway, which is frequently inactivated during tumorigenesis. During my PhD, I have highlighted and characterized a novel level of regulation implicating E4F1 and an other key regulator of the p53 pathway: the Mdm2 oncoprotein.My work have shown for the first time that Mdm2 and E4F1 directly interact in a same multiprotein complex associated with chromatin, and this recruitment occurs independently of p53. The Mdm2-E4F1 complex formation is dependant of some cellular stresses, such as oxidative stresses reponses. Our preliminary data also indicate that E4F1 and Mdm2 ubiquitylate each other without promoting their proteolysis, and influence their stability on chromatin. Our preliminary results indicate that this Mdm2-E4F1 chromatinian complex is involved in the regulation of a transcriptional program dependant of oxidative stresses. These data support the notion that Mdm2 presents unexpected functions on chromatin, independently of its very well described p53 regulation.

3 domaines cellulaires définissent l'épiderme de la queue de Ciona intestinalis. Le domaine des lignes médianes, donne naissance à deux structures différenciées larvaires, la nageoire et système nerveux périphérique. Il a été montré que les voies de signalisation BMP et FGF sont respectivement les inducteurs des lignes médianes ventrale et dorsale. Ce travail a consisté en la caractérisation des mécanismes moléculaires, situés à l'interface des signaux inducteurs et des processus de différenciation, définissant l'identité cellulaire des lignes médianes. L'identification des relations épistatiques reliant 7 facteurs de transcription impliqués dans ce processus suggère un fonctionnement en réseau hiérarchisé et place Msxb comme gène clé. Cette hiérarchie est validée par: un atlas d'expression spatio-temporelle, la perte de fonction de Msxb, l'analyse des régions cis-régulatrices de la transcription. Nous avons identifié 2 types d'enhancers, contrôlant respectivement les gènes précoces et tardifs du réseau. L'expression précoce de Msxb dans les précurseurs dorsaux est controlée par un enhancer distinct régulé par la voie FGF relayée par Otx et Nodal. Cette signature transcriptionnelle est retrouvée dans les enhancers de gènes co-exprimés, et chez l'orthologue de Msxb chez une autre ascidie. Enfin, nous montrons que la partie la plus postérieure des lignes médianes constitue un troisième compartiment, déployant un programme génétique distinct. Ce travail nous renseigne sur la structure, les mécanismes moléculaires de formation des lignes médianes, l'existence d'une signature transcriptionnelle évolutivement conservée pour le gène clé de l'acquisition de ce destin cellulaire. / Ciona intestinalis tail epidermis has 8 rows of cells defining 3 domains. One of them, the midline domain, gives rise to differentiated cells which form the larval fin and part of peripheral nervous system. Previous work has shown that BMP and FGF signalling are the inducers of ventral and dorsal midlines respectively. My work consisted in the identification of molecular events which lead epidermal cells to adopt midline fate, from induction to tail differentiation. We identified 7 transcription factors involved in this process. Identification of epistatic relationships suggest that these genes are in a hierarchical network where Msxb is a key gene. This hierarchy is validated by 1) a spatio-temporal expression atlas, 2) loss of function of Msxb, 3) cis-regulatory regions analysis for each network gene. We identified 2 types of enhancers, one capable to decouple ventral / dorsal signals used by early genes, and the other used by later genes, acting as a global response in both midlines. We showed that the early expression of Msxb in dorsal precursors is controled by a distinct enhancer, regulated by FGF9/16/20 via Otx and Nodal. This transcriptional signature is found in enhancers of co-expressed genes and in Msxb orthologue in another ascidian. Finally, we showed the most posterior part of the midlines is controlled by a distinct genetic program than the one used in dorsal and ventral midlines. This work gives insight into midlines structure, the mechanisms involved in their formation and a conserved transcriptional signature for the key gene involved in midline cell fate.

No description available.

Diabetes é uma doença metabólica caracterizada por hiperglicemia associada a prejuízos na captação e utilização de glicose, em que reduções na expressão da proteína GLUT4 (codificada pelo gene SLC2A4), bem como das enzimas Hexokinase-2 e Glycogen synthase (codificadas pelos genes HK2 e GYS1), desempenham papel importante. Recentemente, um novo elemento vem sendo relacionado à etiopatogenia e à fisiopatologia do diabetes, os microRNAs (miRNAs), que são pequenos RNAs envolvidos na regulação da expressão gênica, geralmente afetando a degradação de mRNAs. Entretanto, a participação de miRNAs envolvidos na redução da expressão de mRNAs relacionados a proteínas envolvidas na captação e utilização de glicose, sobretudo em músculo esquelético, permanece desconhecida. O objetivo desse estudo foi investigar a expressão de miRNAs potencialmente reguladores da expressão de Slc2a4/GLUT4, Hk2/HK2 e Gys1/GYS1 em músculo esquelético de ratos diabéticos. Utilizamos ratos Wistar machos que foram tornados diabéticos pela administração de estreptozotocina. Após 13 dias, 3 grupos foram formados: não-diabético (ND) e diabético tratado com placebo (DP) ou insulina (DI). O tratamento foi conduzido por 7 dias, totalizando 21 dias de diabetes. Variáveis metabólicas foram avaliadas e os músculos sóleos foram removidos para avaliar a expressão de mRNAs, miRNAs e proteínas. Uma abrangente análise in silico foi conduzida para determinar miRNAs candidatos a regularem a expressão de Slc2a4, Hk2 e Gys1. Os animais diabéticos apresentaram perda de peso, poliúria, glicosúria, hiperglicemia e aumento de frutosamina plasmática; a insulinoterapia melhorou estas variáveis. O diabetes reduziu a expressão dos mRNAs Slc2a4 (~55%), Hk2 (~47%) e Gys1 (~45%), e das proteínas GLUT4 (~77%), HK2(~52%) e GYS1 (~49%); a insulinoterapia restaurou essas variáveis. A expressão de 20 miRNAs foi avaliada neste estudo; 8 foram modulados pelo diabetes, sendo três supra-regulados, miR-1 (~28%), miR-29b (~118%) e miR-29c (~51%); e cinco infra-regulados, miR-93 (~39%), miR-199a (~30%), miR-345-3p (~23%), miR-532-3p (~26%) e miR-150 (~32%). Exceto pelo miR-1 e miR-150, a insulinoterapia reverteu as demais alterações. Além disso, miR-29b e miR-29c correlacionaram-se negativamente com GLUT4 e HK2, e positivamente com glicemia, glicosúria e frutosamina, sugerindo uma possível relação causal; enquanto que miR-199a e miR-532-3p correlacionaram-se positivamente com GLUT4 e HK2, e também com as variáveis metabólicas, sugerindo uma regulação indireta sobre os mRNAs dessas proteínas. No último caso, demonstrou-se que o miR-199a tem como alvo o NFKB1, um repressor do gene Slc2a4, o qual diminuiu no diabetes, explicando, pelo menos parcialmente, o efeito indireto sobre o GLUT4. Em suma, o diabetes aumenta a expressão de miR-29b e miR-29c, e reduz a expressão de miR-199a e miR-532-3p; o primeiro efeito, potencialmente age diretamente na tradução do mRNA Slc2a4 e Hk2, e o segundo, potencialmente age indiretamente, via NFKB, na transcrição dos genes. Como consequência, as proteínas GLUT4 e HK2 diminuem, o que reduziria a utilização de glicose pelo músculo, contribuindo para a hiperglicemia do diabetes. / Diabetes is a metabolic disease characterized by hyperglycemia associated with impaired glucose metabolism and uptake, in which reductions of GLUT4, hexokinase 2 (HK2) and glycogen synthase 1 (GYS1) proteins, encoded respectively by SLC2A4, HK2 and GYS1 genes, play an important role. Recently, a new element have been related to etiopathogeny and pathophysiology of diabetes, the microRNAs (miRNAs), which are small RNAs involved in the regulation of gene expression, usually by affecting the degradation of mRNAs. However, the participation of miRNAs diabetes-induced reduction of expression of genes related to glucose uptake and metabolism in skeletal muscle remains unknown. Thus, the objective of this study was to investigate the expression of miRNAs potentially regulators of the Slc2a4/GLUT4, Hk2/HK2 and Gys1/GYS1 in skeletal muscle of diabetic rats. Male Wistar rats were rendered diabetic by receiving streptozotocin. After 13 days, 3 groups were formed: non-diabetic (ND), and diabetic treated with placebo (DP) or insulin (DI) (NPH insulin, 6U/day). Treatment was conducted for 7 days, totalizing 21 days of diabetes. At the end of the experimental period, metabolic variables were evaluated and the soleus muscle was removed for evaluation of mRNA, miRNA and protein expression. A broad in silico analysis was performed to determine candidate miRNAs as potential regulators of Slc2a4, Hk2 and Gys1. Diabetic rats shown weight loss, polyuria, glycosuria, hyperglycemia and increased plasma fructosamine; insulin treatment improved these variables. Diabetes reduced Slc2a4 (~55%), Hk2 (~47%) and Gys1 (~45%) mRNAs, as well as GLUT4 (77%), HK2 (52%) and GYS1 (49%) proteins; insulin treatment restored these variables. Twenty miRNAs were assessed in this study. Eight miRNAs were modulated by diabetes in skeletal muscle; three were upregulated: miR-1 (28%), miR-29b (118%) and miR-29c (51%), whereas five were downregulated: miR-93 (39%), miR-150 (32%), miR-199a (30%), miR-345-3p (23%) and miR-532-3p (26%). Except for miR-1 and miR-150, all regulations were reverted by insulin treatment. Besides, miR-29b and miR-29c were negatively correlated with GLUT4 and HK2 proteins, and positively with glucose, glycosuria and plasma fructosamine suggesting a direct causal relationship; while miR-199a and miR-532-3p were positively correlated with GLUT4 and HK2 proteins, and also with the metabolic variables, suggesting an indirect causal relationship. In the last case, it was demonstrated that miR-199a has the Slc2a4 repressor Nfkb1 as target, which was reduced in muscle from diabetic rats, explaining, at least partially, the indirect effect upon GLUT4. In conclusion, diabetes increase the expression of miR-29b and miR-29c, and reduce the expression of miR-199a e miR-532-3p; the first effect, potentially acts directly in the translation of Slc2a4 and Hk2 mRNAs, and the second one, potentially acts indirectly, via NFKB, in the transcription of these genes. As a result, the expression of GLUT4 and HK2 decreases, which would reduce the muscle glucose uptake and metabolization, contributing to the hyperglycemia of the diabetes.

A fim de contornar a problemática imposta pela recalcitrância da parede celular à hidrólise, o processo de produção de etanol a partir de biomassa vegetal requer um passo denominado de pré-tratamento, que consiste em tornar a biomassa mais acessível à ação de glicosil hidrolases que atacam a parede celular. O melhor conhecimento de processos de degradação de parede operados pela própria planta tem o potencial de direcionar as pesquisas em bioernegia, indicando quais os mecanismos mais eficientes para desmontar o complexo de polímeros da parede celular. Cunhado pré-tratamento biológico, este consiste na hidrólise de polissacarídeos estruturais empregando mecanismos e elementos chave de processos de degradação de parede celular que ocorrem na própria planta. Um exemplo de evento com esta característica é a formação de aerênquima lisígeno, que consiste na abertura de espaços de gás em tecidos parenquimáticos. A formação do aerênquima em cana-de-açúcar se dá de forma modular, sendo o processo constituído por seis etapas: 1) percepção do sinal inicial; 2) separação celular: 3) expansão celular; 4) morte celular programada; 5) hidrólise de hemiceluloses e 6) hidrólise de celulose. O presente trabalho teve como principal foco estudar a regulação das duas etapas iniciais, visando obter conhecimentos que possibilitem para tornar a biomassa de cana de açúcar menos resistente à penetração de enzimas. O aerênquima ocorre na raiz de cana-de-açúcar por um processo constitutivo. Sua independência de um indutor externo foi corroborada mediante tratamento com nutrientes e inibidor da percepção por etileno (1-MCP) pela análise dos cinco centímetros mais apicais da raiz. O atraso na formação do aerênquima após tratamento com nutrientes levou à expressão diferencial de genes relacionados à degradação de parede celular, morte celular programada e sinalização por etileno. Por outro lado, o 1-MCP não afetou visivelmente a formação do aerênquima, porém alterou o balanço hormonal na raiz. O padrão transcricional das raízes tratadas com 1-MCP revelou aumento da expressão de genes relacionados à expansão celular e estresse oxidativo. Tais padrões são discutidos à luz da regulação hormonal da formação do aerênquima através do estabelecimento de um balanço hormonal definido entre auxina e etileno. Ambos os experimentos levaram à seleção de quatro genes candidatos: dois fatores de transcrição (ScRAV1 e ScERF1) e duas glicosil hidrolases (ScEPG1 e ScARA1), sequenciados e analisados quanto à diversidade hom(e)óloga, sequências promotoras e estrutura gênica em comparação a S. bicolor. A topologia das reconstruções filogenéticas e a distribuição diferencial de sítios para RAV e ERF nos promotores de ScEPG1 e ScARA1 não parece refletir padrões de expressão distintos, visto que os diferentes hom(e)ólogos demonstraram ser igualmente expressos. Em sistema heterólogo, o fator de transcrição RAV apresentou atividade repressora sobre o promotor de ScEPG1. Tal papel de RAV sugere regulação negativa sobre a degradação da lamela média e sobre o processo de perda de adesão e separação celular. A identificação de reguladores-chave da formação do aerênquima consiste em importante elo entre a sinalização hormonal e a degradação de parede celular, possibilitando a melhor compreensão dos mecanismos de controle da degradação endógena de parede celular. Os dados produzidos possibilitam a aplicação biotecnológica dos genes sequenciados, além de consistirem em significativo avanço no conhecimento sobre a fisiologia do processo estudado e na dinâmica da regulação da expressão gênica acoplada a aspectos filogenéticos das sequências alvo. / In order to circumvent the problems imposed by the cell wall recalcitrance to hydrolysis, the process underlying biofuel production requires a step called pretreatment, aimed at making biomass more accessible to the action of glycosil hydrolases that attack the cell wall. The increased knowledge of wall degradation processes operated by the plant itself has the potential to influence research in the bioernergy field, pointing out the most efficient mechanisms to disassemble the cell wall complex polymeric structure. The term coined biological pretreatment means taking advantage of key elements and mechanisms of cell wall degradation processes occurring in the plant itself in order to disassemble the entangled polysaccharide structure. One example of endogenous cell wall degradation process is the lysigenous aerenchyma formation, which consists in the opening of gas spaces in parenchyma tissue. The aerenchyma formation in sugarcane is thought to be a modular process that occurs in six steps: 1) target cells perception; 2) cell separation; 3) cell expansion; 4) programmed cell death; 5) hemicellulose hydrolysis and 6) cellulose hydrolysis. This work has as the main objective to study the regulation of the two initial steps, aiming at acquiring knowledge that would afford to turn biomass less resistant to enzyme penetration. The aerenchyma develops within the roots of sugarcane as a constitutive process. Its independence from an external inducer was corroborated in this work by subjecting sugarcane plants to treatment with nutrients and one inhibitor of ethylene perception (1-MCP) when the five more apical centimeters of the root were analyzed. After treatment with nutrients, the delayed aerenchyma formation led to differential expression of cell wall degradation-, programmed cell death- and ethylene signalling-related genes. Under visual inspection, 1-MCP did not show effect on aerenchyma development. Instead, it changed the hormone balance mainly in the two most apical root segments. The transcriptional pattern of 1-MCP treated roots revealed increased expression of cell expansion- and oxidative stress-related genes. Such patterns are discussed in the light of the hormone regulation of the aerenchyma development through the establishment of a balance between auxin and ethylene within root segments. Both experiments led to the selection of four two candidate genes, two transcription factors (ScRAV1 and ScERF1) and two glycosil hydrolases (ScEPG1 and ScARA1), that were sequenced and analyzed regarding homEURologous diversity, promoter sequences and gene structure compared to S. bicolor. The topology of the phylogenetic reconstructions and the differential distribution of sites for RAV and ERF within ScEPG1 and ScARA1 promoters did not reflect distinct expression patterns. Different hom(e)ologous of each target gene were equally expressed. In an heterologous system, RAV transcription factor interacted with ScEPG1 promoter, reducing the activity encoded by the reporter gene. This suggests the repressive role of RAV on pectin degradation within the middle lamella, leading to reduced cell adhesion and cell separation, possibly regulated by this glycosyl hydrolase. The identification of key regulators of the aerenchyma formation is an important link between hormone signaling and the wall degradation within the sugarcane roots. It enables a better understanding of control mechanisms underlying wall modifications when performed by the plant itself. Altogether, the data produced in this work allows biotechnological application of sequenced genes. Moreover, the produced data unveil key aspects regarding physiologycal aspects of aerenchyma development and highlights features related to the dynamics of gene expression in sugarcane coupled to the phylogenetic aspects of the four target sequences.

Embora os glicocorticoides (GCs) sejam os anti-inflamatórios mais prescritos mundialmente, níveis elevados de GCs potencializam alguns aspectos da resposta inflamatória em regiões do encéfalo de ratos, dependentes da ativação dos receptores GR. Neste estudo visamos avaliar os efeitos dos glicocorticoides em alguns aspectos da resposta inflamatória induzida por LPS nas populações de células (neurônio, micróglia e astrócitos) primárias de córtex frontal derivadas de ratos Wistar neonatos. Através de ensaios EMSA e imunofluorescência, avaliamos indicativos da ativação do fator NFKB em culturas mistas e enriquecidas expostas ao pré-tratamento com CORT, seguido do estímulo inflamatório LPS. Verificamos que a CORT não exerceu sua clássica atividade anti-inflamatória, pois não foi capaz de diminuir a ativação do NFKB induzida pelo LPS nas culturas mistas, sugerindo ser parcialmente dependente da ativação de GR, concentração dos GCs e interações celulares, já que os astrócitos em culturas mistas exibem respostas diferentes quanto ao NFKB, mas a microglia e neurônios não. / Although glucocorticoids (GCs) are the most commonly prescribed anti-inflammatory worldwide, high levels of GCs enhance some aspects of the inflammatory response in brain regions of rats, dependent on activation of GR. In this study, we aim to evaluate the effects of glucocorticoids in some aspects of the inflammatory response LPS-induced in populations of cells (neurons, astrocytes, and microglia) in primary frontal cortex derived from newborn rats. Through EMSA and immunofluorescence assays, we evaluated the indicative factor NFKB activation in mixed and enriched cultures exposed to pretreatment with CORT, followed by the LPS inflammatory stimulus. We found that CORT did not exerted its classic anti-inflammatory activity, whereas it was not able to decrease the activation of NFKB LPS-induced in mixed cultures, suggesting be partially dependent on the GR activation, GCs concentration and cellular interactions, since astrocytes in mixed cultures exhibit different responses relative to NFKB, but neurons and microglia did not.

A atividade florestal apresenta-se como um vetor de desenvolvimento social, ambiental e econômico no Brasil. Em 2004 as exportações ligadas ao setor renderam US$ 7 bilhões e contribuíram com US$ 5,5 bilhões em impostos. No mesmo ano, no Brasil, dos 6 milhões de hectares de reflorestamento comercial, mais de 50% dos hectares são ocupados por eucalipto. Esta cultura pode ser utilizada para diversas funções, desde à produção de papel e celulose, até a produção de carvão vegetal. Mas a produtividade desta cultura atualmente tem uma série de fatores limitantes. Destes fatores, um dos mais severos é uma doença denominada ferrugem, causada pelo fungo Puccinia psiddii Winter. Este patógeno entre tanto, não é conhecido nos centros de origem do gênero Eucalyptus, mas, utiliza como hospedeiros vários gêneros das Mirtáceas, infectando folhas, flores e frutos em desenvolvimento. O controle da doença é normalmente realizado mediante o uso de fungicidas. Porém, a utilização de plantas resistentes é o método mais aconselhável por diversos motivos, como o baixo custo, a praticidade e o menor impacto ambiental. Neste trabalho a metodologia do SAGE - Serial Analysis of Gene Expression foi utilizada, para a análise da expressão gênica uma vez que permite detectar e quantificar, de maneira global, a expressão de genes conhecidos e desconhecidos, envolvidos no processo de resistência à ferrugem. Neste trabalho analisou-se a expressão do conjunto de genes diferencialmente expressos e 200 genes sem diferenças no padrão de expressão, em Eucalyptus grandis infectado com Puccinia psidii. Utilizou-se para esta análise, indivíduos susceptíveis e resistentes à infecção por Puccinia psidii Winter. A população amostrada constituída por meios irmãos, apresentava segregação para o carácter de resistência à ferrugem. Os resultados produzidos permitiram identificar 421 genes com expressão diferencial (p ≤ 0,05) nos dois fenótipos. Vários destes genes estavam relacionados a diversos mecanismos de defesa da planta, tais como genes envolvidos na formação de barreiras físicas e na resposta hipersensível, resistência sistêmica adquirida, bem como genes relacionados à polarização celular e resposta ao estresse oxidativo. Os dados em conjunto, corroboram a idéia de que são vários os mecanismos que atuam simultaneamente produzindo o fenótipo resistente. Estes genes apresentam-se para serem utilizados futuramente como marcadores moleculares na seleção de indivíduos resistentes, em cruzamentos naturais ou dirigidos e como fonte de genes para experimentos de transformação em estudos mais profundos de fitopatogênese. / In Brazil the forest activities represent a vector for social, ambiental and economic development. In 2004 the exportations produced US$ 7 billions and contributed with US$ 5,5 billion in taxes. In the same year, in Brasil, of 6 million hectares of commercial reforestation, around 50% is occupied by eucalyptus. This crop can be used as the raw material for several industrial processes, from cellulose and paper production to vegetable charcoal production. However, eucalyptus productivity has several limiting factors. One of the most severe diseases is called rust, caused by the fungus Puccinia psiddii Winter. This pathogen is unknown in the Eucalyptus center of origin; however, it has several Myrtaceae genera as its host, infecting leaves, flowers and developing fruits. Rust management normally involves the use of fungicides, but the use of resistant plants is a much better alternative due the lower costs and minimal ambient impact. In the current study SAGE - Serial Analysis of Gene Expression was used for gene expression analysis because it is a quantitative genome-wide method and that obtains expression profiles from known and unknown genes involved in the rust resistant process. This thesis analyzed the expression profile of the differentially expressed genes and 200 genes without significant statistical differences in their expression patterns, in Eucalyptus grandis infected with Puccinia psidii. For this analysis we used susceptible and resistant individuals to Puccinia psidii Winter infection taken from a population half siblings, which showed segregation for fust resistance. The results indicated 421 genes with differential expression (p ≤ 0.05), 239 were preferentially expressed in the susceptible library and 232 in the resistant. Several of these genes were associated with plant defense mechanisms, such as genes involved in formation of physical barriers, hypersensitive response, systemic adquired resistance, genes related to cellular polarization and oxidative stress responses. All the data support the idea that not one but several mechanisms are acting at the same time producing the resistant phenotype. These genes are future candidates for molecular markers useful in the selection of resistant individuals in breeding programs and a source of genes for transformation experiments or as molecular tools to dissect the phytopathogenic process.

Nesta tese propomos um modelo estocástico multimodal para regulação da expressão gênica em nível de transcrição. A definição de um espaço de parâmetros que contém o conteúdo biológico do sistema aliada à escolha apropriada de uma base para construir a matriz de acoplamento entre os estados do sistema levaram à obtenção de soluções exatas do modelo. Tais soluções são obtidas transformando as equações mestras em equações diferenciais parciais usando a técnica das funções geradoras e escrevendo os coeficientes das equações parciais em termos dos parâmetros biológicos do modelo. No regime estacionário obtivemos uma relação de recorrência para os coeficientes das séries de potências que definem as funções geradoras e a especificação das configurações de equilíbrio do sistema permite que estas séries sejam calculadas exatamente. Com as soluções exatas calculadas não só as distribuições de probabilidade foram obtidas como os momentos das distribuições. As distribuições de probabilidade de equilíbrio apresentam estruturas multimodais com vários picos e a análise do ruído (flutuação) mostra que a existência de um estado intermediário de eficiência transcricional leva a redução do ruído global do sistema. A inspeção dos autovalores da matriz de acoplamento mostrou que existem regiões onde a dinâmica dos momentos é de caráter oscilatória com amortecimento. Diferentes esquemas de acoplamento levam à diferentes regimes transientes, tal característica revela que o sistema multimodal apresentam maior flexibilidade adaptativa quando comparado com sistemas de um ou dois estados. / In this thesis we propose a stochastic model for multimodal regulation of gene expression at the transcriptional level. The definition of a parameter space that contains the contents of the biological system coupled with the appropriate choice of a base to build the coupling matrix between the states of the system led to the exact solutions of the model. Such solutions are obtained by transforming master equations in partial differential equations using the technique of generating functions and writing the coefficients of partial equations in terms of biological parameters of the model. In the steady a recurrence relation for the coefficients of power series defining the generating functions was obtained and specification of the equilibrium configurations of the system allows the exact calculation of these series. With the exact solutions calculated not only the probability distributions were obtained but also the moments of the distributions. The equilibrium distributions probability is multimodal and presents several peaks. Analysis of the noise (fluctuation) shows that the existence of an state with intermediate transcriptional efficiency leads to a reduction of the overall system noise. Inspection of the eigenvalues of the coupling matrix showed that there are regions where the dynamics of the moments is damped oscillating. Different coupling schemes lead to different transient regimes, this feature reveals that the multimodal system have greater adaptive flexibility when compared to systems of one or two states.

La pause transcriptionnelle marquée par les ARN polymérases (RNAP) est un mécanisme clé pour réguler l'expression des gènes dans tous les règnes de la vie et est une condition préalable à la terminaison de la transcription. Le facteur de transcription bactérien essentiel NusA stimule à la fois la pause et la terminaison de la transcription, jouant ainsi un rôle central. Ici, je présente des reconstructions par cryo-microscopie électronique (cryo-EM) à une seule particule de NusA lié à des complexes d'élongation en présence et en absence d’ARN en épingle à cheveux dans le canal de sortie de l'ARN. Les structures révèlent quatre interactions entre NusA et RNAP qui suggèrent comment NusA stimule le repliement de l’ARN, la pause et la terminaison de la transcription. Un intermédiaire de translocation asymétrique de l'ARN et de l'ADN convertit le site actif de l'enzyme en un état inactif, fournissant une explication structurelle pour l'inhibition de la catalyse. La comparaison de RNAP à différentes étapes de la mise en pause donne un aperçu de la nature dynamique du processus et du rôle de NusA en tant que facteur de régulation. / Transcriptional pausing by RNA polymerases (RNAPs) is a key mechanism to regulate gene expression in all kingdoms of life and is a prerequisite for transcription termination. The essential bacterial transcription factor NusA stimulates both pausing and termination of transcription, thus playing a central role. Here, I present single-particle electron cryo-microscopy (cryo-EM) reconstructions of NusA bound to paused elongation complexes with and without a pause-enhancing hairpin in the RNA exit channel. The structures reveal four interactions between NusA and RNAP that suggest how NusA stimulates RNA folding, pausing, and termination. An asymmetric translocation intermediate of RNA and DNA converts the active site of the enzyme into an inactive state, providing a structural explanation for the inhibition of catalysis. Comparing RNAP at different stages of pausing provides insights on the dynamic nature of the process and the role of NusA as a regulatory factor.