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La transcription, la maturation d’ARN, et le remodelage de la chromatine sont tous des processus centraux dans l'interprétation de l'information contenue dans l’ADN. Bien que beaucoup de complexes de protéines formant la machinerie cellulaire de transcription aient été étudiés, plusieurs restent encore à identifier et caractériser. En utilisant une approche protéomique, notre laboratoire a purifié plusieurs composantes de la machinerie de transcription de l’ARNPII humaine par double chromatographie d’affinité "TAP". Cette procédure permet l'isolement de complexes protéiques comme ils existent vraisemblablement in vivo dans les cellules mammifères, et l'identification de partenaires d'interactions par spectrométrie de masse. Les interactions protéiques qui sont validées bioinformatiquement, sont choisies et utilisées pour cartographier un réseau connectant plusieurs composantes de la machinerie transcriptionnelle. En appliquant cette procédure, notre laboratoire a identifié, pour la première fois, un groupe de protéines, qui interagit physiquement et fonctionnellement avec l’ARNPII humaine. Les propriétés de ces protéines suggèrent un rôle dans l'assemblage de complexes à plusieurs sous-unités, comme les protéines d'échafaudage et chaperonnes. L'objectif de mon projet était de continuer la caractérisation du réseau de complexes protéiques impliquant les facteurs de transcription. Huit nouveaux partenaires de l’ARNPII (PIH1D1, GPN3, WDR92, PFDN2, KIAA0406, PDRG1, CCT4 et CCT5) ont été purifiés par la méthode TAP, et la spectrométrie de masse a permis d’identifier de nouvelles interactions. Au cours des années, l’analyse par notre laboratoire des mécanismes de la transcription a contribué à apporter de nouvelles connaissances et à mieux comprendre son fonctionnement. Cette connaissance est essentielle au développement de médicaments qui cibleront les mécanismes de la transcription. / Genomes encode most of the functions necessary for cell growth and differentiation. Gene transcription, RNA processing, and chromatin remodeling are central processes in the interpretation of the information contained in genomic DNA. Although many protein complexes forming the cellular machinery that interprets mammalian genomes have been studied, a number of additional complexes remain to be identified and characterized. Using proteomic approaches, Dr. Benoit Coulombe’s laboratory purified many components of the RNAPII transcription machinery using tandem affinity purification (TAP), a procedure that allows the isolation of protein complexes as they likely exist in live mammalian cells, and the identification of interaction partners using mass spectrometry. High confidence interactions were selected computationally and used to draw the map of a network connecting many components of the mRNA transcriptional machinery. By applying this procedure, our lab has identified, for the first time, a group of proteins, that interacts both physically and functionally with human RNAPII, and whose properties suggest a role in the assembly of multi-subunit complexes, acting as RNAPII-specific scaffolding proteins and chaperones. The aim of my project was to continue the characterization of the network of protein complexes involving transcription factors, and thus, further pursuing our survey of protein complexes in whole cell extracts. Eight novel RNAPII interaction partners (PIH1D1, GPN3, WDR92, PFDN2, KIAA0406, PDRG1, CCT4 and CCT5) were purified using the tandem affinity purification (TAP) method, and their interaction partners were identified by mass spectrometry. Over the years, our lab’s analysis of transcriptional regulation and mechanisms has contributed novel and important knowledge that provided better understanding of mRNA synthesis. This knowledge is paramount to the development of therapeutics that will target transcriptional mechanisms.

Les plantes n'exploitent pas seules les nutriments du sol, mais dépendent de champignons avec lesquels elles forment des symbioses mycorhiziennes dans leurs racines. C'est en particulier vrai pour les 25 000 espèces d'orchidées actuelles qui dépendent toutes de champignons mycorhiziens pour accomplir leur cycle de vie. Elles produisent des graines microscopiques qui n'ont pas les ressources nutritives pour germer, mais qui dépendent de la présence de partenaires adéquats pour nourrir l'embryon (hétérotrophie) jusqu'à l'apparition des feuilles (autotrophie). Les mycorhiziens restent présents dans les racines des adultes où ils contribuent à la nutrition, ce qui permet d'étudier plus facilement la diversité des symbiotes à l'aide des outils génétiques. Conscients des biais des études en faveur des régions tempérées, nous avons étudié la diversité des mycorhiziens d'orchidées tropicales à La Réunion. Nous avons montré que (1) les orchidées tropicales ont des partenaires semblables aux orchidées tempérées et méditerranéennes (Sebacinales, Ceratobasidiaceae et surtout Tulasnellaceae), et que ces taxons de champignons sont largement représentés dans différents biomes et dans différentes plantes hôtes. Nous avons aussi démontré pour la première fois que (2) les orchidées épiphytes (dont les associations étaient peu connues) ont des cortèges mycorhiziens différents de ceux des orchidées terrestres dans les communautés tropicales. De plus, en développant une approche à l'échelle de réseaux d'interactions (78 espèces de La Réunion), nous avons montré que (3) les espèces tropicales ont tendance à être généralistes et que (4) le réseau mycorhizien des orchidées montre des propriétés semblables à celles des réseaux d'interactions mutualistes (nestedness et asymétrie d'interaction), alors que la nature mutualiste de cette symbiose mycorhiziennes fait débat. Dans un second volet de la thèse, nous avons étudié les partenaires des orchidées non chlorophylliennes (mycohétérotrophes) tropicales. Nous avons montré que (5) les espèces tropicales peuvent s'associer à des champignons saprophytes qui les nourrissent en carbone issu de la décomposition de la litière dans les forêts tropicales humides et que (6) les modèles tropicaux (en n'étant pas spécifiques) remettent en question les idées reçues sur la mycohétérotrophie des plantes. Nous avons confirmé que (7) la mycohétérotrophie dérive d'un régime nutritionnel intermédiaire (mixotrophie) mis en place dans des lignées chlorophylliennes. Dans un dernier volet de la thèse, nous avons posé la question du déterminisme phylogénétique des associations orchidées-champignons. En analysant la force du signal dans les phylogénies des deux partenaires, nous avons vérifié que (8) les associations mycorhiziennes sont peu conservées à l'échelle supra-générique dans la phylogénie des orchidées, et qu'elles (9) peuvent être maintenues à une échelle plus récente (cas de certains clades d'angraecoïdes). Ces résultats soulignent l'empreinte relative des processus écologiques et évolutifs sur les patrons d'associations actuels, et remettent en question l'idée qu'un processus de coévolution pourrait guider le système. / Plants generally do not exploit soil nutrients themselves, but they depend upon mycorrhizal symbioses with root-associating fungi. This is the case for the current 25,000 orchid species that depend on the development of a mycorrhizal association to germinate and establish. They produce minute seeds lacking nutritional ressources required to germinate, so that they depend on the presence of suitable fungal partners to obtain carbon (heterotrophy) until the development of leaves (autotrophy). Mycorrhizal fungi remain present in the roots of adult plants where they contribute to the plant nutrition, which makes the molecular identification of fungal partners easier. Given the fact that most studies have been conducted in temperate regions, we have studied the diversity of mycorrhizal fungi in tropical orchids of La Réunion. We have found that (i) tropical orchids have the same partners as temperate and mediterranean orchids (Sebacinales, Ceratobasidiaceae, and above all Tulasnellaceae), and that these fungi are widespread in biomes and host plants. We have also showed for the first time that (ii) epiphytic orchids-that have hardly been studied-have partners that differ from terrestrial orchids' in tropical plant communities. Moreover, by developing an interaction network approach (78 species of La Réunion), we have found that (iii) most tropical species are generalists and that (iv) the mycorrhizal network shows the same properties as the mutualistic interaction networks' (nestedness and interaction asymmetry), whereas the mutualistic nature of the orchid symbiosis is still a current issue. In the second part of our thesis, we have studied the fungal partners of achlorophyllous (i.e. mycoheterotrophic) tropical orchids. We have found that (v) tropical species often associate with saprophytic fungi that provide carbon extracted from decaying wood or leaves in tropical soils, and that (vi) tropical models, because of their lack of specificity, challenge the rule drawn from temperate models. We have also confirmed in tropical models that (vii) mycoheterotrophy evolved from mixotrophic ancestors (i.e. intermediate nutritional mode). In the last part of our thesis, we have dealt with the influence of orchid and fungal phylogenies in explaining the structure of the observed networks. By measuring the phylogenetic signals in both orchid and fungal phylogenies, we have checked that (viii) mycorrhizal interactions are not explained by the phylogenetic placements of either orchid genera or fungal taxa. However, we have noticed that (ix) a phylogenetic signal can occur in recent clades of orchid species (but not in fungal species). These results provide insights in the relative imprint of ecological and evolutionary processes on the current patterns of fungus-orchid associations, and challenge the idea that the coevolutionary process could drive the system.

Les biomolécules exercent leurs fonctions en interagissant avec d'autres molécules. Le recensement de l'ensemble des biomolécules et leurs interactions permet de construire leurs réseaux d'interactions et de les analyser sur le plan structural et fonctionnel par des outils bioinformatiques (BiNGO, DAVID). Cela permet d'identifier les biomolécules clés, de prédire de nouvelles fonctions des protéines et de comprendre et modéliser les mécanismes moléculaires d'un processus biologique ou pathologique donné. Les protéines ou régions intrinsèquement désordonnées, qui possèdent une grande plasticité structurale, sont susceptibles d'interagir avec de nombreux partenaires et d'être importantes dans les réseaux d'interactions. A l'aide du prédicteur IUPred, nous avons dans un premier temps cartographié le désordre intrinsèque des protéines dans le réseau d'interactions de la matrice extracellulaire et dans le réseau extracellulaire des protéoglycanes construits à partir de la base de données MatrixDB développée dans l'équipe. Nous avons montré que les protéines très connectées de ces deux réseaux ne sont pas enrichies en désordre. Les fonctions moléculaires surreprésentées dans le jeu de protéines extracellulaires contenant au moins 50% de désordre intrinsèque sont les interactions avec les facteurs de croissance ou les glycosaminoglycanes. Nous avons étudié un jeu de données d'interactions protéine-héparine comportant 118 valeurs de cinétique et nous avons montré une relation positive entre la vitesse d'association des protéines à l'héparine et le pourcentage de désordre de leurs sites de fixation à l'héparine. Nous avons également étudié les interactions de la matrice extracellulaire avec un pathogène, le parasite Leishmania. Nous avons montré que les protéines sécrétées par les Leishmania ne sont pas enrichies en désordre par rapport au protéome. Nous avons établi une liste de onze protéines parasitaires sécrétées possédant au moins trois motifs d'interaction et susceptibles d'interagir avec l'hôte / Biomolecules perform their functions by interacting with other molecules. The identification of all biomolecules and their interactions is required to build their interaction networks. Their structural and functional analysis with bioinformatics tools (BiNGO, DAVID) allow us to identify the key biomolecules, to predict new protein functions and to understand and model the molecular mechanisms of biological or pathological process. Intrinsically disordered proteins or regions, which are characterized by structural plasticity, may interact with many partners and may play a role in the interaction networks. Using the predictor IUPred we mapped the intrinsic disorder in protein interaction networks of the extracellular matrix and of the proteoglycans constructed from the MatrixDB database developed in the laboratory. We have shown that the highest connected proteins of these two networks are not enriched in disorder. The molecular functions overrepresented in the set of extracellular proteins containing at least 50% of intrinsically disordered residues are interactions with growth factors or glycosaminoglycans. We studied a dataset of heparin-protein interactions including 118 kinetic values and we have shown that the association rate of proteins with heparin is related to the intrinsic disorder of heparin-binding sites. We also studied the interactions of the extracellular matrix with a pathogen, the parasite Leishmania. We have shown that proteins secreted by Leishmania are not enriched in disorder compared to their proteome. We have selected eleven parasite proteins containing at least three interaction motifs, which may interact with the host

Au fil du temps, la vie a évolué pour produire des organismes remarquablement complexes. Pour faire face à cette complexité, les organismes ont développé une pléthore de mécanismes régulateurs. Par exemple, les mammifères transcrivent des milliers d'ARN longs non codants (ARNlnc), accroissant ainsi la capacité régulatrice de leurs cellules. Un concept émergent est que les ARNlnc peuvent servir d'échafaudages aux complexes protéiques, mais la prévalence de ce mécanisme n'a pas encore été démontrée. De plus, pour chaque ARN messager, plusieurs régions 3’ non traduites (3’UTRs) sont souvent présentes. Ces 3’UTRs pourraient réguler la fonction de la protéine en cours de traduction, en participant à la formation des complexes protéiques dans lesquels elle est impliquée. Néanmoins, la fréquence et l’importance ce mécanisme reste à aborder.Cette thèse a pour objectif de découvrir et comprendre systématiquement ces deux mécanismes de régulation méconnus. Concrètement, l'assemblage de complexes protéiques promus par les ARNlnc et les 3'UTRs est étudié avec des données d’interactions protéines-protéines et protéines-ARN à grande échelle. Ceci a permis (i) de prédire le rôle de plusieurs centaines d'ARNlnc comme molécules d'échafaudage pour plus de la moitié des complexes protéiques connus, ainsi que (ii) d’inférer plus d’un millier de complexes 3'UTR-protéines, dont certains cas pourraient réguler post-traductionnellement des protéines moonlighting aux fonctions multiples et distinctes. Ces résultats indiquent qu'une proportion élevée d'ARNlnc et de 3'UTRs pourrait réguler la fonction des protéines en augmentant ainsi la complexité du vivant. / Over time, life has evolved to produce remarkably complex organisms. To cope with this complexity, organisms have evolved a plethora of regulatory mechanisms. For instance, thousands of long non-coding RNAs (lncRNAs) are transcribed by mammalian genomes, presumably expanding their regulatory capacity. An emerging concept is that lncRNAs can serve as protein scaffolds, bringing proteins in proximity, but the prevalence of this mechanism is yet to be demonstrated. In addition, for every messenger RNA encoding a protein, regulatory 3’ untranslated regions (3’UTRs) are also present. Recently, 3’UTRs were shown to form protein complexes during translation, affecting the function of the protein under synthesis. However, the extent and importance of these 3’UTR-protein complexes in cells remains to be assessed.This thesis aims to systematically discover and provide insights into two ill-known regulatory mechanisms involving the non-coding portion of the human transcriptome. Concretely, the assembly of protein complexes promoted by lncRNAs and 3’UTRs is investigated using large-scale datasets of protein-protein and protein-RNA interactions. This enabled to (i) predict hundreds of lncRNAs as possible scaffolding molecules for more than half of the known protein complexes, as well as (ii) infer more than a thousand distinct 3’UTR-protein complexes, including cases likely to post-translationally regulate moonlighting proteins, proteins that perform multiple unrelated functions. These results indicate that a high proportion of lncRNAs and 3’UTRs may be employed in regulating protein function, potentially playing a role both as regulators and as components of complexity.

La compréhension des pathologies humaines et du mode d'action des médicaments passe par la prise en compte des réseaux d'interactions entre biomolécules. Les recherches récentes sur les systèmes biologiques produisent de plus en plus de données sur ces réseaux qui gouvernent les processus cellulaires. L'hétérogénéité et la multiplicité de ces données rendent difficile leur intégration dans les raisonnements des utilisateurs. Je propose ici des approches intégratives mettant en oeuvre des techniques de gestion de données, de visualisation de graphes et de fouille de données, pour tenter de répondre au problème de l'exploitation insuffisante des données sur les réseaux dans la compréhension des phénotypes associés aux maladies génétiques ou des effets secondaires des médicaments. La gestion des données sur les protéines et leurs propriétés est assurée par un système d'entrepôt de données générique, NetworkDB, personnalisable et actualisable de façon semi-automatique. Des techniques de visualisation de graphes ont été couplées à NetworkDB pour utiliser les données sur les réseaux biologiques dans l'étude de l'étiologie des maladies génétiques entrainant une déficience intellectuelle. Des sous-réseaux de gènes impliqués ont ainsi pu être identifiés et caractérisés. Des profils combinant des effets secondaires partagés par les mêmes médicaments ont été extraits de NetworkDB puis caractérisés en appliquant une méthode de fouille de données relationnelles couplée à Network DB. Les résultats permettent de décrire quelles propriétés des médicaments et de leurs cibles (incluant l'appartenance à des réseaux biologiques) sont associées à tel ou tel profil d'effets secondaires / The understanding of human diseases and drug mechanisms requires today to take into account molecular interaction networks. Recent studies on biological systems are producing increasing amounts of data. However, complexity and heterogeneity of these datasets make it difficult to exploit them for understanding atypical phenotypes or drug side-effects. This thesis presents two knowledge-based integrative approaches that combine data management, graph visualization and data mining techniques in order to improve our understanding of phenotypes associated with genetic diseases or drug side-effects. Data management relies on a generic data warehouse, NetworkDB, that integrates data on proteins and their properties. Customization of the NetworkDB model and regular updates are semi-automatic. Graph visualization techniques have been coupled with NetworkDB. This approach has facilitated access to biological network data in order to study genetic disease etiology, including X-linked intellectual disability (XLID). Meaningful sub-networks of genes have thus been identified and characterized. Drug side-effect profiles have been extracted from NetworkDB and subsequently characterized by a relational learning procedure coupled with NetworkDB. The resulting rules indicate which properties of drugs and their targets (including networks) preferentially associate with a particular side-effect profile

A interação entre plantas e seus visitantes florais forma uma rede de interação complexa. Essa rede de interação possui propriedades emergentes que caracterizam a estrutura da rede. A composição, a riqueza e o papel das espécies nas redes de interação podem variar espaço-temporalmente e levar a alteração na estrutura das redes. A estrutura também pode variar de acordo com o grau de dependência entre os pares de espécies. É esperado que interações com elevado grau de dependência mútua variem menos no tempo e no espaço. A interação entre as plantas produtoras de óleo floral e as abelhas coletoras de óleo é de alta dependência mútua, já que as abelhas necessitam do óleo para alimentar as larvas e construir ninhos e são polinizadores mais importantes dessas plantas. O alimento larval destas abelhas é composto por pólen, óleo e néctar. A quantidade de alimento disponível para as larvas pode influenciar o tamanho dos adultos quando emergirem. As espécies de abelhas coletoras de óleo são solitárias, ou seja, não existe contato entre as gerações. Assim uma abelha recém-emergida deve ser capaz de encontrar as fontes de óleo, seguindo algum sinal de atração, que poderia ser através de odores voláteis. No presente trabalho analisamos se a estrutura da rede de interação formada pelas plantas produtoras de óleo floral e seus visitantes coletores de óleo varia espacialmente e temporalmente. Para a análise temporal coletamos dados da interação por dois anos em Itirapina, São Paulo. No estudo de variação espacial comparamos as redes de interação em cinco áreas de cerrado no interior de São Paulo. Em ambos os estudos usamos métricas de redes complexas. Além disso, estudamos a relação entre tamanho de célula de cria e quantidade de alimento disponível para as larvas e tamanho das abelhas adultas ao emergirem, para uma espécie de abelha coletora de óleo: Tetrapedia diversipes. Para isso medimos abelhas e suas células de cria e relacionamos estas medidas. Também manipulamos a quantidade de alimento disponível para as larvas e medimos os adultos emergidos. Por fim, procuramos odores voláteis em Byrsonima intermedia. Coletamos odores voláteis com a técnica de dynamic headspace. Analisamos as coletas com GCMS. A estrutura da rede de interação entre as plantas produtoras de óleo floral e as abelhas coletoras de óleo variou espaço-temporalmente. A análise temporal mostrou que o grau de especialização da rede foi fortemente alterado entre os anos de estudo e que a abundância foi um fator importante para explicar a ocorrência das interações. As espécies que tiveram suas abundâncias alteradas entre os anos também tiveram alteração no número de interações. Na análise espacial observamos que as interações e as espécies compartilhadas entre os locais de estudo foram aquelas com maior grau de interação (generalista). Além das interações e espécies generalistas se manterem nas cinco localidades, as espécies generalistas sustentam as espécies localmente raras. Byrsonima intermedia foi considerada uma espécie super-generalista nas cinco redes analisadas e 77% das interações compartilhadas envolveram esta espécie. A morfometria dos indivíduos de Tetrapedia diversipes estudados foi positivamente correlacionada com o volume da célula de cria e a quantidade de alimento. Quando relacionamos o volume das células de cria com as medidas corpóreas das abelhas verificamos que as abelhas maiores emergiram em células maiores. No entanto essa relação foi fraca. Após o experimento de manipulação de alimento verificamos que as abelhas emergidas de células sem manipulação foram maiores que as abelhas que tiveram o alimento retirado, e que entre as abelhas manipuladas aquelas que receberam mais alimento foram maiores. Nós não encontramos odores voláteis em flores de Byrsonima intermedia. / The plant pollinator interaction forms a complex network. These networks have emergent properties that characterize the structure of network. The number, identity and species role of the network may vary spatio-temporally and lead to changes in the structure of the networks. Moreover, the structure may vary according to the dependence degree of the interacting pairs. It is expected that interactions with a high mutual dependence degree are more stable. The interaction between oil producing plants and oil collecting bees is highly mutual dependence, since bees need oil to feed the larvae and are pollinators of these plants. The larval diet consists of pollen and oil/nectar. The amount of food available to the larvae may influence the adults size. The oil collecting bees are solitary, i.e., there is no contact between generations. Thus, a naive emerged bee should be able to find the oil plants, using some attractive cues that could be volatile odor. In this work we want to answer if the network structure of the interaction between oil producing plants and oil collecting bees varies spatially and temporally. For the temporal analysis we carried out field work for two years at Itirapina, São Paulo. In the study of spatial variation we compare interaction networks in five areas of cerrado. In both studies we use metrics of complex networks. Furthermore, we studied the relation between size of adult bees (Tetrapedia diversipes) and brood cell sizes and the amount of food available for the larvae. We measured the bees and their brood cells and relate it. Also we manipulated the amount of food available for the larvae and we took measures of the adults. Finally, we search for volatile odors in Byrsonima intermedia. We collect volatile odors with the technique of dynamic headspace. We analyzed the samples with GCMS. The network structure of interaction between oil producing plants and oil collecting bees varied spatio-temporally. The temporal analysis showed that the specialization degree of the network changed between the years and that the abundance has a key role to explain the occurrence of interactions. Species that had changed their abundances between years were also changed in the number of interactions. Spatial analysis found out that the interactions and species ubiquitous among the study sites were those that had the greatest interaction degree (generalist specie). Moreover, generalist species maintain the locally rare species. Byrsonima intermedia was regarded a super-generalist specie in the five networks analyzed and 77% of ubiquitous interactions involved this species. The morphometry of Tetrapedia diversipes individuals was positively correlated with the volume of the brood cells and the amount of food. When relating the volume of brood cells with measures of bees we found that larger bees emerged in larger cells. However this relationship was weak. The experiment of handling food showed that bees emerged from control cells were larger than bees that had the food removed, and between the manipulated bees those who received more food became bigger. We did not detect volatile odors in flowers Byrsonima intermedia.

Submitted by Marlene Santos (marlene.bc.ufg@gmail.com) on 2014-09-17T21:01:01Z No. of bitstreams: 2 José Neiva Mesquita Neto.pdf: 2799737 bytes, checksum: 590e98d41066ac6babc47eeb04637796 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Rejected by Luciana Ferreira (lucgeral@gmail.com), reason: Marlene, olhe nas orientações a questão da citação, a qual deve ser de acordo com a NBR 6023: ALCÂNTARA, Guizelle Aparecida de. Caracterização farmacognostica e atividade antimicrobiana da folha e casca do caule da myrciarostratadc.(myrtaceae). 2012. 41 f. Dissertação (Mestrado em Ciências Farmacêuticas) - Universidade Federal de Goiás, Goiânia, 2012. on 2014-09-18T12:33:01Z (GMT) / Submitted by Marlene Santos (marlene.bc.ufg@gmail.com) on 2014-09-18T19:01:02Z No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) José Neiva Mesquita Neto.pdf: 2799737 bytes, checksum: 590e98d41066ac6babc47eeb04637796 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2014-09-19T11:32:20Z (GMT) No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) José Neiva Mesquita Neto.pdf: 2799737 bytes, checksum: 590e98d41066ac6babc47eeb04637796 (MD5) / Made available in DSpace on 2014-09-19T11:32:20Z (GMT). No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) José Neiva Mesquita Neto.pdf: 2799737 bytes, checksum: 590e98d41066ac6babc47eeb04637796 (MD5) Previous issue date: 2013-09-19 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Plant-pollinator interactions in syncronopatric species of Psychotria (Rubiaceae). Psychotria species are commonly found in the understory of forested areas in tropical regions, and are important components for the functioning of these ecosystems. Many species of the genus are sympatric and bloom during the same period, being considered potentially syncronopatrics. For this reason, this genus has been considered as a good model to infer general patterns and mechanisms of speciation in the tropics. In the first paper of this dissertation, the interactions between potentially sympatric species of Psychotria and their pollinators were analyzed in order to verify the possible existence of sharing, specialization or generalization of pollinators and plants in the analyzed system. Then, data of secondary studies that contained identifying pollinators of Psychotria were analyzed From these data, we generated graphs and analyzes of interaction networks and niche overlap. Altogether, nine species of Psychotria and 25 species of pollinators of occurrence in the Atlantic Forest were included in the analyzes. The plant with the most generalist network is Psychotria tenuinervis and the pollinators with the highest proportion of links and consequently with more important role in the network belonged orders Lepidoptera and Hymenoptera. Psychotria species showed niche overlap in pollination and pollinator sharing. The period of greatest intensity of flowering coincided with the rainy season in the Atlantic Forest, with up to seven species coflowering. Because Psychotria species occur in sympatry and have flowering overlap and asymmetric interactions with flower visitors, it is possible that they are involved in a process of facilitation in pollination. The second paper aimed to investigate if four sympatric populations of Psychotria show pollinators sharing and pollination niche overlap . Another objective was to determine if their flowering season are synchronics. We found a dual synchrony in this system, the first in the intensity of flowering among the populations of Psychotria and the second in the increase of the abundance of flowers and pollinators. The species of Psychotria showed sharing fundamental niche of pollination and positive impact on reproductive success. However, we note that the sharing of pollinators can not be generalized to the entire system, but to the modules or pairs of species. Thus, each species of Psychotria has one or more species of preferential pollinator for sharing and that this relationship is not always reciprocal. This reinforces that the interactions among the plant species are asymmetric. / Interação planta-polinizador em espécies sincronopátricas de Psychotria (Rubiaceae). Psychotria contribui de forma importante para a diversidade florística tropical e são comumente encontradas em sub-bosque de áreas florestadas em regiões tropicais, sendo importantes componentes para o funcionamento destes ecossistemas. Muitas espécies do gênero são simpátricas e florescem em um mesmo período do ano, sendo consideradas potencialmente sincronopátricas. Por esta razão, este gênero tem sido considerado como um bom modelo para inferir padrões e mecanismos gerais de especiação nos trópicos. No primeiro artigo, as interações entre espécies potencialmente simpátricas de Psychotria e seus polinizadores foram analisadas com o objetivo de verificar a possível existência de partilha, de especialização ou generalização de polinizadores e plantas no sistema analisado. Para isso, foram incluídas informações secundárias de estudos que continham a identificação em nível de espécie de polinizadores de Psychotria. A partir desses dados, foram gerados grafos e realizadas análises de redes de interação e sobreposição de nicho. Ao todo, foram incluídas nas análises nove espécies de Psychotria com registro de ocorrência para a Mata Atlântica, sendo que elas interagiram com 25 espécies de polinizadores. A planta mais generalista da rede foi Psychotria tenuinervis e os polinizadores que concentraram maior número de links e consequentemente com papel mais importante na rede pertenceram as ordens Lepidoptera e Hymenoptera. As espécies de Psychotria apresentaram sobreposição de nicho na polinização com partilha de polinizadores. O período de maior intensidade de floração coincidiu com o período chuvoso na Mata Atlântica, com até sete espécies apresentando coflorescimento. Assim, pode-se inferir que a polinização em Psychotria demanda polinizadores generalistas. Pelo fato das espécies de Psychotria ocorrerem em simpatria, possuírem floração sobreposta e interações assimétricas com visitantes florais, é possível que estejam envolvidas em um processo de facilitação na polinização. Já o segundo artigo objetivou averiguar se quatro populações simpátricas de Psychotria, em cofloração, apresentaram partilha de polinizadores e sobreposição de nicho na polinização. Além disso, objetivou verificar se existe sincronia entre as populações vegetais e destas com comunidade de polinizadores. Foi encontrada uma dupla sincronia nesse sistema, sendo a primeira na intensidade de floração entres as populações de Psychotria e a segunda no aumento da abundância de flores e de polinizadores. As espécies de Psychotria apresentaram partilha fundamental do nicho da polinização e com reflexo positivo no sucesso reprodutivo. No entanto, verificamos que a partilha de polinizadores não pode ser generalizada a todo o sistema, mas sim a módulos ou pares de espécies. Dessa forma, cada espécie de Psychotria possui uma ou mais espécies preferencias para partilha de polinizadores e que nem sempre essa relação é reciproca. Isso reforça que as interações entre as plantas são assimétricas.

La transcription, la maturation d’ARN, et le remodelage de la chromatine sont tous des processus centraux dans l'interprétation de l'information contenue dans l’ADN. Bien que beaucoup de complexes de protéines formant la machinerie cellulaire de transcription aient été étudiés, plusieurs restent encore à identifier et caractériser. En utilisant une approche protéomique, notre laboratoire a purifié plusieurs composantes de la machinerie de transcription de l’ARNPII humaine par double chromatographie d’affinité "TAP". Cette procédure permet l'isolement de complexes protéiques comme ils existent vraisemblablement in vivo dans les cellules mammifères, et l'identification de partenaires d'interactions par spectrométrie de masse. Les interactions protéiques qui sont validées bioinformatiquement, sont choisies et utilisées pour cartographier un réseau connectant plusieurs composantes de la machinerie transcriptionnelle. En appliquant cette procédure, notre laboratoire a identifié, pour la première fois, un groupe de protéines, qui interagit physiquement et fonctionnellement avec l’ARNPII humaine. Les propriétés de ces protéines suggèrent un rôle dans l'assemblage de complexes à plusieurs sous-unités, comme les protéines d'échafaudage et chaperonnes. L'objectif de mon projet était de continuer la caractérisation du réseau de complexes protéiques impliquant les facteurs de transcription. Huit nouveaux partenaires de l’ARNPII (PIH1D1, GPN3, WDR92, PFDN2, KIAA0406, PDRG1, CCT4 et CCT5) ont été purifiés par la méthode TAP, et la spectrométrie de masse a permis d’identifier de nouvelles interactions. Au cours des années, l’analyse par notre laboratoire des mécanismes de la transcription a contribué à apporter de nouvelles connaissances et à mieux comprendre son fonctionnement. Cette connaissance est essentielle au développement de médicaments qui cibleront les mécanismes de la transcription. / Genomes encode most of the functions necessary for cell growth and differentiation. Gene transcription, RNA processing, and chromatin remodeling are central processes in the interpretation of the information contained in genomic DNA. Although many protein complexes forming the cellular machinery that interprets mammalian genomes have been studied, a number of additional complexes remain to be identified and characterized. Using proteomic approaches, Dr. Benoit Coulombe’s laboratory purified many components of the RNAPII transcription machinery using tandem affinity purification (TAP), a procedure that allows the isolation of protein complexes as they likely exist in live mammalian cells, and the identification of interaction partners using mass spectrometry. High confidence interactions were selected computationally and used to draw the map of a network connecting many components of the mRNA transcriptional machinery. By applying this procedure, our lab has identified, for the first time, a group of proteins, that interacts both physically and functionally with human RNAPII, and whose properties suggest a role in the assembly of multi-subunit complexes, acting as RNAPII-specific scaffolding proteins and chaperones. The aim of my project was to continue the characterization of the network of protein complexes involving transcription factors, and thus, further pursuing our survey of protein complexes in whole cell extracts. Eight novel RNAPII interaction partners (PIH1D1, GPN3, WDR92, PFDN2, KIAA0406, PDRG1, CCT4 and CCT5) were purified using the tandem affinity purification (TAP) method, and their interaction partners were identified by mass spectrometry. Over the years, our lab’s analysis of transcriptional regulation and mechanisms has contributed novel and important knowledge that provided better understanding of mRNA synthesis. This knowledge is paramount to the development of therapeutics that will target transcriptional mechanisms.