ADN subit une série de transformations structurelles complexes au cours de la division cellulaire, ce qui entraîne dans son compactage chromosomes mitotiques par un processus appelé la condensation des chromosomes. Le complexe de condensine pentamérique est fortement impliqué comme un effecteur majeur de ce phénomène. Il s'agit d'un complexe protéine de sous-unités multiples avec deux sous-unités catalytiques [SMC- Structural Maintenance of Chromosomes] et de trois sous-unités de régulation, hautement conservés de la levure à l'homme. Le complexe de condensine dans Saccharomyces cerevisiae est constitué de deux sous-unités de SMC [Smc2 et Smc4] et trois protéines non réglementaires [Brn1, Ycs4, Ycg1]. Malgré son importance, le mécanisme d'action de condensine reste largement inconnu. Par conséquent, l'objectif de cette recherche est de comprendre le mécanisme d'action de condensine et comment elle est affectée par l'interaction entre ses sous-unités réglementaires et non-réglementaires. Cette thèse identifie quatre morphologies dépendants du cycle cellulaire distincts du locus d'ADNr. Cette transformation du phénotype ADNr de G1 à la mitose dépend condensine. Afin de déterminer le rôle de l'interaction entre les sous-unités catalytiques et réglementaires de condensine dans la régulation du complexe condensine, nous avons identifié six résidus positifs sur l'extrémité C-terminale de BRN1 qui affectent la formation du complexe condensine, l'activité de la condensation et l'interaction avec tubuline, ce qui suggère que ces résidus ont un rôle dans la régulation de condensine. Ensemble, nos résultats suggèrent un modèle de règlement du condensine par l'interaction entre les sous-unités de condensine. / DNA undergoes a series of complex structural transformations during cell division, resulting in its compaction into intact mitotic chromosomes called chromosome condensation. The pentameric condensin complex has been strongly implicated as a major effector of this phenomenon. It is a multi-subunit protein complex with two catalytic “Structural maintenance of chromosome” [SMC] subunits and three regulatory subunits, highly conserved from yeast to humans. The condensin complex in Saccharomyces cerevisiae is made up of two SMC subunits [Smc2 and Smc4] and three regulatory non-SMC proteins [Brn1, Ycs4, Ycg1]. Despite its importance, the mechanism of action of condensin remains largely unknown. Hence, the objective of this research is to understand the mechanism of action of condensin and how it is affected by interaction between its regulatory and non-regulatory sub-units. This thesis identifies four distinct cell cycle dependent morphologies of the rDNA locus. The transformation of the rDNA phenotype from G1 to mitosis is condensin dependent. In order to determine the role of the interaction between the catalytic and regulatory subunits of condensin in the regulation of the condensin complex, we have identified six positive residues on the C-terminus of Brn1 which affect complex formation, condensation activity and interaction with tubulin, suggesting that these residues have a role in condensin regulation. Together, our results suggest a model for condensin regulation by interaction between condensin subunits.

Nous montrons dans cette étude que dans les cellules déplétées pour la nucléoline, une plus faible accumulation de pré-ARNr est associée à une augmentation de marques d’hétérochromatine (H3K9me2) et une diminution de marques d’euchromatine (H4K12ac et H3K4me3) sur la chromatine des gènes ribosomiques. Des expériences de ChIP-seq montrent que la nucléoline est enrichie dans la région codante et promotrice de l’ADNr et est préférentiellement associée avec les gènes non méthylés des ARNr. La déplétion de la nucléoline entraîne une accumulation de l’ARN Pol I au début de l’ADNr et une diminution de UBF sur la région codante et promotrice. La nucléoline interfère avec la liaison de TTF-1 sur le promoteur-proximal T0, inhibant ainsi le recrutement de TIP5 du complexe NoRC, et établissant un état d’hétérochromatine répressive. Ces résultats révèlent l’importance de la nucléoline dans le maintien d’un état euchromatinien des ADNr et dans l’élongation de la transcription. Nous montrons aussi dans cette thèse que l’acétylation est une nouvelle modification post-traductionnelle de la nucléoline. Des études d’immunofluorescence utilisant l’anticorps anti nucléoline acétylée montrent que la nucléoline acétylée est exclue des nucléoles. De plus, par ChIP-seq nous n’avons jamais pu détecter d’association significative de la nucléoline acétylée sur la chromatine des ADNr. Aussi, nous n’avons détecté aucune activation de la transcription de Pol II sur des matrices de chromatine avec la nucléoline acétylée. Nous trouvons une distribution de la nucléoline acétylée majoritairement dans le nucléoplasme où elle co-localise parfaitement avec le facteur d’épissage SC35, et partiellement avec les structures marquées avec un anticorps dirigé contre Y12, mais ne co-localise pas avec des structures contenant la coïline, ce qui suggère que cette fraction de la nucléoline pourrait être impliquée dans la synthèse ou le métabolisme des pré-ARNm. / Here we have shown that, in nucleolin depleted cells, lower accumulation of pre-rRNA is associated with the increase in heterochromatin marks (H3K9me2) and decrease of the euchromatin histone marks (H4K12Ac and H3K4me3) in rDNA chromatin. ChIP-seq experiments show that nucleolin is enriched in the coding and promoter region of the rDNA and is preferentially associated with the unmethylated rRNA genes. Nucleolin knockdown results in the accumulation of RNAPI at the beginning of the rDNA and a decrease of UBF in the coding and promoter regions. Nucleolin is able to interfere with the binding of TTF-1 on the promoter-proximal terminator T0 thus inhibiting the recruitment of the NoRC subunit TIP5 and HDAC1 and establishing a repressive heterochromatin state. These results reveal the importance of nucleolin in the maintenance of the euchromatin state of rDNA and transcription elongation.In this thesis we have also shown that acetylation is a novel post-translational modification of nucleolin. Immuno-fluorescence studies using anti-acetylated nucleolin antibody illustrated that acetylated nucleolin is excluded from nucleoli and interestingly, neither could we detect any significant binding of ac-nucleolin on rDNA chromatin by doing ChIP-Seq, nor did we detect any activation of Pol II transcription with ac-nucleolin from DNA and chromatin templates. Moreover, we found acetylated nucleolin had a predominant nucleoplasmic distribution where it associates with the splicing factor SC35 and partially with the structures labeled with Y12 antibody, but not with coilin containing structures.

O mosquito Culex quinquefasciatus tem grande importância médica no mundo devido à sua habilidade como vetor de arboviroses e filarioses. O principal fator limitante dos métodos de controle populacional para Cx. quinquefasciatus é a microevolução dessa espécie, fenômeno que freqüentemente resulta em aumento da sua resistência a inseticidas e da sua tolerância à poluição. No Estado de São Paulo existem duas populações vivendo sob diferentes condições ambientais. Uma delas, próxima ao Rio Pinheiros (PIN), está sujeita à alta poluição e aplicações de piretróides e outra, em Pariquera-Açu (PAR), vive em local semi-rural na ausência de inseticidas. O objetivo deste trabalho foi investigar se essas populações provenientes de ambientes distintos possuem polimorfismos genéticomorfológicos e se a população PIN apresenta variações morfológicas ao longo do tempo. Os parâmetros utilizados nas comparações foram morfometria geométrica alar, análises do DNA ribossômico (DNAr) e de cromossomos politênicos. Não obtivemos sucesso na caracterização cariotípica devido a pouca nitidez das bandas e interbandas. A morfometria geométrica de 286 asas de PIN, amostras coletadas em 2004 (PIN04) e 2007 (PIN07), e 150 asas de PARI, amostras coletadas em 2008 demonstrou variações morfológicas. As duas populações PIN04 e PIN07 revelaram alto dimorfismo sexual de forma e tamanho, sendo asas de fêmeas maiores que asas de machos em ambas as populações. A assimetria bilateral não é significante para tamanho e é tênue para forma, sendo ligeiramente mais pronunciada em machos e em PIN07. Os espécimes de PIN07 são maiores e mais assimétricos que PARI, fenômenos possivelmente relacionados à maior disponibilidade de alimento e à contínua exposição a altos níveis de inseticida, respectivamente. Análises de DNAr revelaram padrões equivalentes para PIN07 e PARI. Em suma, supomos que o fluxo gênico entre as populações geográficas pode ter ocorrido até recentemente. Este estudo mostrou que é possível ocorrer variação de tamanho e forma de asas em Culicidae em um intervalo de tempo de três anos. O próximo passo poderia ser uma investigação aprofundada a respeito da relação entre variação geográfica-temporal e algumas de suas possíveis causas tais como poluição e inseticida. / The mosquito Culex quinquefasciatus has medical importance due to its ability of vectoring arboviruses and filariases. Microevolution resulting in insecticide resistance is a remarkable limiting factor for populational control of this species. In the State of São Paulo there are two populations under different environmental conditions. One, near Rio Pinheiros (PIN), is subjected to pollution and pyrethroids applications and another, in Pariquera-Açu (PAR), lives in a semi-rural place in the absence of insecticides. The objective of this work was to investigate if these populations from different environments have geneticmorphological polymorphisms and if PIN population exhibits morphological variations during the time. Parameters used in the comparisons were wing geometric morphometrics, ribosomal DNA (rDNA), and polytene chromosomes. Karyotypic characterization was unsuccessful due to the poor definition of bands and interbands. Morphometrical analyses of 286 wings of PIN, collected in 2004 (PIN04) and 2007 (PIN07), and 150 wings of PARI, collected in 2008, demonstrate morphological variations. The two populations PIN04 and PIN07 revealed strong intrapopulational sexual dimorphism concerning shape and size, being the wings of females larger than those of males in both populations. The wing asymmetry is non-significant for size and tenuous for shape, being slightly more conspicuous in males and in PIN07. The specimens of PIN07 are larger and more bilaterally asymmetric than PARI, possibly due to higher food availability and to continuous exposition to high level of insecticide, respectively. Analysis of rDNA revealed restriction patterns equivalent for PIN07 and PARI populations. Thus, one may suppose that gene flow may have occurred until recently. This study showed that it is possible to occur size and shape variation of wings in Culicidae in time intervals as short as three years. The next step would be to evaluate in depth the relationship between geographical-temporal variation and some of possible causes like pollution and insecticides.

Os impactos da poluição por hidrocarboneto de petróleo sobre a diversidade e funcionalidade das comunidades microbianas em manguezais não são totalmente conhecidos, principalmente devido às limitações metodológicas para acessar os microrganismos nãocultiváveis. No entanto, vários métodos moleculares independentes de cultivo têm sido utilizados para investigar a diversidade e a estrutura das comunidades microbianas em ecossistemas naturais. O objetivo deste trabalho foi avaliar as variações da estrutura das comunidades de Bacteria e Archaea e a diversidade de Bacteria em uma transeção de solo de mangue do rio Iriri (Bertioga, SP) com um gradiente de contaminação por hidrocarbonetos de petróleo. As análises por eletroforese em gel com gradiente desnaturante (DGGE) mostraram que as comunidades de Bacteria e Archaea nas diferentes posições geográficas foram mais similares entre si do que entre diferentes profundidades ao longo do perfil em uma mesma posição geográfica. A análise das seqüências de clones de rDNA 16S de Bacteria dos diferentes pontos amostrados em abril de 2000, mostrou que a diversidade genética, avaliada pelo índice de Shannon, das comunidades microbianas diferem estatisticamente somente entre ponto o P1 (ponto menos contaminado) e P3 (ponto mais contaminado). As estimativas não-paramétricas da riqueza de espécies mostraram que P1, P2 e P3 possuem mais de 3539, 2524 e 1421 espécies bacterianas, respectivamente. Já, para as amostras do ponto P2 coletadas nos anos 2000 e 2004, muito embora os valores dos índices de Shannon tenham sido semelhantes, houve uma provável dominância de grupos específicos nas amostras coletadas em 2004, verificada pelos altos valores da recíproca do índice de Simpson. Os dados mostraram também que o número estimado de espécies bacterianas no ponto P2 diminuiu com o tempo, sendo menor em amostras de 2004, se comparado com amostras de 2000. No geral, a afiliação filogenética dos clones de rDNA 16S mostrou a grande diversidade de espécies, a maioria não conhecidas. Os dados sugerem que a contaminação do solo de mangue do rio Iriri está selecionando microrganismos mais adaptados às fontes de carbono introduzidas no solo. / The impacts of petroleum hydrocarbon pollution on the diversity and functionality of the microbial communities in mangrove soils are not totally understood, mainly due to the methodological limitations to access unculturable microorganisms. However, several cultureindependent molecular methods have been used to investigate the diversity and structure of microbial communities in natural ecosystems. The aim of this work was to evaluate shifts in Bacteria and Archaea community structures and the diversity of Bacteria in a soil transection of the Iriri river mangrove (Bertioga, SP) showing a petroleum hydrocarbon contamination gradient. The analyses by denaturing gradient gel electrophoresis (DGGE) showed that the communities of Bacteria and Archaea in different geographical positions were more similar among them than the communities in different depths along the soil profile at the same geographical position. Sequence analyses of bacterial 16S rDNA clones from different points sampled in April 2000 showed that the genetic diversity of the bacterial communities, based on the Shannon index, differ statistically only between P1 (less polluted) and P3 (more polluted) locations. Nonparametric estimates of species richness showed that P1, P2 and P3 may have more than 3539, 2524 and 1421 bacterial species, respectively. For P2 sampled in years 2000 and 2004, even though the Shannon indices were similar, there was a probable dominance of specific bacterial groups in year 2004, based on the high values of the reciprocal of Simpson\'s index. The data also showed that the estimated number of bacterial species in P2 decreased with the time, being lower in samples collected in 2004, as compared to samples collected in 2000. In the general, the phylogenetic affiliation of the 16S rDNA clones showed high bacterial species diversity, and most of the bacteria were of unknown species. The data suggest that the contamination of Iriri river mangrove soil with petroleum hydrocarbon is selecting microorganisms more adapted to the introduced carbon sources into the soil.

Made available in DSpace on 2017-07-21T19:59:52Z (GMT). No. of bitstreams: 1 LeonardoGoll.pdf: 2578520 bytes, checksum: e4dc5f18aad72bd8d62d5803fed3c4ef (MD5) Previous issue date: 2012-02-29 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / TheColeopterahave a large variationof the sex determinationsystem, being the Xypmechanism considered ancestorfor thisorder. The presenceof a argyrophilous material associated with thesexbivalent is described asresponsible formaintenance andassociation of thesechromosomes.However,there is no consensus in the literature about the nature ofthisargyrophilous material. In addition,few species have been studied using fluorescence in situhybridization (FISH) tothe location of the multigene families, which areuseful and can elucidate the variation in the karyotype and genomic organization of several species.To date,there is no cytogenetic analysis in the genus Lagria.Thus, theaim of this study was tocharacterizecytogeneticallyLagriavillosa, throughconventional andmolecularcytogenetics, forinvestigating the mechanism ofassociationofsex chromosomesXyp,through analysis of nature of argyrophilous material in the lumen of sex bivalent. Additionally, thechromosomalmapping of 5S rDNA multigene families, 18S rDNA and histone H3 genes in L. villosa, was carried out, and showing for the first timein Coleoptera, that genes 18S and 5S DNAs are interspersed. In addition, the analysis oftesticular cellsshowed 2n=18=16+Xypand formulameiotic 2n=8II+Xyp.In this workwe discuss thepossible mechanismsevolvingin the association ofXypsex chromosomes,where it is consideredthat the combination ofsynaptonemalcomplex proteinsmayparticipate in thecorrect segregationofchromosomes.In addition, the fiber-FISH technique with dual probes (18S and 5S) was used for better resolution mapping of these regions in L. villosa, showing that the two DNAs are closely interspersed with varying amounts of both classes of DNA. Association between the two rDNA families have been reported for other species and different hypotheses are raised about the functional organization in these families. / Os Coleoptera apresentam grande variação quanto aos sistemas de determinação sexual, sendo o mecanismo cromossômico do tipo Xyp considerado ancestral para esta ordem. A presença de uma substância argirofílica associada ao bivalente sexual é descrita como sendo a responsável pela manutenção e associação desses cromossomos, porém não existe um consenso na literatura sobre a natureza desse material. Além disso, poucas espécies têm sido analisadas utilizando a Hibridação in situ Fluorescente (FISH) para a localização das famílias multigênicas, as quaissão úteis marcadores e podem elucidar a variação do cariótipo e a organização genômica de diversas espécies.Até o presente momento, não existe análise citogenética em espécies do gênero Lagria. Desta forma, o objetivo desse trabalho foi caracterizar citogeneticamente Lagria villosa, através da citogenética convencional e molecular, investigando o mecanismo de associação dos cromossomos sexuais Xyp, através da análise da natureza do material argirofílico presente no lúmen do bivalente sexual. Adicionalmente, realizou-se o mapeamento cromossômico das famílias multigênicasDNAr 5S, DNAr18S e genes de histona H3 em L. villosa, mostrando pela primeira vez uma associação intercalar dosgenes DNAs 5S e 18S em Coleoptera. Além disso, aanálise de células testiculares evidenciou 2n=18=16+Xype fórmula meiótica 2n=8II+Xyp.No presente trabalho são discutidos os possíveis mecanismos evolvidos na associação dos cromossomos sexuais Xyp, onde é considerado que a associação de proteínas do complexo sinaptonêmico pode participar da correta segregação desses cromossomos. Além disso, atécnica de fiber-FISH com dupla sonda (18S e 5S) foi utilizada para uma melhor resolução do mapeamento dessas regiões genômicas em L. villosa, mostrando que os dois DNAs estão intimamente intercalados com quantidades variáveis de ambas as classes de DNA. Associação entre as duas famílias DNArtem sido relatadopara outras espécies e diferentes hipóteses são levantadas sobre a organização funcional dessas famílias.

Submitted by Angela Maria de Oliveira (amolivei@uepg.br) on 2017-10-31T12:38:31Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) DISSERTAÇÃO michele.pdf: 3067898 bytes, checksum: 4e684091426aed5eb28e585d5db27abb (MD5) / Made available in DSpace on 2017-10-31T12:38:31Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) DISSERTAÇÃO michele.pdf: 3067898 bytes, checksum: 4e684091426aed5eb28e585d5db27abb (MD5) Previous issue date: 2014-02-25 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Alticinae apresenta características cariotípicas muito interessantes quanto a variação do número diplóide, do sistema de determinação sexual e irregularidades meióticas. O número cromossômico mais frequente é de 11 ou 12 pares. As espécies de Oedionychina estudadas citogeneticamente possuem 2n= 22,10II+X+Y com cromossomos sexuais gigantes. No que se refere à posição sistemática existem muitas divergências entre os estudos e problemas de identificação das espécies pertencentes aos vários gêneros. O estudo com técnicas mais refinadas e o mapeamento do DNA r 5S é recente em Coleoptera, e as poucas espécies de Alticinae estudadas mostram a presença de dois ou três pares autossômicos. Assim, este trabalho tem o objetivo de analisar citogeneticamente e propor as estratégias de diferenciação cariotípica para as espécies de Omophoita communis e Omophoita sexnotata. A análise citogenética dos indivíduos de duas populações de O. communis estudadas mostrou a ocorrência de grande variação no número diploide e morfologia cromossômica, sendo possível separá-las em dois citótipos. O citótipo I possui 2n= 22 e o citótipo II 2n= 12, sendo essa variação é descrita pela primeira vez no gênero. Adicionalmente, o estudo da morfologia do edeago mostrou diferenças, indicando um provável padrão de diferenciação das duas espécies. A análise da árvore consenso da reconstrução filogenética também evidencia que os citótipos mostram agrupamentos diferentes e reforçam a hipótese de duas espécies. O estudo do mapeamento do gene DNAr 5S em O. sexnotata evidenciou a presença desse cluster em todos os cromossomos autossômicos, sendo esse padrão de dispersão nunca descrito em Coleoptera. Na literatura, a dispersão dos genes ribossomais está sempre relacionada com a presença de elementos transponíveis. O resultado do sequenciamento dos fragmentos de 5S ribossomal obtidos de cada cromossomo de O. sexnotata resultaram em sequencias similares a RNAr 5S de Drosophila melanogaster, elemento transponível EnSpm, retropseudogene de 5S e microssatélite. Adicionalmente, a analise da estrutura secundaria do RNAr 5S, mostrou que as sequencias obtidas não são funcionais quando comparadas seus percentuais de energia livre em relação ao percentual da sequencia original do RNAr 5S. / Alticinae presents karyotypic characteristics very interesting as the variation of the diploid number, sex determination system and meiotic irregularities. The most frequent chromosome number is 11 or 12 pairs. The species cytogenetically studied Oedionychina have 2n = 22,10 II + X + Y with giant sex chromosomes. In relation to the systematic position there are many divergence between the studies and problems of identification of species belonging to several genera. The study with more refined techniques and rDNA 5s mapping is recent in Coleoptera , and the few Alticinae species studied show the presence of two or three autosomal pairs. Thus, this study aims to analyze cytogenetically and propose strategies for the species karyotype differentiation of Omophoita communis and Omophoita sexnotata. Cytogenetic analysis of individuals of both populations of O. communis showed the existence of a large variation in diploid number and chromosome morphology, being possible to separate them into two cytotypes. Cytotype I presented 2n = 22 and cytotype II 2n = 12, this variation is described for the first time in the genus. Additionally, the study of the morphology of the aedeagus showed differences, indicating a likely pattern of differentiation of the two species. The phylogenetic reconstruction consensus tree analysis also presented that cytotypes show different groupings and reinforce the hypothesis of two species. The 5S rDNA gene mapping of O. sexnotata showed the presence of this cluster in all autosomes, this dispersal pattern was never described in Coleoptera before. In the literature, the dispersion of ribosomal genes is always associated with the presence of transposable elements. The sequencing of 5S rRNA fragments obtained from each chromosome of O. sexnotata resulted in similar to 5S rRNA sequences of Drosophila melanogaster transposable element EnSpm, retropseudogene 5S and microsatellite. Additionally, analysis of the secondary structure of 5S rRNA showed that the sequences obtained are not functional compared their percentage of free energy with the percentage of the original sequence of the 5S rRNA .

La nucléoline, une des protéines non-ribosomique les plus abondantes du nucléole, semble être impliquée dans de nombreux aspects du métabolisme de l'ADN en plus de son rôle dans la régulation de la transcription par l'ARN polymérase I, la maturation du pré-ARNr et l’assemblage des ribosomes. L'objectif de cette thèse est d'étudier l'interaction de la nucléoline avec la chromatine, et de déchiffrer la fonction de la nucléoline dans la régulation de l’expression génique. Il a été rapporté que la nucléoline est nécessaire pour la transcription des gènes codant pour l'ADN ribosomal in vivo, mais le mécanisme par lequel la nucléoline module la transcription d’ARN polymérase I (Pol I) est inconnue. Dans cette thèse, je montre que l’inhibition de l’expression de la nucléoline par siRNAconduit dans les gènes de l’ADNr à une augmentation de la marque hétérochromatine et une diminution des marques caractéristiques de l’euchromatine. La nucléoline est associée à des gènes ADNr non méthylés et ChIP-seq montrent un fort enrichissement de la nucléoline dans le promoteur et la région codante de l'ADNr. La nucléoline est capable d'interférer avec la liaison de TTF-1 sur le terminateur T0 proches du promoteur inhibant ainsi le recrutement du sous-unité NoRC TIP5 et HDAC1 et la création d'un état répressif hétérochromatine. Cette invasion de macroH2A1 dans le nucléole joue un rôle majeur dans l'inhibition de la transcription par la RNA Polymérase I en l'absence de la nucléoline. Ces résultats révèlent l'importance de la nucléoline pour le maintien de l'état euchromatien de l'ADNr et le rôle de macroH2A1 dans la régulation de la transcription de l'ADNr. / Besides the well-known role of the nucleolus in ribosome biogenesis, nucleoli play important roles in the regulation of many fundamental cellular processes, including cell cycle regulation, apoptosis, telomerase production, RNA processing and therefore it is not surprising that many nucleolar proteins appear to be multifunctional proteins. Nucleolin, one of the most abundant non-ribosomal proteins of the nucleolus, has been the focus of many studies since it was first described 35 years ago. It seems to be involved in many aspects of DNA metabolism, chromatin regulation and appeared to be a good pharmacological target for drug development in addition to its role in RNA polymerase I transcription and pre-ribosomal processing and assembly in pre-ribosomes. In eukaryotic cells, DNA is packed into nucleosomes to form chromatin in the nucleus. The cells develop a variety of strategies to overcome the nucleosomal barriers. These strategies include DNA methylation, histone post-translational modifications, incorporation of histone variants and ATP dependent chromatin remodeling. The aim of this thesis is to study the interaction of nucleolin with chromatin, and to decipher the mechanism of nucleolin in gene regulation. It was reported that nucleolin possesses a histone chaperone activity, helps the transcription through nucleosomes, and it is required for ribosomal DNA gene (rDNA) transcription in vivo, but the mechanism by which nucleolin modulates RNA polymerase I (Pol I) transcription is unknown. In the thesis it is shown that nucleolin knockdown results in an increase of the heterochromatin mark H3K9me2 and a decrease of H4K12Ac and H3K4me3 euchromatin histone marks in rDNA genes. Nucleolin is associated with unmethylated rDNA genes and ChIP-seq experiments identified a strong enrichment of nucleolin in the promoter and coding regions of rDNA. Nucleolin is able to interfere with the binding of TTF-1 on the promoter-proximal terminator T0 thus inhibiting the recruitment of the nucleolar remodeling complex (NoRC) subunit TIP5 and HDAC1 and the establishment of a repressive heterochromatin state. In addition, in absence of nucleolin or after inhibition of Pol I by actinomycin D, a strong relocalization of the histone variant macroH2A1 to the nucleolus and on the rDNA genes was observed. This invasion of macroH2A1 in the nucleolus plays a major role in the inhibition of Pol I transcription in absence of nucleolin, as knockdown of macroH2A1 eliminates the repressive effect of nucleolin depletion. These results reveal the importance of nucleolin for the maintenance of the euchromatin state of rDNA required for an efficient production of ribosomal RNAs and the role of macroH2A1 in rDNA transcription.

- 1 - ABSTRACT Nucleoli are formed on the basis of genes of ribosomal DNA (rDNA) clusters called Nucleolus Organizer Regions (NORs). The essential structural components of the nucleoli, Fibrillar Centers (FC) and Dense Fibrillar Components (DFC), together compose FC/DFC units. These units are centers of rDNA transcription by RNA polymerase I (pol I), as well as the early processing events, in which an essential role belongs to fibrillarin. Each FC/DFC unit probably corresponds to a single transcriptionally active gene. In our work we study changes of FC/DFC units in the course of cell cycle. Correlative light and electron microscopy analysis showed that the pol I and fibrillarin positive nucleolar beads correspond to individual FC/DFC units. In vivo observations showed that at early S phase, when transcriptionally active ribosomal genes were replicated, the number of the units in each cell increased by 60 to 80 %. During that period the units transiently lost pol I, but not fibrillarin. Then, until the end of interphase, number of the units did not change, and their duplication was completed only after the cell division, by mid G1 phase. This peculiar mode of reproduction suggests that a considerable subset of ribosomal genes remain transcriptionally silent from mid S phase to mitosis but become again active...

Dans les écosystèmes marins, les protistes jouent un rôle important en tant que producteurs primaires, prédateurs ou encore symbiontes. De par leur position clef, l'étude de leur diversité est fondamentale afin d'améliorer notre compréhension du fonctionnement des écosystèmes marins. Pour ce faire, des approches moléculaires couplées à la microscopie ont été utilisées afin d'examiner en profondeur la diversité des communautés microbiennes dans deux écosystèmes contrastes (Manche Orientale et Méditerranée). La première étude conduite en Manche Orientale a permis d'établir une vue d'ensemble de la diversité des protistes eucaryotes sur une période de 4 ans (de mars 2011 à Juillet 2015) et d'examiner l'influence des variables environnementales sur leur distribution temporelle. L'approche novatrice utilisée consistait à séquencer simultanément l'ARNr et l'ADNr. Ce double ciblage a permis de calculer le rapport ARNr : ADNr et de démontrer qu'il pouvait être un paramètre utile et significatif pour évaluer l'activité cellulaire globale de la communauté microbienne. Nous avons également montré la faisabilité d'utiliser ce ration ARNr : ADNr comme indicateur des transitions écologiques d'espèces microbiennes emblématiques (e.g. bloom de Phaeocystis globosa). Ainsi, le calcul individuel de ce rapport pour chaque OTU devrait permettre d'apporter une information supplémentaire indispensable pour une meilleure compréhension du fonctionnement de l'écosystème et de l'influence des paramètres biotiques et abiotiques sur la structuration des communautés microbiennes, en particulier dans un contexte de changement global. La seconde étude a également été menée en Manche Orientale sur les données de séquençage obtenues pendant la période 2011-2013. Elle avait pour objectif de mettre en évidence les relations existantes entre les parasites eucaryotes et les autres taxons ainsi que les paramètres environnementaux. Cette érude a montré la diversité insoupçonnée des symbiontes/décomposeurs en Manche Orientale, et leur importance pour la structuration des communautés et la succession saisonnière. En particulier, des analyses en réseaux de corrélations ont montré la prédominance des relations inter-taxa, plutôt qu'entre OTUs et paramètres abiotiques, et la position centrale des symbiontes/décomposeurs dans ces relations. Cette étude a permis de souligner la complexité et l'importance des interactions microbiennes dans la structuration des communautés toute en apportant des informations cruciales permettant de mieux comprendre les mécanismes sous-jacent qui régissent cette structuration. La dernière étude a été menée dans la Méditerranée Occidentale, au niveu de quatre stations situées dans le Golfe du Lion, soumises à l'influence du fleuve Rhône, sur une période de 2 ans (2012-2014). Elle avait pour objectif de réaliser la liste la plus exhaustive possible de la diversité des protistes eucaryotes et d'évaluer leur variabilité spatiale en relation avec les variables environnementales. Cette étude a confirmé l'effet prépondérant du Rhône sur la structuration des communautés, particulièrement dans la partie Est du Golf du Lion. Globalement, ces travaux de thèse ont permis de renforcer, par le biais d'approches combinées de microscopie et de biologie moléculaire, nos connaissances sur le fonctionnement, la diversité taxonomique et la succession des espèces microbiennes, en relation avec les paramètres environnementaux, de deux écosystèmes marins contrastés. / In marine ecosystems, protists play an important role as primary producers, predators or symbionts. Therefore studying their diversity and metabolic activity is fundamental for understanding the functioning of marine ecosystems. In order to achieve this goal, molecular approaches were coupled with microscopy analysis for an in depth examination of microbial communities diversity in two contrasting ecosystems (Eastern English Channel EEC and Mediterranean). The first study, conducted in the EEC, allowed to establish a precise overview of eukaryotic protists diversity over 4 years (from March 2011 to July 2015) and to examine the influence of environmental variables on their temporal distribution. The innovative approach used during this study was to sequence simultaneously rRNA and rDNA. This double targeting allowed calculating the rRNA : rDNA ratio and demonstrate that it could be an useful and significant parameter for measuring the relative cellular activity of the microbial community. We also showed the feasibility of using this rRNA : rDNA ratio as an indicator of ecological transitions of iconic microbial species (e.g. bloom of Phaeocystis globosa). Thus, calculation of this ratio for each individual OTU provided additional information that are essential for a better understanding of the functioning of the ecosystem and the influence of biotic parameters on the structuring of microbial communities, in particular in the context of global change. The second study was also conducted in the EEC on sequencing data obtained during the period 2011-2013. It aimed to highlight the relationships between eukaryotic parasites and other taxa as well as environmental parameters. This study revealed un unsuspected diversity of symbionts / decomposers in the EEC, and their importance for structuring microbial community and influence seasonal succession. More specifically, correlation network analysis showed the predominance of inter-taxa relations, over those between OTUs and abiotic parameters, and the central position of symbionts / decomposers in these relationships. This study highlighted the complexity and importance of microbial inteactions in the structuring of microbial communities while providing crucial information to better understand underlying lechanisms. The last study was conducted in the Western Mediterranean , at four stations located in the Golfe du Lion, subject to the influence of the Rhône River, over a period of two years. This study aimed to provide the most exhaustive list of eukaryotic protist diversity and to evaluate its spatial variability in relation with environmental variables. This study further underlined the effect of the Rh^ne on the structuring of microbial communities, particularly in the eastern part of the Golfe du Lion. Overall, thesis workhas reinforced, through combined aproaches of microscopy and molecular biology, our knowledge of the functioning, taxonomic diversity and succession of microbial species, in relation to environmental parameters, of two contrasted marine ecosystems.

Traditionally, bacterial culture has been used for bacterial detection, allowing study of living microorganisms. Molecular methods are rapid and allow simultaneous identification of numerous species and uncultivated phylotypes. The objective of this doctoral thesis was to investigate the role of the oral microbiota, including poorly characterized and uncultivated bacteria, in dental caries and periodontitis, by comprehensive molecular, clinical, and statistical methods. The microbiota of 275 pre-school children (75 with caries and 200 caries-free) was examined by whole genomic DNA probes, 16S rDNA cloning and sequencing, and PCR. Streptococcus mutans, exhibiting a combined association with Streptococcus sobrinus, was significantly associated with Early Childhood Caries (ECC). Plaque from children with Severe Early Childhood Caries (S-ECC) was diverse with 138 identified and 107 unidentified taxa, which possibly included novel phylotypes. Other species/phylotypes associated with childhood caries included Lactobacillus gasseri (p<0.01), Lactobacillus fermentum, Actinomyces israelii, and Actinomyces odontolyticus (all p<0.05, ECC), Veillonella parvula (p<0.01), Veillonella atypica (p<0.05), and Veillonella sp. HOT-780 (p<0.01, S-ECC). Lactobacillus acidophilus and Lactobacillus reuteri, both used as probiotic therapy species, were detected more frequently in caries-free children than those with ECC. Fastidious periodontal species, including Parvimonas micra, Aggregatibacter actinomycetemcomitans, Eubacterium brachy, Filifactor alocis (all p <0.05), and Porphyromonas gingivalis (p<0.01), were also more frequently detected in children with dental caries than in caries-free children. Other variables associated with ECC were race, dental visit, snacking (all p<0.05), and visible dental plaque (p<0.01). The oral microbiota of early periodontitis in young adults (N=141) was analyzed by whole genomic and oligonucleotide DNA probes, and PCR. Species detected more frequently in early periodontitis than periodontal health included Treponema denticola, F. alocis, Porphyromonas endodontalis, Bacteroidetes sp. HOT-274 (oral clone AU126), and A. odontolyticus (p<0.01) by oligonucleotide DNA probes, and P. gingivalis (p<0.001) and T. forsythia (p=0.03) by PCR. Subgingival samples exhibited a higher prevalence of periodontitis-associated species than samples from tongue surface, including A. actinomycetemcomitans, T. denticola, T. forsythia (all p<0.05), and uncultivated TM7, Treponema, and Actinobaculum clones (all p<0.05). P. gingivalis (p<0.01) by PCR was associated with periodontal disease progression. Early periodontitis was associated with older age (p=0.01), male gender (p=0.04), and cigarette smoking (p=0.05). The role of bacterial subgroups in periodontitis was examined by studying the serotypeability of 313 genotyped clinical A. actinomycetemcomitans isolates (189 subjects). A total of 95 strains (30 subjects) remained non-serotypeable, although PCR revealed presence of the serotype- specific genes. The absence of the immunodominant serotype-specific antigen was confirmed by immunoblot assays. No major DNA rearrangement in the studied serotype-specific gene clusters was found. In summary, detection of previously cultured species and uncultivated phylotypes revealed the diversity of the oral microbiota in dental diseases and health already early in life. Bacterial species have insufficiently characterized subgroups that may have attributes to evade the host response. Molecular approaches used in this study enable comprehensive, culture-independent characterization of the oral microbiome that may in the future lead to identification of diagnostic bacterial profiles for dental diseases.