Les systèmes toxine-antitoxines (TA) sont abondants dans la majorité des génomes bactériens séquencés à ce jour. Ces systèmes codent une toxine stable qui inhibe soit la transcription, soit la traduction, et une antitoxine qui contrecarre l’effet de la toxine par formation d’un complexe avec celle-ci. L’antitoxine est instable suite à sa dégradation continue par les protéases ATP-dépendantes. Afin de maintenir un ratio antitoxine :toxine constant en condition normale de croissance, l’expression des systèmes TA est régulée négativement au niveau transcriptionnel par le complexe toxine-antitoxine.<p><p>Au début de notre travail, cinq systèmes TA étaient identifiés dans le chromosome d’E. coli. Il avait été montré par notre laboratoire que parmi ces systèmes, seul yefM-yoeB était activé en condition de surproduction de la protéase ATP-dépendante Lon. Ce résultat était surprenant puisque Lon était connue pour dégrader également l’antitoxine RelB du système chromosomique relBE. Un des objectifs de notre travail était de comprendre les mécanismes sous-jacents à cette spécificité. Nous avons montré que l’antitoxine YefM était dégradée à la fois par Lon et les protéases ClpAP et ClpXP. Nous avons également montré qu’en condition de surproduction de Lon, YefM était fortement instable (t1/2~ 10 min. vs 60 min en condition normale). Cette instabilité accrue permet donc l’activation du système yefM-yoeB, c’est-à-dire la libération de la toxine YoeB du complexe qu’elle forme avec YefM. Nous avons également avons montré que le t1/2 de RelB n’était pas affecté par la surproduction de Lon, ce qui explique pourquoi le système relBE n’est pas activé dans ces conditions. Notre hypothèse était qu’un cofacteur soit nécessaire à la dégradation de RelB par Lon et que celui-ci serait limitant dans nos conditions expérimentales. Le crible génétique que nous avons réalisé n’a cependant pas permis d’identifier de cofacteur de dégradation ni de régulateur transcriptionnel en trans du système relBE. <p><p>Un deuxième volet de notre travail de thèse a consisté en l’étude de la fonction des systèmes TA chromosomiques. L’hypothèse prévalente au début de notre travail était que les systèmes TA soient intégrés dans les voies adaptatives de réponses au stress. Cependant, le résultat de leur activation était controversé. L’hypothèse du groupe de Gerdes était que leur activation mène à un état bactériostatique réversible alors que le groupe d’Engelberg-Kulka montrait que le système mazEF était un système de mort programmée. Afin d’éclaircir le rôle des cinq systèmes TA dans la physiologie d’E. coli, nous avons testé l’effet de nombreux stress sur la croissance et la viabilité de souches sauvages et de souches délétées de ces systèmes. Aucune des conditions que nous avons testées n’a entraîné une diminution de la viabilité excluant de manière définitive l’hypothèse de la mort programmée. De plus, l’inhibition de croissance causée par ces différents stress s’est avérée être indépendante des cinq systèmes, de même que la phase de récupération suivant les différents stress. Enfin, nos expériences de compétition ont clairement démontré que les cinq systèmes ne procuraient aucun avantage sélectif aux bactéries dans des conditions de compétition en carence nutritive. Les systèmes TA étudiés dans ce travail ne jouent donc aucun rôle dans l’adaptation aux stress que nous avons testé puisqu’ils n’améliorent ni l’aptitude (fitness), ni la compétitivité des bactéries dans ces conditions. <p><p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Les infections néonatales tardives (INT, survenant après 3 jours de vie) sont fréquentes et sont associées à une mortalité et une morbidité importantes chez les nouveau-nés prématurés. Dans ce contexte, il a été récemment décrit un clone de Staphylococcus capitis, appelé NRCS-A, impliqué spécifiquement dans ces INT dans différents services de réanimation néonatale (RN) à travers la France, et présentant un profil multirésistant atypique chez cette espèce, incluant notamment une sensibilité diminuée à la vancomycine, qui est pourtant l'antibiotique de première ligne en cas de suspicion d'INT. Dans le cadre de ce travail, nous avons démontré la distribution endémique du clone NRCS-A dans au moins 17 pays à travers le monde, spécifiquement dans les services de RN. De plus des données épidémiologiques issues des services de RN français ont identifié une prévalence élevée du clone dans certains services, illustrant sa capacité à s'implanter puis à persister dans ces services. Une caractérisation génétique du clone NRCS-A a été réalisée afin de mettre en évidence d'éventuels facteurs génétiques pouvant favoriser son implantation dans les services de RN. Cette analyse a démontré le rôle des éléments génétiques mobiles dans l'émergence du phénotype multirésistant du clone NRCS-A. En revanche aucun gène de virulence spécifique du clone n'a pu être mis en évidence. L'analyse des gènes spécifiques du clone a toutefois permis d'identifier le gène nsr codant pour la résistance à la nisine, bactériocine active sur de nombreuses bactéries à Gram positif et sécrétée par les bactéries de la flore commensale digestive. Ce gène pourrait donc conférer un avantage sélectif au clone NRCS-A pour s'implanter dans le microbiote des nouveau-nés prématurés. La persistance du clone dans les services de RN évoque la présence de réservoirs inertes ou humains au sein de ces services. Grâce à la mise au point d'une technique d'identification de S. capitis par gélose chromogénique sélective, nous avons pu démontrer la diffusion et la persistance de S. capitis dans un service de RN, sans toutefois identifier un réservoir unique responsable de cette colonisation. Nous avons également observé une inefficacité partielle des mesures de décontamination. Il n'existe en revanche pas de portage chronique chez le personnel soignant, ni de colonisation vaginale chez les femmes enceintes. Par ailleurs, nous avons pu mettre en évidence par repiquages successifs in vitro une capacité particulière du clone NRCS-A à acquérir de façon rapide et stable une résistance à la vancomycine sous pression de sélection par cet antibiotique. Cette capacité constitue un avantage sélectif majeur pour ce clone et pourrait avoir favorisé son implantation et sa persistance dans les services de RN où la pression de sélection par la vancomycine est élevée. Pour compléter ces résultats, une étude de cohorte prospective menée en RN a permis de démontrer que l'administration de vancomycine constituait un facteur de risque indépendant de survenue d'INT à S. capitis. Au-delà de la problématique spécifique des INT à S. capitis en RN, nos travaux illustrent plus largement un des enjeux majeurs de santé publique qui est l'impact écologique potentiel de l'utilisation des antibiothérapies probabilistes à large spectre sur l'émergence et la sélection de bactéries multirésistantes impliquées secondairement dans des infections nosocomiales. Ces travaux ouvrent de nouveaux axes de recherche concernant d'une part la meilleure compréhension de la physiopathologie des INT à S. capitis, et d'autre part plus largement les modalités de prévention des INT en RN et d'amélioration du diagnostic précoce des INT / Pas de résumé en anglais

La propagation de bactéries intracellulaires invasives peut entrainer celle des génomes mitochondriaux qui leur sont liés génétiquement au sein du cytoplasme. Cette sélection par autostop peut conduire à une réduction de la taille efficace (Ne) pour le génome mitochondrial. Elle peut également favoriser l'introgression d'une mitochondrie introduite dans une espèce suite à une hybridation. Le principal objectif de ma thèse est de quantifier ces différents effets, de manière globale, au moyen d'un large échantillonnage d'Arthropodes de Polynésie française. Les événements d'introgressions mitochondriales sont à l'origine de discordances entre les histoires évolutives des génomes mitochondriaux et nucléaires. Afin de rechercher de telles discordances, nous avons développé des marqueurs génomiques nucléaires de type RADseq, permettant de reconstruire l'histoire des populations étudiées. J'ai pu montrer au moyen de simulations que ce type de données pouvait être utilisé pour inférer des relations phylogénétiques entre espèces (Cariou et al. 2013). Des améliorations du protocole RADseq nous ont également permis de démontrer l'applicabilité de cette méthode à de nombreux spécimens au sein de librairies hautement multiplexées (Henri et al. 2015). A partir d'analyses in silico, j'ai par ailleurs évalué l'importance de différents biais liés à l'utilisation de marqueurs RADseq pour estimer les diversités génétiques et proposé une méthode permettant de corriger certains d'entre eux. A partir de ces développements, j'ai pu démontrer que sur 30 espèces de Diptères et de Lépidoptères testées à ce jour, la proximité génétique mitochondriale est systématiquement confirmée par les marqueurs nucléaires, rejetant ainsi l'hypothèse d'une introgression mitochondriale récente. Sur un plus large échantillon, nous avons en revanche mis en évidence une réduction significative du Ne mitochondrial dans les lignées infectées par Wolbachia, suffisante pour réduire le polymorphisme, mais insuffisante pour générer une réduction notable de l'efficacité de la sélection naturelle / The spread of endosymbiotic bacteria can drive that of the linked mitochondrial genomes within the cytoplasm. This hitchhiking selection can lead to a reduction of the effective population size of the mitochondrial genomes (Ne). 1t can also facilitate mitochondrial introgression, following the introduction of exogenous mitochondria in a species by hybridization. The main objective of my thesis is to quantify these different effects, on a global scale, using a large sample of Arthropods. Mitochondrial introgressions can lead to discrepancies between the evolutionary histories of mitochondrial and nuclear genomes. To investigate such patterns, we used RADseq genomic markers, that allow reconstructing population histories, and developed improvements for the library preparation and data analysis. Using in silico experiments, 1 showed that RADseq data is suitable for phylogenetic inferences (Cariou et al. 2013). Adjustments in the RADseq protocol also allowed us to demonstrate the applicability of this method for highly multiplexed libraries (Henri et al. 2015). The impact of various biases related the estimation of population genetic diversity using RADseq was also investigated in silico, which lead me to propose an ABC method to correct some of them. Following these developments, 1 showed on 30 species of Diptera and Lepidoptera that nuclear markers always confirmed the mitochondrial genetic relatedness, ruling out the hypothesis of recent mitochondrial introgressions. On a larger sample, we detected a reduction of the mitochondrial Ne in Wolbachia infected lineages. This reduction caused a significant decrease in the polymorphism of infected populations, but appeared insufficient to reduce the efficacy of natural selection

Viruses are the most abundant organisms on Earth, yet their collective evolutionary history, biodiversity and functional capacity is not well understood. Viral metagenomics offers a potential means of establishing a more comprehensive view of virus diversity and evolution, as vast amounts of new sequence data becomes available for comparative analysis.Metagenomic DNA from virus-sized particles (smaller than 0.2 microns in diameter) was isolated from approximately 20 liters of sediment obtained from Boiling Springs Lake (BSL) and sequenced. BSL is a large, acidic hot-spring (with a pH of 2.2, and temperatures ranging from 50°C to 96°C) located in Lassen Volcanic National Park, USA. BSL supports a purely microbial ecosystem comprised largely of Archaea and Bacteria, however, the lower temperature regions permit the growth of acid- and thermo-tolerant Eukarya. This distinctive feature of the BSL microbial ecosystem ensures that virus types infecting all domains of life will be present. The metagenomic sequence data was used to characterize the types of viruses present within the microbial ecosystem, to ascertain the extent of genetic diversity and novelty comprising the BSL virus assemblage, and to explore the genomic and structural modalities of virus evolution.Metagenomic surveys of natural virus assemblages, including the survey of BSL, have revealed that the diversity within the virosphere far exceeds what has currently been determined through the detailed study of viruses that are relevant to human health and agriculture. The number of as-yet-uncharacterized virus protein families present in the BSL assemblage was estimated by clustering analysis. Genomic context analysis of the predicted viral protein sequences in the BSL dataset indicates that most of the putative uncharacterized proteins are endemic or unique to BSL, and are largely harbored by known virus types. A comparative metagenomic analysis approach identified a set of conserved, yet uncharacterized BSL protein sequences that are commonly found in other similar and dissimilar environments.New sequence data from metagenomic surveys of natural virus assemblages was also used to better characterize and define known virus protein families, as some of the viruses found in the BSL environment represent distant relatives of well-characterized isolates. By comparing viral genes and protein sequences from these highly divergent species, it is possible to better understand the dynamics of adaptation and evolution in the virosphere. Additionally, as structures of virus proteins continue to be experimentally determined by X-ray crystallography and cryo-electron microscopy, a merger of structural and metagenomic sequence data allows the opportunity to observe the structural dynamics underlying virus protein evolution.Capsid (structural) proteins from two distinct Microviridae strains; a globally ubiquitous and highly sequence-diverse virus family, were identified in, and isolated from the BSL metagenomic DNA sample. These BSL capsid protein sequences, along with several other homologous sequences derived from metagenomic surveys and laboratory isolates, were mapped to the solved structure of a closely related capsid protein from the Spiroplasma phage-4 microvirus. Patterns of amino acid sequence conservation, unveiled by structure-based homology modeling analysis, revealed that the protein sequences within this family exhibit a remarkable level of plasticity, while remaining structurally and functionally congruent.Lateral gene transfer is thought to have had a significant impact on the genomic evolution and adaptation of virus families. Genomic context analysis was also utilized to identify interviral gene transfer within the BSL virus assemblage. An ostensibly rare interviral gene transfer event, having transpired between single-stranded RNA and DNA virus types, was detected in the BSL metagenome. Similar genomes were subsequently detected in other ecosystems around the globe. The discovery of this new virus genome dramatically underscores the scope and importance of genetic mobility and genomic mosaicism as major forces driving the evolution of viruses.The analyses conducted herein demonstrate the many ways in which viral metagenomic sequence data may be utilized to not only evaluate the composition of a natural virus assemblage, but to discover new viral genes, and to better understand the dynamics of both genomic and structural evolution within the virosphere.

Die Anordnung von Tiergenomen in topologisch assoziierten Domänen (TADs) spielt eine entscheidende Rolle bei der Regulation von Genen. Diese TADs sind Bereiche mit erhöhter Interaktion, die durch kontaktarme Zonen getrennt sind. In Wirbeltieren erfolgt die Bildung von TADs durch die Bindung von Kohäsin und CTCF (CCCTC-bindender Faktor) im Rahmen eines dynamischen Prozesses namens Loop-Extrusion. Dieser Prozess erzeugt Chromatinschleifen, die gestoppt werden, wenn sie auf CTCF-Proteine in einer spezifischen Ausrichtung treffen. Obwohl CTCF in den meisten Bilaterien stark konserviert ist, wurde seine globale architektonische Funktion in Fliegen bisher nicht erforscht. In dieser Studie wurde ein innovativer Ansatz entwickelt, um die evolutionären Aspekte der CTCF-vermittelten 3D-Chromatinorganisation zu untersuchen. Die Auswirkungen des Austauschs von CTCF-Orthologen innerhalb der Bilateriengruppe auf Lebensfähigkeit, Phänotypen, Genexpression, Genomarchitektur und genomweite Bindungsmuster wurden analysiert. Die Ergebnisse zeigen, dass die nicht-vertebraten Chordatiere C. robusta, unabhängig von der Anwesenheit von CTCF, keine herkömmlichen TAD-Strukturen aufweisen. Dennoch kann das Ciona-Ortholog als Transkriptionsfaktor fungieren, um die Expression bestimmter Gene und die Lebensfähigkeit wiederherzustellen, die bei vollständigem CTCF-Verlust in embryonalen Stammzellen der Maus dysreguliert sind. Dies deutet darauf hin, dass CTCF eine konservierte Rolle als Transkriptionsregulator hat, die über seine bekannte Funktion als architektonisches Protein in einigen Arten hinausgeht. Weitere Untersuchungen sind erforderlich, um festzustellen, ob CTCF in Ciona das Genom in seiner nativen Umgebung bindet und die Bindung von Kohäsin aufrechterhält. Die Unfähigkeit des CTCF-Orthologs der Maus, Chromatinschleifen im Genom der Fruchtfliege zu erzeugen, legt nahe, dass die Wirbeltier-Version von CTCF allein nicht für eine funktionelle Schleifen-Extrusion ausreicht. Es könnte notwendig sein, dass sie mit Fliegen-Kohäsin oder spezifischen Kofaktoren kompatibel ist. Die Studie zeigt auch subtile Unterschiede in den Bindungsmotiven von CTCF zwischen den Arten. Während die Orthologe der Chordatiere ähnliche Motivstrukturen aufweisen, zeigt das Fliegen-Ortholog eine abweichende Musterpräferenz. Diese Erkenntnisse verdeutlichen die evolutionären Verschiebungen in den Bindungsvorlieben von CTCF in pan-chordaten Linien. Zusammenfassend bietet diese Forschung wertvolle Einblicke in die evolutionäre Bewahrung und funktionelle Divergenz von CTCF-vermittelten Chromatin-Kontakten bei Bilaterien. Sie betont die Bedeutung artspezifischer Faktoren und koevolutionärer Dynamiken bei der Gestaltung der Chromatinorganisation und Genregulation. Weitere Untersuchungen an verschiedenen Arten sind entscheidend, um die Entstehung und Bewahrung der CTCF-vermittelten Chromatinarchitektur im Verlauf der Evolution genau zu verstehen. / The three-dimensional organization of animal genomes, known as topologically associating domains (TADs), is crucial for controlling gene activity. TADs are regions with increased genetic interactions, separated by zones with fewer contacts. In vertebrates, the formation of TADs involves a dynamic process called loop extrusion, where cohesin and CTCFs bind to the chromatin. This process creates chromatin loops, with cohesin complexes pausing when they encounter CTCF molecules in a specific orientation. However, although CTCF is highly conserved among bilaterian species, its vital role in organizing genomes spatially has not been observed in invertebrates like flies. This study investigates the chromatin structure in Ciona robusta, a chordate species situated evolutionarily between well-studied organisms like mice and fruit flies. A unique approach was developed to explore the evolution of CTCF as a mediator of three-dimensional chromatin organization. By swapping CTCF orthologs from representative species across the bilaterian group, the research examined their impact on viability, traits, gene expression, genome architecture, and binding patterns across the genome. The findings indicate that Ciona robusta, a non-vertebrate chordate, lacks typical TAD structures, even in the presence of CTCF. However, although the Ciona ortholog cannot create TADs in mouse embryonic stem cells, it can act as a transcription factor, restoring the expression of specific genes and viability in cases of complete CTCF loss. This suggests that CTCF serves a conserved role as a transcription regulator, beyond its recognized role as a structural component in some species. Furthermore, when the mouse ortholog of CTCF was introduced into the fruit fly genome, it failed to induce the formation of chromatin loops, suggesting that the vertebrate version of CTCF alone is insufficient for effective loop extrusion. Additionally, the study revealed subtle differences in CTCF's binding motif preferences between species. While chordate orthologs shared similar motif structures, the fly ortholog had a distinct pattern. These findings underscore the evolutionary changes in CTCF binding preferences among chordate lineages. In summary, this research offers valuable insights into the evolutionary preservation and functional differences in CTCF-mediated chromatin interactions in bilaterian species. It highlights the significance of species-specific factors and co-evolutionary dynamics in shaping chromatin organization and gene regulation.

An unprecedented use of high-throughput sequencing for routine monitoring of SARS-CoV-2 viruses in patient samples has created a dataset of over 6 million SARS-CoV-2 genomes. To monitor genomes, deposited in the GISAID database, and to track the continuous sequence evolution of molecular assay oligonucleotide target sequences. A simple pipeline tool for non-experts was developed to mine this database for nucleotide changes in oligonucleotides and tested with the long oligonucleotides of a Recombinase polymerase amplification (RPA) assay targeting the RNA-dependent RNA polymerase (RdRP) gene of the SARS-CoV-2. Results indicate the emergence of a single nucleotide change in the reverse oligonucleotide from 0.03 to 26.23% (January to May 2021) in Alpha variant genomes, which however reduced to 17.64% by September after which the Alpha variant was completely displaced by the Delta variant. For all other variants, no relevant nucleotide changes were observed. The oligonucleotide screening pipeline allows efficient screening of nucleotide changes in oligonucleotides of all sizes in minutes.

A identidade na segmentação do corpo de diversos organismos, durante o desenvolvimento, é devida, em grande parte, à ação das proteínas homeóticas. Em especial, dois grupos de proteínas, Trithorax (trxG) e Polycomb (PcG) têm um papel fundamental na manutenção, respectivamente, da ativação e da repressão da transcrição gênica, associando-se à cromatina. A importância das PcG nos estimulou a buscar a caracterização das proteínas humanas ortólogas ao \"Enhancer of Polycomb\" (Epc) de Drosophila, até então não descritas no genoma humano. Para tanto, buscamos: - obter a sequência completa e mapear o cDNA do novo gene humano homólogo ao \"Enhancer of Polycomb\" de Drosophila; - analisar sua expressão em tecidos fetais, adultos e tumorais e fazer estudos buscando sua caracterização funcional. Encontramos, mapeamos e obtivemos a seqüência completa de dois genes humanos, ortólogos de Epc1 (10p11-22) e de Epc2 (2q21-23) de camundongo, publicando estes dados em 2001 (Camargo et al., 2001). Ambos os genes são bastante conservados entre várias espécies, sendo que o cDNA de hEPC2 humano, por exemplo, é 94% idêntico ao Epc2 de camundongo e possui 96% de identidade ao nível de proteína, sugerindo que a função do gene deve ter sido mantida durante a evolução. No entanto, as seqüências protéicas de hEPC1 e hEPC2 humanos possuem apenas 68% de identidade entre si. Portanto, é provável que após a duplicação dos parálogos, estes tenham divergido funcionalmente. A expressão de ambos os genes foi avaliada utilizando \"dot-blots\" contendo 76 mRNAs de amostras de tecidos fetais, adultos e tumorais, mostrando-se fraca e ubíqua. Análises in silico sugeriram a existência de 4 isoformas de splicing para hEPC2, as quais foram validadas por RT-PCR ou \"Northern blots\". Uma das isoformas (de 2.7 Kpb) se mostrou mais abundante em todas as linhagens tumorais estudadas através de análises de \"Northern blot\", principalmente nas linhagens de linfoma de Burkitt\'s Raji e na linhagem de leucemia pró-mielocítica HL-60. Esta isoforma é gerada através de um sítio alternativo de poli-adenilação, que reduz sua porção 3\'UTR, retirando 4 dos 5 \"elementos ricos em adenilatos e uridilatos\" (AREs), envolvidos com a degradação de mRNAs lábeis que codificam proteínas regulatórias. Estes resultados se encontram em um manuscrito recentemente submetido à publicação (anexo à tese). Interação entre hEPC2 e SMADs e sua modulação por TGF-&#946;. Durante a montagem da seqüência completa de hEPC2, verificamos que duas ESTs patenteadas mostravam alta identidade com o gene. Estas seqüências foram descritas como sendo parte de uma nova proteína de interação com as proteínas da família SMAD, envolvidas com transdução de sinais desencadeados por TGF-&#946;. Esta citocina por sua vez, regula a proliferação, diferenciação e morte celular. Partimos para a avaliação da possível interação entre hEPC2 e as SMADs, em colaboração com o grupo do Dr. Aristidis Moustakas, do Ludwig Institute for Cancer Research de Uppsala, Suécia. Os resultados de co-imunoprecipitação sugeriram que as SMADs 2, 3, 4, 7 e 8 interagem com hEPC2, sendo que a interação entre as SMAD2, SMAD3, SMAD4 e hEPC2 nas células tratadas com TGF-&#946;1, mostraram uma redução na co-imunoprecipitação. Este resultado sugere que TGF-&#946;1 modula negativamente a interação entre essas proteínas. Da mesma maneira, foi observada uma redução na interação de hEPC2 com SMAD8 após o tratamento com BMP-7. Esse resultado é ainda mais destacado para as SMADs 2 e 3. Estes dados foram observados para ambas as construções de hEPC2, o que sugere fortemente a veracidade da interação entre estas proteínas. A localização celular de hEPC2, e também sua co-localização com SMAD2 foram investigadas através de imunofluorescência indireta e confirmaram a predição do programa PSORTII, de que hEPC2 se localiza no núcleo. No entanto, não foi possível observar a co-localização entre hEPC2 e SMAD2. É possível que hEPC2 não se ligue diretamente ao DNA, necessitando se associar como parceiro de um fator de transcrição. Esta foi uma das hipóteses para a atuação de hEPC2, como um co-fator que se associe com uma das SMADs e se ligue a um elemento específico de ligação a SMAD (SBE). Para investigar essa hipótese um ensaio de gene repórter foi feito utilizando uma construção de um repórter contendo 12 repetições da seqüência CAGA (seqüência específica de ligação das SMADs 2,3 e 4) fusionado com o gene da luciferase. No entanto, este ensaio não demonstrou que a transcrição de SMAD2 é dependente de hEPC2 e o experimento deverá ser repetido. Para confirmar a interação entre hEPC2 e as SMADs, será feito um experimento de \"pull-down\". Para tal o cDNA de hEPC2 foi clonado no vetor pET-32A de expressão indutível em bactérias. A proteína recombinante já foi produzida, tendo sido induzida e posteriormente purificada em condições desnaturantes. Apesar de dezenas de genes PcG terem sido caracterizados em Drosophila, poucos destes genes foram estudados em mamíferos. Portanto, a descrição do gene hEPC2 e seus transcritos alternativos, contribui para o conhecimento de PcG humanos, indicando a associação de maior expressão de uma de suas isoformas em linhagens celulares tumorais. Em relação à interação de hEPC2 com as SMADs, é interessante observar que nenhuma outra proteína foi descrita por possuir a particularidade de interagir com as SMADs de diferentes categorias. Talvez este seja um dado importante, que indique o papel singular de hEPC2 na sinalização de TGF-&#946;1. / The identity of body segmentation in several organisms during development is, to a large extent, due to the action of the homeotic proteins. In particular, two groups of proteins, the Trithorax (trxG) and Polycomb (PcG), have a major role in maintenance of respectively, transcription activation and repression, when associated to the chromatin. The importance of PcGs has motivated us to pursue the isolation and characterization of two new human proteins that are orthologs of the \"Enhancer of Polycomb\" (Epc) of Drosophila. To achieve this goal we undertook the task of the cloning and mapping of complete cDNA sequence of the novel genes hEPC1 and hEPC2, analyzing its expression in fetal, adult and tumoral tissues and functionally characterizing the hEPC2 protein. In 2001, we published the mapping and cloning of the complete cDNA sequences of both genes, as being orthologs of the mouse Epc1 (10p11-22) and Epc2 (2q21-23), together with the strategy used to obtain the full-length cDNAs (Camargo et al., 2001). Both genes are shown to be highly conserved among several species. Thus, the human hEPC2 cDNA is 94% identical to the mouse Epc2 and displays 96% identity at the protein level, suggesting maintenance of its function during the evolution. However, the protein sequences of the human hEPC1 and hEPC2 display only 68% identity. Therefore, it is likely that they have undergone a functional divergence after their duplication. The expression of both genes was evaluated using \"dot-blots\" containing 76 mRNAs samples from fetal, adult and tumoral tissues and is shown to be weak and ubiquitous. \"In silico\" analysis suggested the existence of 4 hEPC2 splicing isoforms that were validated by RT-PCR and/or Northern-blots. One of the isoforms (of 2.7 Kbp) is shown to be more abundant in all of the tumoral cell lines evaluated using Northern-blot analysis, mainly in the Burkit\'s Raji lymphoma and in the promyelocytic leukemia HL-60. This isoform results from the use of an alternative polyadenylation site that reduces the 3\'UTR, abolishing 4 of 5 \"adenylates and urilates rich elements\" (AREs), involved in the degradation of labile mRNAs that codify to regulatory proteins. These results have been recently submitted to publication (manuscript attached to this thesis). Interaction between the hEPC2/SMADs and its modulation by TGF-&#946;. During the assembly of the hEPC2 full-length cDNA sequence, we found two patented ESTs that tagged a portion of the gene. These sequences were described as partial sequences of a \"new SMAD interacting protein\", involved in signal transduction of TGF-&#946;, a cytokine that regulates cell proliferation, differentiation and death. To evaluate this putative interaction between hEPC2 and the SMADs proteins, we begun a collaboration with the TGF-&#946; signalling group of the Dr. Aristidis Moustakas, from the Uppsala Ludwig Institute for Cancer Research, Sweden. The results of co-imunoprecipitation assays suggested that SMADs 2, 3, 4, 7 e 8 interact with hEPC2. Moreover, the interaction among SMAD2, SMAD3, SMAD4 and hEPC2 in cells treated with TGF-&#946;1 showed decreased co-imunoprecipitation. This result suggests that TGF-&#946;1 negatively modulates the interaction of these proteins. Likewise, we observed a reduction in hEPC2 interaction with SMAD8 upon BMP-7 treatment. This effect was even more dramatic for SMADs 2 and 3. These data were observed for both hEPC2 plasmid constructs, which strongly suggest the veracity of these proteins interaction. The cell localization of the hEPC2 protein, as well as its co-localization with the SMAD2, were investigated through indirect immunofluorescence assay, confirming the predicted localization of hEPC2 in the cell nucleus using the PSORTII program. However, we were not able to confirm the co-localization of hEPC2 and SMAD2. It is possible that hEPC2 does not bind directly to the DNA, requiring an association with a partner such as a transcription factor. This raises the hypothesis of hEPC2 having a role as a co-factor associated to one of the SMADs and binding to a \"SMAD binding element\" (SBE). To investigate this hypothesis, gene reporter assays were undertaken using a reporter construct containing 12 CAGA sequence repetitions (specific binding sequence of the SMADs 2, 3 and 4) fused to the luciferase gene. However, this assay could not demonstrate that the transcription of the SMAD is dependent on hEPC2. This experiment must be repeated. To confirm the interaction of hEPC2 and SMADs, a pull-down assay will be performed. To this end, the coding region of hEPC2 was cloned into the pET-32A bacterial inducible expression vector. The recombinant protein was already produced, having been induced and purified under denaturing conditions. Despite the dozens of PcG genes that were described in Drosophila, only a few of these genes have been characterized in mammals. Therefore, the description of the hEPC2 and its alternative transcripts is a contribution to better knowledge of the human PcGs. Regarding the hEPC2 and SMADs interaction, it\'s it is noteworthy that this is the first protein described to interact with SMADs of distinct categories. This may be an important indication of a unique role for hEPC2 in the TGF-&#946;1 signaling pathway.

A evolução molecular, impulsionada pela Teoria Sintética da Evolução, tornou um assunto indispensável na compreensão da evolução da vida. O crescimento genômico, etapa responsável pelo maior potencial de armazenamento de informação e estabilidade, também foi submetido à indelével ação da seleção natural. Utilizando a metodologia dos ciclos de amplificação-mutação-seleção, tal como o SELEX (systematic evolution of ligands by exponential enrichment), que mimetizam a seleção natural, e ferramentas da Teoria de Informação, foram desenvolvidos e implementados programas para simular a evolução, considerando, além de outros, um parâmetro pouco explorado na literatura: a variação do tamanho do genoma. Foram estudados dois cenários distintos; no primeiro a seleção era dependente da busca exata de uma sequência pré-determinada (o filtro). Além disso, a entropia de Shannon considerada era referente ao alinhamento da molécula toda. Avaliando configurações simples desse modelo, foi possível desenvolver uma equação analítica que descreveu bem os resultados (para tamanho de genoma constante). No segundo cenário, foram exploradas a seleção não específica de uma sequência, o número máximo de bases constante, a entropia apenas das regiões de interesse e a presença de até cinco filtros de seleção. A entropia da molécula toda se mostrou pouco significativa (primeiro cenário), diferentemente da avaliada em apenas uma região. Foi possível observar que o crescimento do genoma foi pouco acentuado, predominando as moléculas menores, mesmo com grande quantidade de filtros, o que indica que o sistema está sob \"seleção por compressão\", sendo, pois, necessário atribuir explicitamente vantagens às moléculas mais complexas para poder haver aumento no crescimento médio. / Molecular Evolution, stimulated and supported by Evolution Synthetic Theory, became essential to understand evolution of life. Genomic growth was responsible to increase the capacity of storage information and to stability of the molecule; besides, it was also submitted to natural selection. Using amplification-mutation-selection methodology, such as SELEX (systematic evolution of ligands by exponential enrichment), and tools of Information Theory, it was developed computer program to simulate macromolecule evolution taking into account, besides other, a less study parameter in specialized journal: the genome size variation. It was studied two different scenarios. In the first one, selection was dependent of a specific sequence to be searched; moreover, Shannon entropy took into account all nucleotides of all molecules and it was studied with until two sequence target. It was possible develop an analytical equation to well describe simple settings of this model. In the second scenario, in another hand, selection depends on a specific sequence, but is not required to match the whole sequence. Also, to compute Shannon entropy it was taking into account only the least Hamming distance sequence of each molecule. It was studied until five sequence target in this scenario. Entropy was not significant in first scenario as it was in the second one. Size genome evolution shows the system were under compression selection, being necessary to get other advantage to become possible an increase in genome size.

O Cancro Cítrico, um dos mais graves problemas fitossanitários da citricultura atual, é uma doença causada pelo fitopatógeno Xanthomonas axonopodis pv. citri (Xac). Um estudo funcional do genoma de Xac foi iniciado com o intuito de identificar interações proteína-proteína envolvidas em processos de patogenicidade de Xac. Através da utilização do sistema duplo-híbrido de levedura, baseado nos domínios de ligação ao DNA e ativação da transcrição do GAL4, nós analisamos os principais componentes dos mecanismos de patogenicidade de Xac, incluindo o Sistema de Secreção do Tipo III (TTSS), Sistema de Secreção do Tipo IV (TFSS) e Sistema de \"Quorum Sensing\" composto pelas proteínas Rpf. Componentes desses sistemas foram utilizados como iscas na triagem de uma biblioteca genômica de Xac. O TTSS é codificado pelos genes denominados hrp (\"hypersensitive response and pathogenicity\"), hrc (\"hrp conserved\") e hpa (\"hrp associated\") localizados no locus hrp do cromossomo de Xac. Esse sistema de secreção é capaz de translocar proteínas efetoras do citoplasma bacteriano para o interior da célula hospedeira. Nossos resultados mostraram novas interações proteínaproteína entre componentes do próprio TTSS além de associações específicas com uma proteína hipotética: 1) HrpG, um regulador de resposta de um sistema de dois componentes responsável pela expressão dos genes hrp, e XAC0095, uma proteína hipotética encontrada apenas em Xanthomonas spp; 2) HpaA, uma proteína secretada pelo TTSS, HpaB e o domínio C-terminal da HrcV; 3) HrpB1, HrpD6 e HrpW, 4) HrpB2 e HrcU e 5) interações homotrópicas envolvendo a ATPase HrcN. Em Xac, foram encontrados dois loci vir que codificam proteínas que possuem similaridade com componentes do TFSS envolvido em processos de conjugação/secreção bacteriana: TFSS-plasmídeo localizado no plasmídeo pXAC64 e TFSS-cromossomo localizado no cromossomo de Xac. O TFSS-plasmídeo, o qual possui maior similaridade com sistemas de conjugação, mostrou interações envolvendo proteínas cujos genes estão localizados na mesma região do plasmídeo pXAC64: 1) interação homotrópica da TrwA; 2) XACb0032 e XACb0033; 3) interações homotrópicas da proteína XACb0035; 4) VirB1 e VirB9; 5) XACb0042 e VirB6; 6) XACb0043 e XACb0021b. O TFSS-cromossomo apresentou interações envolvendo as proteínas: 1) VirD4 e um grupo de 12 proteínas que contém similaridade entre si, incluindo XAC2609 cujo gene encontra-se no locus vir, 2) XAC2609 e XAC2610; 3) Interações homotrópicas da VirB11; 4) XAC2622 e VirB9. A análise do sistema de \"Quorum-Sensing\" composto pelas proteínas Rpf mostrou interações envolvendo componentes do próprio sistema: 1) RpfC e RpfF; 2) RpfC e RpfG; 3) interações homotrópicas da RpfF; 4) RpfC e CmfA, uma proteína similar a Cmf de Dictyostelium discoideum que, neste organismo, é fundamental para processos de \"quorum-sensing\". As interações proteína-proteína encontradas permitiram-nos entender melhor a composição, organização e regulação dos fatores envolvidos na patogenicidade de Xac. / Citrus Canker, caused by the bacterial plant pathogen Xanthomonas axonopodis pv. citri (Xac) presents one of the most serious problems to Brazilian citriculture. We have initiated a project to identify protein-protein interactions involved in pathogenicity of Xac. Using a yeast two-hybrid system based on GAL4 DNA-binding and activation domains, we have focused on identifying interactions involving subunits, regulators and substrates of: Type Three Secretion System (TTSS), Type Four Secretion System (TFSS) and Quorum Sensing/Rpf System. Components of these systems were used as baits to screening a random Xac genomic library. The TTSS is coded by the hrp (hypersensitive response and pathogenicity), hrc (hrp conserved) and hpa (hrp associated) genes in the chromosomal hrp locus. This secretion system can translocate efector proteins from the bacterial cytoplasm into the host cells. We have identified several previously uncharacterized interactions involving: 1) HrpG, a two-component system response regulator responsible for the expression of Xac hrp operons, and XAC0095, a previously uncharacterized protein encountered only in Xanthomonas spp; 2) HpaA, a protein secreted by the TTSS, HpaB and the C-terminal domain HrcV; 3) HrpB1, HrpD6 and HrpW; 4) HrpB2 and HrcU; 5) Homotropic interactions were also identified for the ATPase HrcN. Xac contains two virB gene clusters, one on the chromosome and one on the pXAC64 plasmid, each of which codes for a unique and previously uncharacterized TFSS. Components of the TFSS of pXAC64, which is most similar to conjugation systems, showed interactions involving proteins coded by the same locus: 1) Homotropic interactions of TrwA; 2) XACb0032 and XACb0033; 3) XAC0035 homotropic interactions; 4) VirB1 and VirB9; 5) XACb0042 and VirB6; 6) XACb0043 and XACb0021 b. Components of the chromosomal TFSS exhibited interactions involving: 1) VirD4 and a group of 12 uncharacterized proteins with a common C-terminal domain motif, include XAC2609 whose gene resides within the vir locus; 2) XAC2609 and XAC261 O; 3) Homotropic interactions of VirB11; 4) XAC2622 and VirB9. Analysis of Quorum Sensing/Rpf System components revealed interactions between the principal Rpf proteins which control Xanthomonas quorum sensing: 1) RpfC and RpfF; 2) RpfC and RpfG; 3) RpfF homotropic interactions; 4) RpfC and CmfA, a protein that presents similarity with Cmf (conditioned medium factor) of Dictyostelium discoideum, which contrais quorum sensing in this organism. The protein-protein interactions that we have detected reveal insights into the composition, organization and regulation of these important mechanisms involved in Xanthomonas pathogenicity.

Xylella fastidiosa (Xf) e Xanthomonas axonopodis pv. citri (Xac) são gama proteobactérias gram negativas, responsáveis por grandes perdas econômicas no setor citrícola brasileiro. Com seus genomas seqüenciados e anotados, fizemos uma análise comparativa entre suas composições gênicas e seus ambientes de vida. Xac apresenta um genoma de 5.2Mb contra 2.7Mb de Xf Isto reflete no número de genes (4432 contra 2838) que acabam refletindo em uma maior complexidade metabólica de Xac, caracterizada por: uma extensa gama de genes de degradação de parede celular (44), biossíntese de proteases (92), genes de funções regulatórias (296), um completo metabolismo energético (209), quimiotático (inexistente em Xf) e secretório (presença dos tipos I, II, III e IV , sendo o II em duplicata), além de um grande número de genes envolvidos com captação de ferro (65), fazem de Xac um patógeno de alto poder invasivo e de rápida propagação e virulência Em contrapartida, Xf por não possuir a complexidade supracitada, parece ter seus recursos adaptados ao ambiente em que vive, como por exemplo um alto número de genes envolvidos com biossíntese de pili, que associado à biossíntese de goma, favorecem sua adesão nas glândulas salivares do vetor (cigarrinha) e a formação de aglomerados celulares responsáveis pelo entupimento dos vasos que levam às patologias decorrentes do evento. / Xylella fastidiosa (Xf) and Xanthomonas axonopodis pv. citri Xac are gram negative gamma proteobacteria, responsible for great economical losses in the Brazilian citrus sector. With their sequenced and annotated genomes, we have done a comparative analysis between their genetic composition and life habitat. Xac displays a genome of 5.2Mb against 2. 7Mb of Xf. This reflects the number of genes (4432 against 2838) which results in a greater metabolic complexity of Xac, characterized by: a wide range of genes of cell wall degradation (44), biosynthesis of proteases (92), many genes of regulatory functions (296), a complex energy metabolism (209), chemotatic (absent in Xf) and secretory systerns (presence of types I, II, III and IV, type II in duplicate), besides a great number of genes involved in iron acquisition (65), make of Xac a pathogen of high invasive power and of quick spreading and virulence. In the other hand Xf, due to the lack of the complexity just cited, seems to have its resources adapted to the habitat in which it lives, as for example a large number of genes involved in pili biosynthesis, that associated with gum biosynthesis, favor its adhesion to the salivary glands of the vector (sharpshooter) and the formation of cellular agglomerations responsible for the blockage of the vessels which leads to the pathologies resulted from this event.