Ce travail de thèse a eu pour objectif d’identifier les mécanismes majeurs intervenant dans la régulation de l’adiposité musculaire chez la truite arc-en-ciel. Pour cela, nous avons analysé les effets combinés de la sélection génétique et de l’alimentation, facteurs prépondérants de variation de l'adiposité. Deux lignées de truites arc-en-ciel sélectionnées sur la teneur en lipides du muscle dorsal ("muscle gras" et "muscle maigre"), ont été nourries pendant 6 mois avec un régime contenant 10 ou 23% de lipides (% de la matière sèche). Nous avons mesuré l'activité et/ou l'expression d’enzymes clé des principales voies métaboliques intervenant dans l'utilisation de l'énergie, puis développé une analyse différentielle globale à l’échelle du transcriptome (microarray nylon) et du protéome (électrophorèse bidimensionnelle). Ces analyses portent sur le muscle blanc, tissu cible de la sélection, et le foie, carrefour métabolique et site majeur de la lipogenèse chez les poissons. Les résultats obtenus confirment l’effet inhibiteur d’un apport alimentaire riche en lipides sur la lipogénèse et la désaturation des acides gras dans le foie, déjà observé chez des individus de plus grande taille, et fournissent de nouvelles connaissances sur l’effet exercé sur les autres voies, en particulier la protéolyse. Ces analyses ont également permis de mettre en évidence des différences métaboliques existant entre lignées, qui concernent non seulement le métabolisme des lipides mais aussi celui des autres substrats énergétiques. Il apparaît que les deux moyens utilisé pour augmenter la teneur en lipides du muscle mettent en jeu des mécanismes moléculaires différents. Nos travaux ont permis d’identifier deux gènes dont l’expression est augmentée dans le muscle en réponse à un apport alimentaire riche en lipides et par la sélection génétique en faveur d’un indice d’adiposité musculaire élevé, et qui pourraient être des marqueurs moléculaires de l’adiposité musculaire. / The objective of the study was to identify genes and proteins that are involved in the control of muscle fat deposition in rainbow trout. We analyzed the combined effects exerted by genetic selection and dietary treatment, which are the two main factors that can be used to manage body fat content. Two lines of rainbow trout, obtained after 3 generations of divergent selection for high or low muscle fat content, were fed diets containing either 10% or 23% lipids (% dry matter), for six months. We analyzed the activity and gene expression of key enzymes involved in energy utilization, and performed a more global approach through transcriptome (nylon microarray) and proteome (two- dimensional electrophoresis) analysis. We analyzed the liver, which is the centre of intermediary metabolism and the main site of lipogenesis in fish, and the muscle, the target tissue of the selection provedure. The results confirmed the depressing effect exerted by a lipid rich diet on lipogenesis and fatty acid desaturation, already described in larger size fish, and provided new insight about the effect exerted on the other metabolic pathways, in particular the proteolysis. These analyses pointed out metabolic differences existing between lines. They involved not only lipid metabolism, but also the other pathways of nutrient utilization. With regard to their muscle-fattening effect, the dietary treatment and the genetic selection appear to act through different molecular mechanisms. These analyses allowed the identification of two genes that are over-expressed in muscle upon both high dietary lipid supply and upward selection for muscle fat content, suggesting that these two genes could be relevant molecular markers of muscle fattening.

Regeneration, the replacement of lost or damaged body parts, and biomineralisation, the biologically controlled formation of minerals, are important and widespread abilities in the animal kingdom. Both phenomena have a complex evolutionary history; thus their study benefits from investigations in diverse animals. Spirobranchus (formerly Pomatoceros) lamarcki is a small tube-dwelling polychaete worm of the serpulid family. Serpulids have evolved a novel head appendage, the operculum, which functions as a defensive tube plug and regenerates readily when lost. In S. lamarcki, the end of the operculum is reinforced by a calcareous plate; thus, the operculum is a good system in which to study both regeneration and biomineralisation. This thesis explores several aspects of these important processes in the adult operculum. First, a time course of normal regeneration is established. Next, cell proliferation patterns are described, suggesting a combination of proliferation-dependent and proliferation-independent elements in opercular regeneration. The formation of the calcareous opercular plate is examined using both microscopic observations of whole opercular plates and X-ray diffraction analysis of isolated plate mineral, revealing a large shift in mineralogy over the course of regeneration. Histochemical study of alkaline phosphatase enzyme activity indicates the importance of these enzymes in the operculum, although their precise functions are as yet unclear. Finally, a preliminary survey of three opercular transcriptomic datasets is presented, with a broad sampling of gene families with regeneration- or biomineralisation-related roles in other animals. The opercular transcriptome constitutes the first biomineralisation transcriptome from any annelid, and one of the first transcriptomic datasets related to annelid regeneration. Many of the candidate genes examined here display interesting behaviour and suggest targets for further investigation. The work presented here establishes the S. lamarcki operculum as a promising model system in the field of evolutionary developmental biology.

A Biologia de Sistemas proporciona um olhar holístico sobre os processos biológicos, integrando os diversos componentes intracelulares através de redes altamente complexas. Em particular, redes de co-expressão tem permitido nos últimos anos uma compreensão cada vez maior dos sistemas biológicos e dos mecanismos moleculares que os regem. Por outro lado, as ferramentas matemáticas e estatísticas já desenvolvidas para a análise destas redes e sistemas são, em geral, densas e pouco familiares para profissionais das áreas biológicas e da saúde. Portanto, a fim de possibilitar uma análise ao mesmo tempo relevante e facilitada, nosso grupo criou a ferramenta CEMiTool, que tem por objetivo identificar módulos de coexpressão de genes de modo automático, de maneira fácil e intuitiva para usuários com pouca ou nenhuma experiência com linguagens de programação. A fim de demonstrar a facilidade de uso da ferramenta, aplicamos o CEMiTool a mais de 1000 estudos de transcriptômica, cujos resultados foram utilizados para a confecção de um banco de dados, permitindo a integração de informações entre estudos. Além disso, para facilitar ainda mais o acesso a este tipo de análises, foi criada uma versão online da ferramenta, denominada webCEMiTool, que permite realizar as análises no navegador. Finalmente, criou-se também a ferramenta annotator, permitindo a definição automática de grupos de amostras de estudos de transcriptômica a partir do agrupamento de cadeias de caracteres presentes em dados de anotação. Todo o código está livremente disponível à comunidade. / System biology methods provide a holistic view of biological processes, integrating the several intracellular molecular components via the use of highly complex networks. In particular, co-expression networks have allowed for an increasing understanding of biological systems and the complex molecular mechanisms driving them. On the other hand, previously described tools for the analysis of biological networks are in general relatively difficult to use for life and health scientists given their high mathematical and computational demand. Therefore, in order to provide at the same time a relevant and easy-to-use analysis, we have developed the CEMiTool package, which aims to identify gene coexpression modules in an automatic, easy and intuitive way for users with little to no prior computational expertise. We applied CEMiTool to over 1000 transcriptomics studies and used the results to create a new gene coexpression database, which allows users to integrate information across analyses. Moreover, to further facilitate analyses we developed an online version of the tool named webCEMiTool, which permits users to run coexpression analysis easily via browser. Finally, we also developed annotator, a package for automatically determining experimental groups based on sample annotation string similarity. All code is freely available to the community.

No description available.

L'aquaculture est en pleine expansion et fournit aujourd'hui la moitié des produits aquatiques destinés à la consommationhumaine. Elle constitue ainsi un secteur clé pour le maintien et l'amélioration de la sécurité alimentaire dans lemonde. Le développement de l'aquaculture est étroitement lié à celui des formules alimentaires. Ces dernières années,la part des farines de poisson dans la formulation des aliments a particulièrement diminué au profit des farines d'originevégétale pour répondre aux nombreuses contraintes économiques et environnementales. Néanmoins, ces matièrespremières sont moins adaptées aux besoins nutritionnels des poissons carnivores et leur utilisation peut entraîner unebaisse des performances de croissance et d'efficacité alimentaire. L'ajout d'additifs et d'ingrédients fonctionnels devientalors indispensable. Les hydrolysats protéiques issus des co-produits de la pêche et de l'aquaculture sont des ingrédientsà fort potentiel appétence, nutritionnel et bioactif. Ces ingrédients sont des mélanges complexes riches en peptides hydrolytiqueset en acides aminés libres, mais dont la composition varie en fonction de l'origine de la matière première etdes paramètres d'hydrolyse appliqués lors de leur fabrication. Au cours de ces travaux de thèse, nous avons développéet mis en pratique des outils permettant d'approfondir la caractérisation structurale et les propriétés fonctionnellesdes hydrolysats de protéines. Dans un premier temps, nous avons développé une méthode analytique basée sur unenormalisation des échantillons suivie d'une détermination de l'abondance et de la richesse en peptides par chromatographied'exclusion stérique et chromatographie liquide couplée à la spectrométrie de masse de type électro-spray,respectivement. Les résultats présentés sous forme d'un diagramme 2D permettant de classer et comparer facilementles hydrolysats de protéines de forme galénique, d'origine et de process différents. Nous avons également développé unoutil in vitro sur l'intestin de bar européen, Dicentrarchus labrax, permettant de déterminer les activités myotropesdes hydrolysats. Nous avons pu notamment démontrer que l'hydrolysat de crevettes possède une plus forte activitémyotrope que les hydrolysats de poissons et que cette activité est portée par un pentapeptide KNPEQ clivé à partirde l'hémocyanine lors du process d'hydrolyse appliqué sur les co-produits de crevette. Enfin, dans un second temps,un conditionnement alimentaire de 65 jours a été conduit chez le bar européen nourri avec un aliment pauvre en farinede poisson supplémenté en hydrolysat de différentes origines et couplé à une analyse d'expression génique (approcheen RNA-Seq Illumina). Cette étude a permis de montrer que les hydrolysats de protéines appliqués sur un alimentfaible en farine de poisson (5%) sont capables de restaurer des performances de croissance équivalentes à celles d'unrégime contenant 20% de farine de poisson mais qu'ils portent par ailleurs des propriétés fonctionnelles spécifiques.Il a également été montré que le mélange des deux hydrolysats permet de moduler les transcriptomes intestinal ethépatique de façon plus profonde que lorsque que les hydrolysats sont utilisés séparément. Ces résultats confirmentl'intérêt des hydrolysats de protéines pour la formulation d'aliment à faible teneur en farines de poisson et apportentde nouveaux outils de caractérisation de ces ingrédients complexes, qui seront utiles pour leur optimisation et leurstandardisation ainsi que pour la compréhension de leurs mécanismes d'action chez les poissons. / Aquaculture is a key sector for supporting and improving the food security worldwide. The global production of farmedfish and shrimp has grown dramatically over the past decades and now contributes to half of the aquatic productsintended for human consumption. Aquaculture will require feeds to support its growth but availability of some raw materialssuch as fish meal are limited. The use of fish meal in aquafeed has particularly declined in favor of plant proteinsources to fit with economic and environmental constraints. But plant proteins do not meet perfectly the nutritionalrequirements of carnivorous fish and their utilization often results in lower growth and feed performances. Proteinhydrolysates manufactured from fishing and aquaculture co-products are ingredients with a high palatable, nutritionaland bioactive potential. They are rich in hydrolytic peptides and free amino acids, but they are complex mixtureswhose composition could vary according to raw material origin and hydrolysis parameters. During this PhD study, wedeveloped and implemented tools to further characterize the structure and functional properties hydrolysates. On afirst step, we developed a fast methodological tool based on sample standardization, followed by the determination ofthe abundance and richness of peptides using size exclusion chromatography and liquid chromatography coupled toelectro-spray mass spectrometry, respectively. We merged the results into a 2D diagram that made it easy to classifyand compare hydrolysates having different galenic, origin and process. We also developed a tool on isolated intestinefrom European seabass (Dicentrarchus labrax ) to rank protein hydrolysates according to their myotropic property.We demonstrated that shrimp hydrolysate showed a higher myotropic activity than fish hydrolysates and that thisactivity was carried by a unique pentapeptide KNPEQ produced by the enzymatic clivage of haemocyanin during thehydrolysis process applied on shrimp co-products. On a second step, a 65-day feeding trial was conducted in Europeanseabass fed a low fish meal diet supplemented with protein hydrolysates of different origin, and coupled to a studyof the intestine and liver transcriptomic response (Illumina RNA sequencing). It has been shown that protein hydrolysatesincluded in a low fish meal diet (5%) restored growth performances to the same level than a diet containing20% of fishmeal, and that they exhibited very specific functional properties. These results showed that a mixture oftwo hydrolysates impacted more deeply the intestine and liver transcriptomes than hydrolysate tested alone. ThisphD study confirmed that protein hydrolysates are very interesting candidates for formulating low fish meal feed andoffer new tools for characterizing such complex ingredients, which will be useful to optimize and standardize proteinhydrolysate while understanding their mechanisms of action in fish.

Estudos prévios demonstraram que as atividades biológicas do veneno da serpente Bothrops jararaca sofrem significantes modificações ontogenéticas. Neste estudo é apresentada uma análise comparativa do proteoma, peptidoma e transcriptoma da glândula de veneno de filhotes e adultos de B. jararaca, correlacionando os resultados obtidos com algumas características funcionais dos venenos. Venenos de 694 filhotes de até duas semanas de idade e 110 adultos, provenientes do Estado de São Paulo foram extraídos e liofilizados para as análises proteômicas/peptidômicas e funcionais. O mRNA de glândulas de veneno de 20 filhotes e 10 adultos foi obtido para a contrução de bibliotecas de cDNA e a análise de Expressed Sequence Tag (ESTs). Demonstramos que a atividade hemorrágica é similar para os venenos de filhotes e adultos, enquanto que o veneno de adultos é discretamente mais letal para camundongos; entretanto, o veneno de filhotes mostrou-se extremamente mais letal para aves, uma característica que pode garantir proteção contra potenciais predadores nas fases iniciais de vida da espécie. A atividade coagulante do veneno de filhotes é cerca de 10 vezes mais alta que aquela verificada para o veneno de adultos e é atribuída sobretudo à atividade de metaloproteinases. Essas diferenças nas atividades funcionais se refletiram nos diferentes perfis verificados por eletroforese bidimensional e identificação de spots de proteínas por digestão tripsínica in-gel seguida de análise por cromatografia líquida acoplada à espectrometria de massas em tandem, zimografia com gelatina, imunocoloração utilizando anticorpos específicos anti-proteinases, e glicoproteínas com afinidade pela concanavalina -A. A comparação dos venenos por derivatização com tags isóbaros (iTRAQ) e a análise das ESTs revelaram diferenças claras entre os níveis de toxinas presentes nos venenos e as metaloproteinases foram a classe de toxinas mais expressa, além de serem as toxinas cujos perfis estruturais apresentaram maior mudança, como ilustrado pelo quociente PIII/PI, que é maior nos venenos de filhotes. Dimorfismo sexual foi detectado em diversas classes de toxinas no veneno de adultos por análises proteômicas e transcriptômicas e, surpreendentemente, o fator de crescimento de nervo foi detectado apenas no veneno/glândula de veneno de machos. A análise glicoproteômica mostrou que N-glicosilações parecem ser as modificações pós-traducionais mais proeminentes nas toxinas de B. jararaca, e os perfis de N-glicosilação apresentaram-se distintos para as proteínas dos venenos de filhotes e adultos. Entretanto, a composição de N-glicanos não variou entre as amostras, indicando que diferenças na utilização de motivos de N-glicosilação poderiam explicar as diferenças nos níveis de glicosilação observados pelos diferentes perfis eletroforéticos dos venenos. A análise da fração peptídica dos venenos de filhotes e adultos por espectrometria de massas resultou num perfil similar de Peptídeos Potenciadores de Bradicinina (BPPs), que foram detectados em suas formas canônicas e também com novas seqüências, cujas estruturas primárias sugerem um processamento da proteína precursora em sítios até então não descritos. Acreditamos que este seja o estudo mais abrangente sobre a variabilidade do veneno de uma serpente já realizado e os resultados demonstram que há uma relação clara entre as alterações ontogenéticas na dieta e tamanho corporal e o proteoma/peptidoma do veneno desta espécie. / Previous studies have demonstrated that the biological activities displayed by the venom of the snake Bothrops jararaca undergo a significant ontogenetic shift. In this investigation, we performed comparative proteomic, peptidomic and transcriptomic analyses of venoms and venom glands from newborn and adult specimens of B. jararaca and correlated the results with the evaluation of functional venom features. Venoms from 694 two-week old newborns and 110 adults from São Paulo state were milked and lyophilized for functional and proteomic/peptidomic analyses. Additionally, mRNA was obtained from the venom glands of 20 newborns and 10 adults and used for the construction of cDNA libraries and Expressed Sequence Tag (ESTs). We demonstrate that newborn and adult venoms have similar hemorrhagic activities, while the adult venom has a slightly higher lethal activity upon mice; however, the newborn venom is extremely more potent to kill chicks, a feature that might ensure protection against potential predators in early stages of B. jararaca life. Interestingly, the coagulant activity of the newborn venom upon human plasma is ten times higher than that of the adult venom and is contributed mainly by metalloproteinases. Differences in functional activities were clearly reflected in the venom different profiles of two-dimensional gel electrophoresis (2D-PAGE) and protein spot identification by in-gel trypsin digestion followed by liquid chromatography and tandem mass spectrometry (LC-MS/MS), gelatin zimography, immunostaining using specific anti-proteinase antibodies, and concanavalin A-binding proteins. The venom comparison by isobaric tag peptide labeling (iTRAQ) and ESTs analysis revealed clear differences in toxin levels. The metalloproteinases were detected as the toxin class most expressed in the venoms in addition to being the toxins whose structural profile most changed, as illustrated by the ratio P-III/P-I class being higher in newborn venoms. Sexual dimorphism has been detected in various adult venom toxin classes by proteomic and transcriptomic analyses and, interestingly, the nerve growth factor was detected only in the male venom gland/venom. The glycoproteomic analysis showed that N-glycosylation seems to be the most prominent post-translational modification in B. jararaca toxins and the N-glycosylation profiles differed for newborn and adult venom toxins. Nevertheless, the N-glycan composition between the samples did not vary indicating that differences in the utilization of the N-glycosylation motif could be the explanation for the differences in the glycosylation levels indicated by the differential electrophoretic profiles of venom proteins. The analysis of the peptide fraction of newborn and adult venoms by mass spectrometry revealed a similar profile of Bradykinin Potentiating peptides (BPPs), however these were detected in the venoms showing their canonical sequences and also novel sequences corresponding to BPPs processed from their precursor protein at sites so far not described. To the best of our knowledge, this is the most comprehensive study on a snake venom variation and the results clearly demonstrate a relationship between the ontogenetic shift in diet and animal size, and the venom proteome/peptidome in B. jararaca species

Les médicaments sont fréquement retrouvés dans les effluents de STEP relargués dans l’environnement aquatique. Dans le but de prévenir et de mieux comprendre les impacts des médicaments sur les écosystèmes aquatiques, il semble pertinent d’évaluer les perturbations comportementales, physiologiques et transcriptomiques des psychotropes sur les organismes aquatiques. Dans ce contexte, l’objectif de cette étude a été d’évaluer les perturbations induites par 4 psychotropes (oxazépam, carbamazépine, cyamémazine et sertraline) chez deux organismes, Radix balthica et Schmidtea polychroa. Pour ce faire, des embryons de Radix au stade trochophore et des planaires adultes ont été exposés à chaque psychotrope (du μg/L jusqu’à 100 μg/L). Il en ressort que les psychotropes allongent la durée du développement embryonnaire du Radix et perturbent le déplacement, la reproduction et la digestion, mais pas la régénération de la planaire. D’un point de vue transcriptomique, nous avons réalisé le séquençage du transcriptome en conditions différentielles chez le Radix. Ainsi, nous avons obtenu d’une part les séquences du transcriptome et d’autre part, après analyse en différentiel, 144 contigs différentiellement exprimés par l’oxazépam parmi lesquels 94 ont été vérifiés en RT-qPCR chez des Radix exposés à chaque psychotrope. Il ressort de cette analyse que les psychotropes impactent principalement la voie de signalisation Notch, mais aussi les voies de biosynthèse des polyamines et des catécholamines. Les psychotropes modulent aussi l’expression de gènes codant des protéines de la Matrice Extra Cellulaire (MEC), comme la Matriline ou encore la Dentine sialophosphoprotéine. Chez la planaire, nous avons analysé l’expression de 87 gènes impliqués dans différentes fonctions. Il ressort de cette étude que les 4 psychotropes modulent l’expression de nombreux gènes impliqués dans la mobilité ciliaire et musculaire et dans les systèmes nerveux, reproducteur, excréteur et digestif. / Pharmaceuticals are often found in WWTP effluents realesed in surface water. In order to prevent and to understand the pharmaceuticals impact on aquatic ecosystems, it seems relevant to evaluate behavioural, physiological and transcriptomic disturbances of psychotropics on freshwater organisms. The aim of this study was thus to analyse disturbances of 4 psychotropics (oxazepam, carbamazepine, cyamemazine and sertraline) on 2 freshwater organisms, Radix balthica and Schmidtea polychroa. In our experiments, both Radix embryos at the trochophore stage and mature planarian were exposed to each psychotropic (from 1 to 100 μg/L). This psychotropic exposure results in an increase of the duration of Radix embryonic development and a disturbance of the mobility, the reproduction and the digestion but not the regeneration of planarian. Regarding the transcriptomic impact, we performed RNA sequencing in differential conditions of Radix embryos exposed or not to oxazepam. On one hand, this analysis allowed us to obtain the transcriptome sequences and, on the other hand, 144 contigs differentially expressed among which 94 genes were verified by RT-qPCR. The results showed that psychotropics impact mainly the Notch signalling pathways, but also the biosynthetic pathways of polyamines and catecholamines. Psychotropics also disturb the gene expression encoding some Extra Cellular Matrix (ECM) protein such as Matrilin and Dentin sialophosphoprotein. Regarding the molecular study of the psychotropics impact on planarian, we analysed the expression of 87 genes involved in different functions. The results showed that the 4 psychotropics modulate expression of genes involved in ciliary and muscular motility and in the nervous, reproductive and excretory systems. Genes from the digestive system are also impacted by the psychotropics. All the observed impacts on the 2 organisms suggest a possible disturbance on the population fitness and therefore on the freshwater ecosystems.

No Brasil, o eucalipto é uma das principais fontes de matéria-prima para a produção de celulose e papel. A crescente demanda por sua madeira tem levado à expansão dos plantios florestais para regiões com déficit hídrico e baixa fertilidade do solo, resultando no interesse pela obtenção de plantas tolerantes à condições ambientais estressantes. Nas plantas, o déficit hídrico acarreta diversas respostas fisiológicas, bioquímicas e moleculares. Estudos fisiológicos observaram que plantas com um adequado suprimento de potássio (K+) são mais tolerantes à seca. Contudo, na nutrição de algumas plantas, o sódio (Na+) pode substituir parcialmente o K+. Como muitas áreas agricultáveis do mundo são deficientes em potássio, essa pode ser uma solução mais econômica para a fertilização dos plantios florestais. Assim, é de sumo interesse compreender à nível molecular os mecanismos de interação entre esses minerais com a água e suas consequências para o metabolismo da planta, visando a sustentabilidade dos plantios florestais e o desenvolvimento de programas de melhoramento genético florestal. Neste contexto, o presente trabalho objetivou realizar o estudo do transcriptoma foliar de Eucalyptus grandis visando identificar e categorizar funcionalmente transcritos diferencialmente expressos para os quais o efeito de interação entre os fatores fertilização e água foi significativo, considerando três regimes de fertilização (controle (C) ou fertilização com potássio (+K) ou sódio (+Na)) associados a dois regimes hídricos (sem exclusão da precipitação interna (+H2O) ou 37% de exclusão da precipitação interna (-H2O)). Transcritos relacionados às funções osmótica, fotossíntese e metabolismo do carbono foram selecionados para estudos mais detalhados. Para a análise do transcriptoma foi utilizada a técnica de RNA-Seq através da plataforma Illumina e diversos programas de bioinformática como o Bowtie 2, TopHat, pacote DEseq2 (programa R) para as análises estatísticas e Blast2GO para anotação e a categorização funcional. Seis comparações estatísticas foram realizadas: (I) +K+H2O vs C+H2O, (II) +Na+H2O vs C+H2O, (III) +K+H2O vs +Na+H2O, (IV) +K-H2O vs C-H2O, (V) +Na-H2O vs C-H2O e (VI) +K-H2O vs +Na-H2O. Nas comparações entre os tratamentos +H2O, transcritos relacionados ao metabolismo energético e ao processo biológico \"Crescimento\" foram mais abundantes em árvores fertilizadas com potássio e sódio. Nas comparações entre os tratamentos -H2O, transcritos que podem estar relacionados ao ajuste osmótico e transcritos associados ao processo biológico \"Resposta ao estresse\" foram mais abundantes em árvores fertilizadas com potássio. Ainda, foram associados ao tratamento +K-H2O transcritos que podem afetar a biossíntese da parede celular. Em ambos os regimes hídricos, transcritos associados ao fechamento estomático foram menos abundantes nas árvores fertilizadas com potássio. Verificou-se também que vias bioquímicas relacionadas ao metabolismo de açúcares e do carbono foram impactadas. Tais dados observados abrem novos caminhos para pesquisas futuras relacionadas à substituição parcial do potássio pelo sódio na fertilização de plantas, à melhor compreensão molecular do controle estomático e do metabolismo energético e à formação da parede celular influenciados por diferentes condições ambientais visando o melhoramento de tal espécie florestal. / In Brazil, eucalypts is one of the main sources of raw material to produce pulp and paper. The increasing demand for this wood has induced the expansion of forest plantations to lands presenting water deficit and low fertility soils, becoming interesting the obtainment of tolerant plants to stressful environmental conditions. In plants, water deficit causes several physiological, biochemical and molecular responses. Some physiological studies showed that plants with an adequate potassium (K+) supply are more drought tolerant. Nevertheless, for the nutrition of some plants, sodium (Na+) can partially replace the K+. As many agricultural fields in the world are potassium deficient, this can be a cheaper solution for forest plantations fertilization. Understanding at the molecular level the mechanisms of interaction between these minerals and water as well as its consequences for the plant metabolism is very important for the sustainability of forest plantations and the development of forest breeding programs. Thus, the present work aimed the study of Eucalyptus grandis leaf transcriptome to identify and functionally categorize differentially expressed transcripts for which the interaction effect between fertilization and water was significant, given three fertilization regimes (control (C), potassium fertilization (+K) or sodium fertilization (+Na)) associated to two water regimes (regime without throughfall exclusion (+H2O) or regime with 37% of throughfall exclusion (-H2O)). Transcripts related to osmotic functions, photosynthesis and carbon metabolism were selected for more detailed studies. RNA-seq via Illumina platform and many bioinformatic softwares, like Bowtie 2, TopHat, DEseq2 package for R (for statistical analyzes) and Blast2GO tool (for annotation and functional categorization) were used in the transcriptome analysis. Six statistical comparisons were performed: (I) +K+H2O vs C+H2O, (II) +Na+H2O vs C+H2O, (III) +K+H2O vs +Na+H2O, (IV) +K-H2O vs C-H2O, (V) +Na-H2O vs C-H2O and (VI) +KH2O vs +Na-H2O. In the comparisons between +H2O treatments, transcripts related to energetic metabolism and \"Growth\" biological process were more abundant in potassium or sodium fertilized trees. In the comparisons between -H2O treatments, transcripts possibly associated to osmotic adjustment and transcripts related to \"Response to stress\" biological process were more abundant in potassium-fertilized trees. Besides, transcripts that could affect cell wall biosynthesis were associated to +K-H2O treatment. In both water regimes, transcripts associated to stomatal closure were less abundant in potassium-fertilized trees. In addition, biochemical pathways related to carbohydrates and carbon metabolism were impacted. These data provide new perspectives for future researches related to partial replacement of potassium by sodium in plant fertilization, to a better molecular understanding of stomatal control and energetic metabolism and to the cell wall synthesis under different environmental conditions, aiming the breeding of this forest specie.

Orchids are characterized by producing minute endosperm-lacking seeds, which depend on mycorrhizal fungi for germination and embryo development. Some aclorophyllous orchids remain dependent on the mycorrhizal association for carbon acquisition during their whole life history, whereasother orchids develop photosynthesis. Despite the biological significance of orchid mycorrhiza, gene expression studies are lacking. We have used different highthroughput approaches in order to understanding the mechanisms regulating orchid mycorrhiza development and functioning. Firstly, we have used a 2D-LC-MS/MS approach coupled to isobaric tagging for relative and absolute quantification (iTRAQ) to identify proteins with differential accumulation in Oncidium sphacelatum at different stages of mycorrhizal protocorm development (achlorophyllous and green protocorms) after seed inoculation with a Ceratobasidium sp. isolate. Quantitative analysis showed that the expected changes in carbon metabolism in green protocorms were accompanied by enhanced accumulation of proteins involved in the modulation of reactive oxygen species homeostasis, defense related responses, phytoalexins and carotenoid biosynthesis, suggesting that orchid protocorms undergo profound metabolic changes during the switch from the fully mycoheterotrophic to the photosynthethic stage. Secondly, three different proteomic techniques were carried out in independent experiments aiming to identify changes in protein accumulation in mycorrhizal roots of the terrestrial orchid Oeceoclades maculata.Finally, O. maculatamycorrhizal roots were used for transcriptome analyses. The data revealed a strong increase in general stress responses, accompanied by changes in signaling pathways possibly related to fungal recognition and establishment of a compatible interaction. Some of the upregulated genes may be involved in the reorganization of cell structure, likely related to accommodation of the fungal symbiont in the plant roots. We have also observed in mycorrhizal roots up-regulation of genes involved in carbon metabolism, including glycolysis/gluconeogenesis and amino sugars metabolism, as well as genes involved innitrogen assimilation. The down-regulation of genes involved in the jasmonate and ABA transduction pathways, and key genes encoding anti-fungal proteins, such as chitinase and a mannose-specific binding lectin, strongly suggests an alleviation of plant defense responses in O. maculata mycorrhizal roots. In general, our data suggest that the physiology of an orchid mycorrhiza is more similar to a compatible interaction than to an arm-race between plant and fungi. Overall orchid mycorrhiza have proved to be a promising model for investigating plantfungal interactions and further studies should now address the specific roles of the genes showing differential regulation in this study. / As orquídeas são caracterizadas por produzirem sementes diminutas, que não possuem endosperma. Necessitam, portanto, da interação com fungos micorrízicos para germinação e desenvolvimento do embrião. Algumas orquídeas aclorofiladas se mantêm dependentes dos fungos micorrízicos para a aquisição de carbono, enquanto outras desenvolvem a maquinaria fotossintética. Apesar do significado biológico das micorrizas de orquídeas, alterações na expressão gênica e no acúmulo de proteínas foram altamente negligenciads nos últimos anos. Neste trabalho, foram utilizadas diferentes técnicas sequenciamento e identificação de genes e proteínas em larga-escala para acessar as alterações moleculares responsáveis pela regulação das micorrizas de orquídeas. Uma abordagem baseada em 2D-LC MS/MS acoplada a técnica de quantificação absoluta e relativa iTRAQ, foiutilizada para identificar proteínas com acúmulo diferencial em Oncidium sphacelatum em diferentes estágios do desenvolvimento do protocormo (protocormos aclorofilados versus protocormos fotossintetizantes), após inoculação com um fungo do gênero Ceratobasidium. As análises mostraram que, as alterações esperadas no metabolismo do carbono foram acompanhadas de um acúmulo aumentado de proteínas envolvidas na modulação de espécies reativas de oxigênio, respostas de defesa, biossíntese de fitoalexinas e carotenóides, sugerindo que os protocormos de orquídeas passam por profundas alterações metabolicas durante a transição do metabolismo micoheterotrófico para o fotossintético. Posteriormente foram utilizadas três diferentes técnicas de proteômica quantitativa para explorar alterações fisiológicas em raízes micorrizadas e não-micorrizadas de Oeceoclades maculata.Este estudo foi ampliado, pela utilização de uma abordagem transcritômica ao mesmo modelo biológico. Em conjunto, os dados revelaram um forte aumento em respostas relacionadas ao estresse, acompanhadas de alterações em vias de transdução de sinal possivelmente relacionadas ao reconhecimento do simbionte fúngico e estabelecimento de uma interação compatível. Alguns genes com expressão aumentada devem estar envolvidos na reorganização celular, provavelmente ligada a acomodação do simbionte fúngico nas raízes das plantas. Também foi observado o aumento de genes envolvidos no metabolismo do carbono e de açúcares aminados, juntamente a genes relacionados a assimilação de nitrogênio em raízes micorrizadas. A expressão diminuída de genes envolvidas nas vias do jasmonato e ácido abscícico, juntamente a genes-chave que codificam para proteínas anti-fúngicas sugerem fortemente uma atenuação das respostas de defesa da planta em raízes micorrizadas de Oeceoclades maculata. No geral, parece que as micorrizas de orquídeas são fisiológicamente mais próximas de uma simbiose compatível do que de uma interação unilateral em favor da planta. Sobretudo, este sistema biológico provou ser promissor para investigação de interações planta-fungo e, próximas pesquisas devem agora ser focadas em funções específicas dos genes que mostraram regulação diferencial neste estudo.

Orchids are characterized by producing minute endosperm-lacking seeds, which depend on mycorrhizal fungi for germination and embryo development. Some aclorophyllous orchids remain dependent on the mycorrhizal association for carbon acquisition during their whole life history, whereasother orchids develop photosynthesis. Despite the biological significance of orchid mycorrhiza, gene expression studies are lacking. We have used different highthroughput approaches in order to understanding the mechanisms regulating orchid mycorrhiza development and functioning. Firstly, we have used a 2D-LC-MS/MS approach coupled to isobaric tagging for relative and absolute quantification (iTRAQ) to identify proteins with differential accumulation in Oncidium sphacelatum at different stages of mycorrhizal protocorm development (achlorophyllous and green protocorms) after seed inoculation with a Ceratobasidium sp. isolate. Quantitative analysis showed that the expected changes in carbon metabolism in green protocorms were accompanied by enhanced accumulation of proteins involved in the modulation of reactive oxygen species homeostasis, defense related responses, phytoalexins and carotenoid biosynthesis, suggesting that orchid protocorms undergo profound metabolic changes during the switch from the fully mycoheterotrophic to the photosynthethic stage. Secondly, three different proteomic techniques were carried out in independent experiments aiming to identify changes in protein accumulation in mycorrhizal roots of the terrestrial orchid Oeceoclades maculata.Finally, O. maculatamycorrhizal roots were used for transcriptome analyses. The data revealed a strong increase in general stress responses, accompanied by changes in signaling pathways possibly related to fungal recognition and establishment of a compatible interaction. Some of the upregulated genes may be involved in the reorganization of cell structure, likely related to accommodation of the fungal symbiont in the plant roots. We have also observed in mycorrhizal roots up-regulation of genes involved in carbon metabolism, including glycolysis/gluconeogenesis and amino sugars metabolism, as well as genes involved innitrogen assimilation. The down-regulation of genes involved in the jasmonate and ABA transduction pathways, and key genes encoding anti-fungal proteins, such as chitinase and a mannose-specific binding lectin, strongly suggests an alleviation of plant defense responses in O. maculata mycorrhizal roots. In general, our data suggest that the physiology of an orchid mycorrhiza is more similar to a compatible interaction than to an arm-race between plant and fungi. Overall orchid mycorrhiza have proved to be a promising model for investigating plantfungal interactions and further studies should now address the specific roles of the genes showing differential regulation in this study. / As orquídeas são caracterizadas por produzirem sementes diminutas, que não possuem endosperma. Necessitam, portanto, da interação com fungos micorrízicos para germinação e desenvolvimento do embrião. Algumas orquídeas aclorofiladas se mantêm dependentes dos fungos micorrízicos para a aquisição de carbono, enquanto outras desenvolvem a maquinaria fotossintética. Apesar do significado biológico das micorrizas de orquídeas, alterações na expressão gênica e no acúmulo de proteínas foram altamente negligenciads nos últimos anos. Neste trabalho, foram utilizadas diferentes técnicas sequenciamento e identificação de genes e proteínas em larga-escala para acessar as alterações moleculares responsáveis pela regulação das micorrizas de orquídeas. Uma abordagem baseada em 2D-LC MS/MS acoplada a técnica de quantificação absoluta e relativa iTRAQ, foiutilizada para identificar proteínas com acúmulo diferencial em Oncidium sphacelatum em diferentes estágios do desenvolvimento do protocormo (protocormos aclorofilados versus protocormos fotossintetizantes), após inoculação com um fungo do gênero Ceratobasidium. As análises mostraram que, as alterações esperadas no metabolismo do carbono foram acompanhadas de um acúmulo aumentado de proteínas envolvidas na modulação de espécies reativas de oxigênio, respostas de defesa, biossíntese de fitoalexinas e carotenóides, sugerindo que os protocormos de orquídeas passam por profundas alterações metabolicas durante a transição do metabolismo micoheterotrófico para o fotossintético. Posteriormente foram utilizadas três diferentes técnicas de proteômica quantitativa para explorar alterações fisiológicas em raízes micorrizadas e não-micorrizadas de Oeceoclades maculata.Este estudo foi ampliado, pela utilização de uma abordagem transcritômica ao mesmo modelo biológico. Em conjunto, os dados revelaram um forte aumento em respostas relacionadas ao estresse, acompanhadas de alterações em vias de transdução de sinal possivelmente relacionadas ao reconhecimento do simbionte fúngico e estabelecimento de uma interação compatível. Alguns genes com expressão aumentada devem estar envolvidos na reorganização celular, provavelmente ligada a acomodação do simbionte fúngico nas raízes das plantas. Também foi observado o aumento de genes envolvidos no metabolismo do carbono e de açúcares aminados, juntamente a genes relacionados a assimilação de nitrogênio em raízes micorrizadas. A expressão diminuída de genes envolvidas nas vias do jasmonato e ácido abscícico, juntamente a genes-chave que codificam para proteínas anti-fúngicas sugerem fortemente uma atenuação das respostas de defesa da planta em raízes micorrizadas de Oeceoclades maculata. No geral, parece que as micorrizas de orquídeas são fisiológicamente mais próximas de uma simbiose compatível do que de uma interação unilateral em favor da planta. Sobretudo, este sistema biológico provou ser promissor para investigação de interações planta-fungo e, próximas pesquisas devem agora ser focadas em funções específicas dos genes que mostraram regulação diferencial neste estudo.