Les communautés du coralligène dominées par des gorgonaires ont été sévèrement affectées par des évènements de mortalités massives liés au réchauffement de la Méditerranée. Pour évaluer la contribution des bactéries associées à l’holobionte Paramuricea clavata à son fonctionnement et sa santé, il est apparu primordial de caractériser le compartiment microbien naturel de ce gorgonaire tempéré.Dans ce contexte, l’objectif général de cette thèse était de décrire les interactions existant entre la gorgone rouge P. clavata et ses bactéries associées en Méditerranée nord-occidentale. Les analyses entreprises par des techniques culture-indépendantes basées sur l’analyse des ADN ribosomiques 16S bactériens ont inclus (i) la caractérisation de la variation spatio-temporelle des communautés bactériennes, (ii) la localisation des bactéries dans les tissus de l’hôte, (iii) l’évaluation de la stabilité des associations gorgones-bactéries en conditions de stress et (iv) la détermination de la spécificité d’hôte des bactéries dominantes entre différentes espèces de gorgonaires sympatriques (Eunicella singularis, Eunicella cavolini et Corallium rubrum). Les résultats obtenus ont établi que P. clavata et son microbiote forment un holobionte au sein duquel hôte et bactéries vivent en étroite association, stable dans le temps et l’espace ou en conditions de stress. Les communautés bactériennes associées sont principalement endosymbiotiques et dominées par un ribotype bactérien appartenant à un genre nouveau de la famille des Hahellaceae qui semble présenter une forte spécificité d’hôte. Ces résultats suggèrent un rôle particulier de ce genre bactérien chez les holobiontes gorgonaires. / Coralligenous communities dominated by gorgonian species have been severely affected by diseases and mass mortality events linked to the current warming trends reported for the Mediterranean Sea. The characterization of the natural microbial compartment of this temperate gorgonian species becomes a crucial step in the evaluation of the bacterial contribution to health and functioning of the Paramuricea clavata holobiont.Under these circumstances, the global aim of this PhD work was to describe the interactions existing between the red gorgonian P. clavata and its associated bacteria in the Northwestern Mediterranean basin. The culture-independent analyses based on the bacterial 16S ribosomal DNA included (i) the characterization of spatiotemporal variation of the bacterial communities, (ii) the localization of the bacteria within host tissues, (iii) the evaluation of the stability of gorgonian-bacterial associations under stress conditions and (iv) the determination of the host-specificity of dominant bacteria in different sympatric gorgonian species (Eunicella singularis, Eunicella cavolini and Corallium rubrum).The results of this study highlighted that P. clavata and its microbiota form a holobiont in which host and bacteria live in close association. This association is spatiotemporally stable and maintained under stress conditions. Associated bacterial communities are mostly endosymbiotic and dominated by a bacterial ribotype belonging to a new genus within the Hahellaceae family that seems to be host-specific. These results suggest a particular role of this bacterial genus in the gorgonian holobionts.

In eukaryotic cells, genomic DNA exists as chromatin in association with histone octamers called nucleosomes, and various other chromatin proteins. Chromatin structure varies along the chromosome and this influences the state of gene expression. Based on such variations in structure and gene expression, chromatin can be broadly classified into euchromatin (transcriptionally active) and heterochromatin (silent or transcriptionally repressed). In the budding yeast, Saccharomyces cerevisiae, there are four canonical transcriptionally silent regions, namely, the HMR, the HML (cryptic mating loci), the telomeres and the RDN1. Silencing at the HM loci and the telomeres is very well characterized. The repressive structure at the HMR spans around 3.5 Kb and extends between the two silencers E and I. It is well established that silencing in HMR is due to a specialized chromatin organization brought about by Orc1p, Rap1p, Abf1p and Sir proteins. Following recruitment, the Sir proteins spread along the DNA to form a repressive chromatin domain believed to arise from the deacetylation of amino-terminal tails of histones H3 and H4 by Sir2p (an NAD dependent deacetylase) and the interaction of Sir3p and Sir4p with the histones. The bi-directional spreading of silencing at HMR is restricted by barrier or boundary elements that flank the silencers. A tRNAThr gene in the right boundary of HMR acts as a strong barrier. Mutations in the promoter of this tRNA gene (tDNA) or in RNA polymerase III subunits/ transcription factors weaken the barrier activity of this tDNA. The barrier activity of this tDNA is also dependent on histone acetyltransferases like Sas2p and Gcn5p. Silencing in HML is uniformly high between the silencers E and I and falls sharply outside I. Recently, barriers to HML silencing have been discovered. A 0.71Kb sequence near E, which maps to the upstream activating sequence of YCL069W, acts as a robust barrier to spread of HML silencing. This is effectively the left boundary of silent HML. The right boundary maps to the promoter of CHA1 gene though silencing is believed to terminate at HML-I. An unusual form of silencing occurs at the RDN1, which contains 100-150 copies of tandemly repeated rRNA genes. Some RNA polymerase II transcribed genes integrated within the array are silenced by a Sir2p dependent mechanism whereas genes driven by RNA polymerases III and I are transcriptionally active. Though all the three forms of silencing (RDN1, HM and telomere) require Sir2p, RDN1 silencing differs from the others in its relative strength and factors responsible for repression. Several trans-acting factors required for RDN1 silencing are known. However, it is still unclear as to what limits the spread of RDN1 heterochromatin into neighbouring essential genes. RDN1 silencing spreads unidirectionally in its left hand side sequence. However, the zone of RDN1 heterochromatin does not engulf the essential gene, ACS2, which is present ~3 kb away from NTS1. This implies that there is a mechanism by which rDNA heterochromatin is contained. There could be several ways by which this is accomplished. Firstly, the cell could be critically maintaining the levels of Sir2p, the protein required for silencing at all the four silenced loci, such that silencing in the left flank of RDN1 does not spread beyond 300 bp of NTS1 (Buck et al, 2002). There is a ~2.5 kb gene free intervening sequence between NTS1 of the rDNA array and the Ty1 LTR, in which interval Sir2p level could fall below the threshold mark required for causing repression. In fact Buck et al. have demonstrated that Sir2p is bound to upto 1.5 kb from the NTS1 in the left flank but there is no accompanying silencing of the mURA3 reporter in these regions (1200L and 2000L), suggesting that the level of Sir2p at these sequences could be lower than the threshold required for initiation of silencing. Secondly, there could be cis-acting boundary elements or barriers as in the case of HMR, which prevents the propagation of RDN1 silencing. The third option is that termination of RNA polymerase I transcription at the terminator sites automatically halts the spread of rDNA silencing since Buck et al. have demonstrated that progression of rDNA heterochromatin is dependent on RNA pol I transcription. This however, does not seem to be the case as deletion of both the terminator sites within NTS1 does not lengthen the zone of silencing. Finally, there could be an euchromatin organizing center further from the array, which creates an “open” chromatin configuration required to confront the Sir2p mediated condensed chromatin. The balance of these two opposing activities, much like that at the telomeres, could set up a molecular boundary for containing rDNA silent chromatin. We have attempted to identify whether there are any sequences in the unique left flank of RDN1 that can act as a heterochromatin barrier. Towards that end we tested four overlapping fragments from NTS1 of RDN1 to the promoter of ACS2 for boundary activity in a quantitative mating assay. We have found that of all the four fragments tested, only a 0.427 kb tRNAGln-Ty1 LTR fragment, which is present 2.4 Kb from the NTS1 acts as a robust barrier in this assay. Further mapping revealed that the barrier activity of this sequence resides in the tRNAGln gene and that its activity is orientation-independent. tDNAs are transcribed by RNA polymerase III from internal promoters termed Box A and Box B. It has been shown for the HMR-tRNAThr that the transcriptional potential of the tDNA is crucial for its barrier function. Mutations in genes encoding various subunits of the RNA polymerase III complex, or transitions in the conserved bases within Box B known to disrupt transcription complex assembly and subsequent transcription, abrogate the barrier activity of HMR-tRNAThr. Similarly, loss of transcriptional ability of the tRNAAla in the centromere of S. pombe also abolishes its barrier activity, enforcing the fact that RNA polymerase III transcription is a decisive factor for a tDNA barrier. Contrary to the above observations, we report that barrier activity of tRNAGln is very negligibly dependent on RNA polymerase III mediated transcription. Mating assays done with the RNA pol III mutants and promoter point mutants, G18C and C55G in boxes A and B respectively, underline the fact that for this tDNA barrier, RNA pol III driven transcription is dispensable. We also show by RT-PCR analysis that in the C55G tRNAGln mutant there is loss of transcription as expected, whereas other wild type copies of tRNAGln are transcribed. Studies with another tDNA barrier, TRT2-tRNAThr, yielded similar results, again emphasizing the point that transcription through the tDNA, which leads to nucleosome displacement and therefore barrier activity, may not be applicable for all tDNA barriers. Acetylation of amino terminal tails of histones is known to influence the epigenetic state of chromatin. Addition of acetyl moiety to histones H3 and H4 initiates a cascade of events, which involves recruitment of a host of other chromatin modifiers to the target sequence, and ultimately culminates in the formation of an euchromatin-favouring environment. As reported for the HMR right boundary, we find that barrier activity of tRNAGln depends on two histone acetyl transferase complexes, SAS-I (comprised of Sas2p, Sas4p and Sas5p) and SAGA (contains Gcn5p HAT). Contrary to the HMR boundary, the barrier activity of tRNAGln is independent of two other nucleoplasmic HATs, NuA3 (Sas3p being the HAT) and NuA4 (Esa1p is the HAT). Barrier function of TRT2-tRNAThr also depends on HATs. Therefore it appears that requirement of HATs for boundary activity is a conserved theme, albeit with differential effects at different barrier sequences. We next attempted to determine the function of tRNAGln in its natural location on chromosome XII. As mentioned earlier, RDN1 silencing spreads upto ~0.3 kb in its left flanking sequence. However, Sir2p occupancy has been observed till 1.5 kb although there is no silencing of reporter genes observed beyond 0.3 kb of NTS1. This lead us to speculate that there could be a boundary sequence in the left flank that stops silencing, or a euchromatin-organizing element, which counters the propagation of silencing by a long-range effect. Since over expression of Sir2p extends the domain of silencing from 0.3 kb to 2.0 kb and the tRNAGln is present at 2.3 kb from NTS1, it was a good candidate for a heterochromatin barrier/ euchromatiniser. However, deletion of tRNAGln does not affect the zone of RDN1 silencing as is evident from our cell viability assays (which is a measure of the expression of the essential gene ACS2 situated further to the left of tRNAGln). Deletion of SAS2 and GCN5, factors that are required for barrier activity of tRNAGln in mating assays, also have no effect on the extent of spreading of RDN1 silencing in normal or Sir2p over expression conditions. Together, these observations imply that in situ, tRNAGln does not act as a barrier or an element with long-range euchromatin inducing properties. It still remains unclear as to what contains RDN1 silencing. It is possible that the cell critically monitors the level of Sir2p in order to maintain boundaries of silencing at the rDNA locus. Telomeres also nucleate the formation of silenced domain which spreads along the subtelomeric region upto ~ 2Kb. The key players in the formation of telomeric heterochromation are the Sir proteins, Sir2p, Sir3p and Sir4p, Rap1p, yKu complex and ORC. Protein-protein interactions between the telosome and the subtelomeric repeat bound silencing proteins create a domain of core heterochromatin that spreads in the adjacent sequences. While Sir2p deacetylates H4K16, Sir3p interacts with the hypoacetylated histone tails and helps in the spreading of the repressive chromatin structure. As a result telomere proximal genes are silent whereas the ones further away are expressed. There is a gradient of acetylation of histone H4, with the hypoacetylated histones at the telomeric ends and the hyperacetylated ones distant from the telomere. Recently it has been shown that this gradient is maintained by the concerted and antagonistic actions of Sir2p and Sas2p. In a sas2Δ strain Sir3p spreads to ~15 kb in the subtelomeric regions and there is increase in the levels of hypoacetylated histones. Though the molecular mechanism by which telomeric silencing is restrained is beginning to be understood, it remains unanswered whether there are any cis-acting sequences, capable of recruiting euchromatin-inducing factors such as Sas2p, near the telomeres. We have identified a RNA polymerase II driven gene, AAD3, in the subtelomeric region of chromosome III that has robust anti-silencing activity. Deletion mapping revealed that only 0.381 kb in the 5′ portion of the gene (excluding the promoter) is sufficient for barrier activity and that this property is orientation-independent (henceforth referred to as TEL-B). The barrier acivity of TEL-B depends strongly on Sas2p and Esa1p but not on Gcn5p and Sas3p, and is independent of cohesin. Previous investigations have shown that acetylation of H4K16 by Sas2p at subtelomeric regions of chromosome VI leads to deposition of HTZ1 in the nucleosome and its subsequent acetylation by Esa1p of NuA4. All these events together are required to contain the onslaught of telomeric core heterochromatin on neighbouring active regions. Since barrier activity of TEL-B depends on Sas2p and Esa1p, it is possible that TEL-B has the potential to act as a bona fide barrier in situ in its endogenous context. Our hypothesis is further cemented by the observation that there is a physical association between Sas2p, the molecule at the top of the entire cascade of events, with TEL-B by yeast one hybrid analysis. Further experiments will shed light on the role of this sequence in its natural location. In summary, I have identified and characterized two different barrier sequences in S. cerevisiae. Not many barriers are known in budding yeast and there is extensive ongoing research dedicated to understand the mechanism(s) of barrier function. In chapter I of my thesis I present a review of current literature regarding silencing barriers in yeast and other systems. In chapter II I have outlined a detailed characterization of a tDNA barrier element, tRNAGln, present near the silenced rDNA array on chromosome XII. My work addresses the various models for barrier activity and their applicability to the tRNAGln barrier. I have also attempted to understand the role of this tDNA in its natural location on the chromosome with respect to limitation of RDN1 silencing. In chapter III I have described an intensive study of a RNA polymerase II transcribed gene, AAD3, present near the right telomere of chromosome III, which acts as a robust barrier to silencing. I have attempted to answer which mechanism(s) is/are operational at this sequence so as to endow it with barrier potential. My studies with the two barrier elements highlight novel trans-acting factors required for barrier function, differential and selective requirements of certain factors for different barriers, and provide a mechanistic view of the boundary activity of these sequences.

Le nucléole est le site de biogenèse des ribosomes, qui commence par la transcription des gènes d’ARN ribosomique (ARNr). Cependant, le nucléole est également impliqué dans d'autres processus cellulaires, comme l’organisation 3D du génome. Ainsi, des régions génomiques appelées NADs pour Nucleolus-Associated chromatin Domains, ont été identifiées dans des cellules animales et végétales. Ces régions sont surtout hétérochromatiques et les gènes associés ont tendance a être peu ou pas transcrits. Un des objectifs de ma thèse a été d’étudier l’implication du nucléole dans l’organisation de la chromatine au sein du noyau et la régulation transcriptionnelle de gènes transcrits par l’ARN Polymérase II chez Arabidopsis thaliana. Par ailleurs, parmi les centaines de copies de gènes d’ARNr, uniquement une fraction participe au processus de biogenèse des ribosomes. Dans un second temps, j’ai donc étudié le rôle de ces copies inactives. On a pu démontrer que l’absence des gènes d’ARNr inactifs n’engendre pas de changements majeurs dans la fonction nucléolaire. Par contre, ces copies participent à la stabilité du génome. En effet, en leur absence, des duplications génomiques allant jusqu’à plusieurs centaines de kilobases s’accumulent, entraînant des duplications de gènes et des différences du niveau d’expression de ces derniers. Finalement, les effets de ces changements structuraux sur la biologie de la plante sont discutés. / The nucleolus is the site of ribosome biogenesis, which begins with the transcription of ribosomal RNA (rRNA) genes. However, the nucleolus is also involved in other cellular processes, such as the 3D genome organization. Thus, genomic regions called NADs for Nucleolus-Associated chromatin Domains, have been identified in animal and plant cells. These regions are mostly heterochromatic and the associated genes tend to be poorly transcribed. One of the objectives of my thesis was to study the involvement of the nucleolus in the 3D genome organization and the transcriptional regulation of genes transcribed by RNA Polymerase II in Arabidopsis thaliana. In addition, only a fraction of rRNA gene copies participates in the process of ribosome biogenesis. In a second time, I studied the role of the inactive rRNA gene copies. We show that in their absence, there is no major changes in the nucleolus function. However, these copies contribute to genome stability. Indeed, in their absence, up to several hundred of kilobases long duplication events accumulate, resulting in the duplication and the differential expression of hundreds of genes. Finally, the impact of these structural changes on the plant biology are discussed.

La réparation par excision de nucléotides (NER) garantit l'intégrité du génome lors de l'exposition aux rayons UV. Après irradiation aux UV, un des premiers problèmes rencontrés par la cellule est l'arrêt général de la transcription dû au blocage de l'ARN polymérase II (ARNP2) au niveau des lésions UV. Pour régler ce problème, le NER possède une voie de réparation spécifiquement couplée à la transcription (TCR). Les connaissances concernant le NER ont été obtenu via des études sur la transcription par l'ARNP2. Cependant, dans les cellules à fort métabolisme, plus de 60% de la transcription correspond à la transcription, dans le nucléole, de l'ADN ribosomique (ADNr) par l'ARN polymérase I (ARNP1). De nombreuses protéines sont absence du nucléole, c'est pourquoi certains processus nucléaires ne peuvent avoir lieu dans cette structure. Afin d ‘être répliqué et réparé, l'ADNr se déplace à la périphérie du nucléole. Malgré l'importance de la transcription par l'ARNP1, la réparation de l'ADNr a été peu étudiée chez l'homme. De plus, à notre connaissance, aucune étude ne s'est penchée sur le mécanisme moléculaire du déplacement de l'ADNr à la périphérie du nucléole. Notre étude démontre l'implication de la TCR dans la réparation de l'ADNr après lésions UV induites. De plus, nos recherches ont démontré que l'ARNP1 reste accrochée à l'ADNr et sont tous les deux délocalisés à la périphérie du nucléole après irradiation aux UV. Enfin, nous avons identifié l'actin et la moysine I nucléaires comme facteurs protéiques nécessaire à cette délocalisation / Nucleotide excision repair (NER) guarantees genome integrity and proper cellular functions against UV-induced DNA damage. After UV irradiation, one of the first burden cells have to cope with is a general transcriptional block caused by the stalling of RNA polymerase II (RNAP2) onto distorting UV lesions. To insure UV lesions repair specifically on transcribed genes, NER is coupled with transcription in an extremely organized pathway known as Transcription-Coupled Repair (TCR). Most of the knowledge about TCR has been gathered from RNAP2 transcription. However, in highly metabolic cells, more than 60% of total cellular transcription results from ribosomal DNA (rDNA) transcription, by the RNA polymerase I (RNAP1), which takes place in the nucleolus. Many nuclear proteins are excluded from the nucleolus and because of this some nucleolar processes cannot occur inside this structure. In order to be replicated and repaired rDNAs need to be displaced at the nucleolar periphery. Despite the importance of RNAP1 transcription, repair of the mammalian transcribed rDNA has been scarcely studied. Moreover, to the best of our knowledge no molecular mechanism has been proposed for rDNA displacement. Our study clearly demonstrated that the full TCR machinery is needed to repair UV-damaged rDNA and restart RNAP1 transcription. Our results show that UV lesions block RNAP1 transcription and that RNAP1 is firmly stalled onto rDNAs without being degraded. Our study also describes the displacement of the RNAP1/rDNA complex to the nucleolar periphery after UV irradiation and identifies both nuclear ß-actin and nuclear myosin I as factors required for this displacement

Resumo: Os triatomíneos são insetos hematófagos de grande importância para a parasitologia humana, pois são transmissores do Trypanosoma cruzi, protozoário causador da doença de Chagas. Além de sua importância médico-sanitária, os triatomíneos destacam-se pela sua citogenética, pois possuem cromossomos holocêntricos e um modelo de meiose incomum, com meiose invertida para os cromossomos sexuais. Recentes pesquisas com marcadores moleculares em triatomíneos tentam compreender a ancestralidade do grupo. Uma das formas de se compreender a evolução entre espécies é a partir da análise de seqüências do DNA ribossômico (DNAr). Por pertencer a famílias multigênicas, cópias individuais do DNAr não acumulam mutações independentemente, resultando em pequena variação intra-específica e relevante diferenciação interespecífica. No presente trabalho foi realizado um estudo comparativo entre as espécies Triatoma maculata e Triatoma pseudomaculata, com base no uso das técnicas citogenéticas convencionais de orceína lacto-acética, impregnação por íons prata, bandamento-C, reação de Feulgen; da técnica de citogenética molecular de bandamento- C CMA/DAPI; e também por meio da análise da região ITS-1 do DNAr, com base no sequenciamento, com o objetivo de avaliar o grau de homologia entre as espécies estudadas. Os cariogramas das duas espécies indicaram dez pares de autossomos (um deles de tamanho maior) e um par de cromossomos sexuais (2n = 22). No ciclo meiótico foi possível observar a fragmentação da região nucleolar no final do estágio difuso. Corpúsculos nucleolares foram observados em alguns dos núcleos em metáfases meióticas de T. pseudomaculata, evidenciando a persistência nucleolar. A técnica de bandamento-C revelou que o cromossomo Y é heterocromático em ambas as espécies. O sequencimento da região ITS-1, indicou que as espécies apresenta... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The triatomines are hematophagous insects of great concern in public health because they are vectors of Trypanosoma cruzi, a protozoan that causes Chagas disease. Triatomines are also of great genetic interest, because that they present holocentric chromosomes and an unusual form of meiosis with post-reductional segregation of sex chromosomes. Recent studies based on molecular markers try to understand the evolutionary history of triatomines. To understand the evolution of a given species, ribosomal DNA (rDNA) analyses are frequently used, which can help to infer evolutionary relationships among species. Individual copies of rDNA do not accumulate mutations independently because they belong to multigene families, resulting in slight intraspecific and important interspecific variation. In this study, a comparative analysis was performed between the species Triatoma maculata and Triatoma pseudomaculata, based on the cytogenetic techniques of lacto-acetic orcein, silver ion impregnation, Cbanding, Feulgen reaction; and CMA/DAPI C-banding. We also compared the species by sequencing the ITS-1 rDNA internal transcribed region in order to evaluate the degree of homology among the studied species. The cariograms of the two species revealed ten autosomes and one pair of sexual chromosomes (2n= 22). In the meiotic cycle, nucleolar fragmentation during the final stages of meiotic prophase I was found. Nucleolar corpuscles were found in some meiotic metaphases of T. pseudomaculata, which is evidence of nucleolar persistence. The C-banding technique revealed that the Y chromosome is heterochromatic in both species. The ITS-1 rDNA sequences showed that the species presented a discharge proximity to each other, and had a high degree of homology (98.5%). The knowledge obtained in this study contributes to the understanding of the interrelation and distribution of those species, and offers... (Complete abstract click electronic access below) / Orientador: Maria Tercília Vilela de Azeredo Oliveira / Coorientador: Lilian Castiglioni / Banca: João Aristeu da Rosa / Banca: Carlos Roberto Ceron / Mestre

Resultados anteriores sugerem que existem diferenças biológicas entre espécimens de Anopheles oswaldoi capturados no Estado do Acre e os do Estado de Rondônia, Brasil. Esta espécie tem sido apontada como um importante vetor de malária em localidades do Peru e Acre. Entretanto, em Rondônia, somente um número pequeno de A. oswaldoi alimentados em pacientes com malária desenvolveram infecção nas glândulas salivares. Além disso, há suspeita de que espécimens identificados como A. oswaldoi, capturados em áreas abertas em Costa Marques, Rondônia, são na verdade A. konderi, e que A. oswaldoi sensu stricto estaria restrito a áreas de florestas. Estes dados, juntamente com as dificuldades encontradas na identificação de anofelinos do grupo Oswaldoi, baseadas em critérios morfológicos, sugerem que espécimens de A. oswaldoi são membros de um complexo de espécies crípticas. A distinção de espécies crípticas de insetos vetores é de grande importância, já que diferentes membros em um complexo podem exibir diferenças na ecologia, capacidade vetorial e resposta a medidas de controle. Análise de sequências de DNA, particularmente da região do ITS2 do cistron do DNA ribossômico, tem sido usada como um caracter diagnóstico em alguns grupos de espécies crípticas, tornando-se um instrumento para estudos taxonômicos e filogenéticos. A primeira parte deste estudo teve o objetivo de determinar as diferenças encontradas nas sequências de ITS2 de espécimens de A. oswaldoi capturados em várias localidades da América do Sul. As regiões dos ITS2 destes anofelinos foram amplificadas usando oligonucleotídeos iniciadores conservados das regiões 5.8S e 28S e os produtos de PCR foram clonados e sequenciados. Os ITS2 de todos os mosquitos capturados tiveram tamanho aproximado de 350 nucleotídeos, com aproximadamente 53% de conteúdo de GC. Análise do alinhamento destas sequências, mostrou similaridade variando entre 87% e 100%, e a análise de uma árvore de similaridade, neighbor-joining, produzida com p-distance usando as diferenças nas sequências de ITS2, separou estes espécimens em quatro grupos. Um deles está provavelmente relacionado ao A. oswaldoi sensu stricto, e um outro pode estar relacionado à espécie A. konderi. Os outros dois grupos podem corresponder a espécies cuja identificação morfológica permanece para ser esclarecida no complexo A. oswaldoi. Estes dados são evidências de que espécimens de A. oswaldoi estão incluídos em um complexo de espécies crípticas e que identificação por DNA pode resolver estas questões taxonômicas. A. konderi tem sido considerado uma sinonímia de A. oswaldoi. Embora estágios adultos e imaturos destas espécies de anofelinos apresentem características morfológicas idênticas, aspectos encontrados na genitália masculina podem distinguir estes taxa. A segunda parte deste estudo foi conduzida com o objetivo de comparar a susceptibilidade de A. oswaldoi s.s. e de A. konderi à infecção com Plasmodium vivax. A susceptibilidade foi baseada na proporção de mosquitos com oocistos e esporozoítos. Os anofelinos foram capturados no Acre e em Rondônia para obtenção de progênies F1. Após a emergência dos adultos, as genitálias masculinas dos mosquitos de cada progênie foram dissecadas e examinadas. Todas progênies originadas dos mosquitos capturados em Rondônia corresponderam a A. konderi, enquanto que cerca de 85,0% das progênies do Acre eram A. oswaldoi s.s.. Estas progênies F1 de A. oswaldoi s.s., A. konderi e A. darlingi foram alimentadas simultaneamente com sangue infectado com P. vivax. Estes mosquitos foram dissecados 10-12 dias após infecção e examinados para verificação da presença de oocistos e esporozoítos. Tanto A. oswaldoi s.s. como A. konderi apresentaram oocistos nos tratos digestivos, entretanto, a porcentagem de tratos digestivos positivos para oocistos foi maior em A. oswaldoi s.s. (13,8%) do que em A. konderi (3,3%). Esporozoítos foram encontrados somente nas glândulas salivares de A. oswaldoi s.s., com 6,9% de positividade. As taxas de infecção nos controles A. darlingi foram de 22,5% a 30,0%, para ambos oocistos e esporozoítos. Estes resultados indicam que A. oswaldoi s.s. pode transmitir P. vivax e sugerem que esta espécie é mais susceptível que A. konderi. Embora A. oswaldoi s.s. seja uma espécie exofílica e zoofílica, este anofelino pode estar envolvido na transmissão da malária humana como parece estar ocorrendo no Estado do Acre. / Previous results have suggested that biological differences could exist between specimens of Anopheles oswaldoi captured in the State of Acre and those from the State of Rondônia, Brazil. This species has been appointed as an important malaria vector in localities of Peru and Acre. However, in Rondônia, only a very low percentage of A. oswaldoi fed on malaria patients developed salivary gland infections. In addition, it was suspected that specimens identified as A. oswaldoi captured in open clearings in Costa Marques, Rondônia, were actually A. konderi, and that A. oswaldoi sensu stricto would be restricted to forested areas. These data, together with the difficulties found to identify anophelines of the Oswaldoi group based on morphologic criteria suggest that specimens of A. oswaldoi are members of a complex of cryptic species. The distinction of cryptic species of insects is of critical importance, since different members in a Complex could exhibit differences in ecology, vectorial capacity and response to control measures. DNA sequence analysis and particularly that of the ITS2 region of the rDNA cistron has provided diagnostic characters in some groups of cryptic species becoming a general tool for taxonomic and phylogenetic studies. The first part of the present study was undertaken to determine the extent of differences over the ITS2 sequence of specimens of A. oswaldoi s.l. captured in several localities of South America. The ITS2 of these anophelines were amplified using conserved primers of the 5.8S and 28S regions, cloned and sequenced. The lengths of ITS2 of all mosquitoes captured were about 350 nucleotides, with about 53% GC content. Analysis of the alignment of the sequences, which showed that the similarity varied between 87% and 100%, and analysis of a neighbor-joining tree produced with p-distance using the ITS2 sequences, separated these specimens in four groups. One of them is probably related to A. oswaldoi sensu stricto, and another one can possibly be related to A. konderi. The other two groups may correspond to species the morphologycal identification of which remains to be clarified in the A. oswaldoi complex. These data are evidences that specimens of A. oswaldoi are included in a complex of cryptic species and that the DNA identification could solve some taxonomic questions. A. konderi has been currently considered to be a synonym of A. oswaldoi. Although adults and immature stages of both these anopheline species have identical morphological characters, features of the male genitalia can distinguish these taxa. The second part of this study was conducted in order to compare the susceptibility of A. oswaldoi s.s. and of A. konderi to infection by Plasmodium vivax. The susceptibility was based on the proportion of mosquitoes with oocysts and sporozoites. Anophelines were captured in the State of Acre and Rondônia and used to obtain F1 progenies. After emergency of adults, male genitalia of mosquitoes of each family were dissected. All families originated from mosquitoes captured in Rondônia corresponded to A. konderi, while about 85.0% of the families from Acre were A. oswaldoi s.s.. F1 progeny of field-captured A. oswaldoi s.s., A. konderi and A. darlingi were fed simultaneously on P. vivax infected blood. Mosquitoes were dissected on day 10-12 after infection and examined for the presence of oocysts and sporozoites. Both A. oswaldoi s.s. and A. konderi developed oocysts in midguts, however, the percentage of oocyst-positives in A. oswaldoi s.s. (13.8%) was higher than in A. konderi (3.3%), and only A. oswaldoi s.s. developed salivary infection with sporozoites (6.9% of positivity). Infection rates in A. darlingi ranged from 22.5% to 30.0% for both oocysts and sporozoites. These results indicate that A. oswaldoi s.s. can transmit P. vivax and suggest that it is more susceptible than A. konderi. Although A. oswaldoi s.s. is an exophilic and zoophilic species, it may be involved in human malaria transmission as it seems to be occuring in the State of Acre.

L’intégrité de l’ADN est continuellement remise en question par divers agents endogènes et exogènes (p. ex., la lumière ultraviolette, la fumée de cigarette, la pollution de l’environnement, les dommages oxydatifs, etc.) qui causent des lésions de l’ADN qui interfèrent avec les fonctions cellulaires correctes. Le mécanisme de réparation par excision de nucléotides (NER) supprime les adduits d’ADN déformantes l’hélice tels que les lésions induites par les UV et il existe dans deux sous voies distinctes selon l’endroit où les lésions de l’ADN sont situées dans le génome. L’une de ces sous voies est directement liée à la transcription de l’ADN (TCR) par l’ARN Polymérase 2 (ARNP2). Dans la première partie de ce travail, nous avons démontré qu’un mécanisme NER entièrement compétent est également nécessaire pour la réparation de l’ADN ribosomique (ADNr), transcrite par ARN Polymérase 1 (ARNP1) et représentant 60 % de la transcription cellulaire totale. De plus, nous avons identifié et clarifié le mécanisme de deux protéines responsables du repositionnement nucléolaire dépendant des UV de l’ARNP1 et de l’ADNr observé pendant la réparation. Dans la deuxième partie de ce travail, nous avons étudié la fonctionne moléculaire de la protéine XAB2 lors de la réparation NER et nous avons démontré son implication dans le processus TCR. De plus, nous avons également montré la présence de XAB2 dans un complexe d’épissage du pré-ARNm. Enfin, nous avons décrit l’impact de XAB2 sur la mobilité de l’ARNP2 lors des premières étapes de la réparation TCR, suggérant ainsi un rôle de XAB2 dans le processus de reconnaissance des lésions / The integrity of DNA is continuously challenged by a variety of endogenous and exogenous agents (e.g. ultraviolet light, cigarette smoke, environmental pollution, oxidative damage, etc.) that cause DNA lesions which interfere with proper cellular functions. Nucleotide Excision Repair (NER) mechanism removes helix-distorting DNA adducts such as UV-induced lesions and it exists in two distinct sub-pathways depending where DNA lesions are located within the genome. One of these sub pathways is directly linked to the DNA transcription by RNA Polymerase 2 (TCR). In the first part of this work, we demonstrated that a fully proficient NER mechanism is also necessary for repair of ribosomal DNA, transcribed by RNA polymerase 1 and accounting for the 60 % of the total cellular transcription. Furthermore, we identified and clarified the mechanism of two proteins responsible for the UV-dependent nucleolar repositioning of RNAP1 and rDNA observed during repair. In the second part of this work, we studied the molecular function of the XAB2 protein during NER repair and we demonstrated its involvement in the TCR process. In addition, we also shown the presence of XAB2 in a pre-mRNA splicing complex. Finally, we described the impact of XAB2 on RNAP2 mobility during the first steps of TCR repair, thus suggesting a role of XAB2 in the lesion recognition process

Made available in DSpace on 2014-06-11T19:26:03Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-02-19Bitstream added on 2014-06-13T20:33:40Z : No. of bitstreams: 1 mendonca_pp_me_sjrp.pdf: 681812 bytes, checksum: d7353da4e0742a18d8d505b0587fda7b (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Os triatomíneos são insetos hematófagos de grande importância para a parasitologia humana, pois são transmissores do Trypanosoma cruzi, protozoário causador da doença de Chagas. Além de sua importância médico-sanitária, os triatomíneos destacam-se pela sua citogenética, pois possuem cromossomos holocêntricos e um modelo de meiose incomum, com meiose invertida para os cromossomos sexuais. Recentes pesquisas com marcadores moleculares em triatomíneos tentam compreender a ancestralidade do grupo. Uma das formas de se compreender a evolução entre espécies é a partir da análise de seqüências do DNA ribossômico (DNAr). Por pertencer a famílias multigênicas, cópias individuais do DNAr não acumulam mutações independentemente, resultando em pequena variação intra-específica e relevante diferenciação interespecífica. No presente trabalho foi realizado um estudo comparativo entre as espécies Triatoma maculata e Triatoma pseudomaculata, com base no uso das técnicas citogenéticas convencionais de orceína lacto-acética, impregnação por íons prata, bandamento-C, reação de Feulgen; da técnica de citogenética molecular de bandamento- C CMA/DAPI; e também por meio da análise da região ITS-1 do DNAr, com base no sequenciamento, com o objetivo de avaliar o grau de homologia entre as espécies estudadas. Os cariogramas das duas espécies indicaram dez pares de autossomos (um deles de tamanho maior) e um par de cromossomos sexuais (2n = 22). No ciclo meiótico foi possível observar a fragmentação da região nucleolar no final do estágio difuso. Corpúsculos nucleolares foram observados em alguns dos núcleos em metáfases meióticas de T. pseudomaculata, evidenciando a persistência nucleolar. A técnica de bandamento-C revelou que o cromossomo Y é heterocromático em ambas as espécies. O sequencimento da região ITS-1, indicou que as espécies apresenta... / The triatomines are hematophagous insects of great concern in public health because they are vectors of Trypanosoma cruzi, a protozoan that causes Chagas disease. Triatomines are also of great genetic interest, because that they present holocentric chromosomes and an unusual form of meiosis with post-reductional segregation of sex chromosomes. Recent studies based on molecular markers try to understand the evolutionary history of triatomines. To understand the evolution of a given species, ribosomal DNA (rDNA) analyses are frequently used, which can help to infer evolutionary relationships among species. Individual copies of rDNA do not accumulate mutations independently because they belong to multigene families, resulting in slight intraspecific and important interspecific variation. In this study, a comparative analysis was performed between the species Triatoma maculata and Triatoma pseudomaculata, based on the cytogenetic techniques of lacto-acetic orcein, silver ion impregnation, Cbanding, Feulgen reaction; and CMA/DAPI C-banding. We also compared the species by sequencing the ITS-1 rDNA internal transcribed region in order to evaluate the degree of homology among the studied species. The cariograms of the two species revealed ten autosomes and one pair of sexual chromosomes (2n= 22). In the meiotic cycle, nucleolar fragmentation during the final stages of meiotic prophase I was found. Nucleolar corpuscles were found in some meiotic metaphases of T. pseudomaculata, which is evidence of nucleolar persistence. The C-banding technique revealed that the Y chromosome is heterochromatic in both species. The ITS-1 rDNA sequences showed that the species presented a discharge proximity to each other, and had a high degree of homology (98.5%). The knowledge obtained in this study contributes to the understanding of the interrelation and distribution of those species, and offers... (Complete abstract click electronic access below)

Resultados anteriores sugerem que existem diferenças biológicas entre espécimens de Anopheles oswaldoi capturados no Estado do Acre e os do Estado de Rondônia, Brasil. Esta espécie tem sido apontada como um importante vetor de malária em localidades do Peru e Acre. Entretanto, em Rondônia, somente um número pequeno de A. oswaldoi alimentados em pacientes com malária desenvolveram infecção nas glândulas salivares. Além disso, há suspeita de que espécimens identificados como A. oswaldoi, capturados em áreas abertas em Costa Marques, Rondônia, são na verdade A. konderi, e que A. oswaldoi sensu stricto estaria restrito a áreas de florestas. Estes dados, juntamente com as dificuldades encontradas na identificação de anofelinos do grupo Oswaldoi, baseadas em critérios morfológicos, sugerem que espécimens de A. oswaldoi são membros de um complexo de espécies crípticas. A distinção de espécies crípticas de insetos vetores é de grande importância, já que diferentes membros em um complexo podem exibir diferenças na ecologia, capacidade vetorial e resposta a medidas de controle. Análise de sequências de DNA, particularmente da região do ITS2 do cistron do DNA ribossômico, tem sido usada como um caracter diagnóstico em alguns grupos de espécies crípticas, tornando-se um instrumento para estudos taxonômicos e filogenéticos. A primeira parte deste estudo teve o objetivo de determinar as diferenças encontradas nas sequências de ITS2 de espécimens de A. oswaldoi capturados em várias localidades da América do Sul. As regiões dos ITS2 destes anofelinos foram amplificadas usando oligonucleotídeos iniciadores conservados das regiões 5.8S e 28S e os produtos de PCR foram clonados e sequenciados. Os ITS2 de todos os mosquitos capturados tiveram tamanho aproximado de 350 nucleotídeos, com aproximadamente 53% de conteúdo de GC. Análise do alinhamento destas sequências, mostrou similaridade variando entre 87% e 100%, e a análise de uma árvore de similaridade, neighbor-joining, produzida com p-distance usando as diferenças nas sequências de ITS2, separou estes espécimens em quatro grupos. Um deles está provavelmente relacionado ao A. oswaldoi sensu stricto, e um outro pode estar relacionado à espécie A. konderi. Os outros dois grupos podem corresponder a espécies cuja identificação morfológica permanece para ser esclarecida no complexo A. oswaldoi. Estes dados são evidências de que espécimens de A. oswaldoi estão incluídos em um complexo de espécies crípticas e que identificação por DNA pode resolver estas questões taxonômicas. A. konderi tem sido considerado uma sinonímia de A. oswaldoi. Embora estágios adultos e imaturos destas espécies de anofelinos apresentem características morfológicas idênticas, aspectos encontrados na genitália masculina podem distinguir estes taxa. A segunda parte deste estudo foi conduzida com o objetivo de comparar a susceptibilidade de A. oswaldoi s.s. e de A. konderi à infecção com Plasmodium vivax. A susceptibilidade foi baseada na proporção de mosquitos com oocistos e esporozoítos. Os anofelinos foram capturados no Acre e em Rondônia para obtenção de progênies F1. Após a emergência dos adultos, as genitálias masculinas dos mosquitos de cada progênie foram dissecadas e examinadas. Todas progênies originadas dos mosquitos capturados em Rondônia corresponderam a A. konderi, enquanto que cerca de 85,0% das progênies do Acre eram A. oswaldoi s.s.. Estas progênies F1 de A. oswaldoi s.s., A. konderi e A. darlingi foram alimentadas simultaneamente com sangue infectado com P. vivax. Estes mosquitos foram dissecados 10-12 dias após infecção e examinados para verificação da presença de oocistos e esporozoítos. Tanto A. oswaldoi s.s. como A. konderi apresentaram oocistos nos tratos digestivos, entretanto, a porcentagem de tratos digestivos positivos para oocistos foi maior em A. oswaldoi s.s. (13,8%) do que em A. konderi (3,3%). Esporozoítos foram encontrados somente nas glândulas salivares de A. oswaldoi s.s., com 6,9% de positividade. As taxas de infecção nos controles A. darlingi foram de 22,5% a 30,0%, para ambos oocistos e esporozoítos. Estes resultados indicam que A. oswaldoi s.s. pode transmitir P. vivax e sugerem que esta espécie é mais susceptível que A. konderi. Embora A. oswaldoi s.s. seja uma espécie exofílica e zoofílica, este anofelino pode estar envolvido na transmissão da malária humana como parece estar ocorrendo no Estado do Acre. / Previous results have suggested that biological differences could exist between specimens of Anopheles oswaldoi captured in the State of Acre and those from the State of Rondônia, Brazil. This species has been appointed as an important malaria vector in localities of Peru and Acre. However, in Rondônia, only a very low percentage of A. oswaldoi fed on malaria patients developed salivary gland infections. In addition, it was suspected that specimens identified as A. oswaldoi captured in open clearings in Costa Marques, Rondônia, were actually A. konderi, and that A. oswaldoi sensu stricto would be restricted to forested areas. These data, together with the difficulties found to identify anophelines of the Oswaldoi group based on morphologic criteria suggest that specimens of A. oswaldoi are members of a complex of cryptic species. The distinction of cryptic species of insects is of critical importance, since different members in a Complex could exhibit differences in ecology, vectorial capacity and response to control measures. DNA sequence analysis and particularly that of the ITS2 region of the rDNA cistron has provided diagnostic characters in some groups of cryptic species becoming a general tool for taxonomic and phylogenetic studies. The first part of the present study was undertaken to determine the extent of differences over the ITS2 sequence of specimens of A. oswaldoi s.l. captured in several localities of South America. The ITS2 of these anophelines were amplified using conserved primers of the 5.8S and 28S regions, cloned and sequenced. The lengths of ITS2 of all mosquitoes captured were about 350 nucleotides, with about 53% GC content. Analysis of the alignment of the sequences, which showed that the similarity varied between 87% and 100%, and analysis of a neighbor-joining tree produced with p-distance using the ITS2 sequences, separated these specimens in four groups. One of them is probably related to A. oswaldoi sensu stricto, and another one can possibly be related to A. konderi. The other two groups may correspond to species the morphologycal identification of which remains to be clarified in the A. oswaldoi complex. These data are evidences that specimens of A. oswaldoi are included in a complex of cryptic species and that the DNA identification could solve some taxonomic questions. A. konderi has been currently considered to be a synonym of A. oswaldoi. Although adults and immature stages of both these anopheline species have identical morphological characters, features of the male genitalia can distinguish these taxa. The second part of this study was conducted in order to compare the susceptibility of A. oswaldoi s.s. and of A. konderi to infection by Plasmodium vivax. The susceptibility was based on the proportion of mosquitoes with oocysts and sporozoites. Anophelines were captured in the State of Acre and Rondônia and used to obtain F1 progenies. After emergency of adults, male genitalia of mosquitoes of each family were dissected. All families originated from mosquitoes captured in Rondônia corresponded to A. konderi, while about 85.0% of the families from Acre were A. oswaldoi s.s.. F1 progeny of field-captured A. oswaldoi s.s., A. konderi and A. darlingi were fed simultaneously on P. vivax infected blood. Mosquitoes were dissected on day 10-12 after infection and examined for the presence of oocysts and sporozoites. Both A. oswaldoi s.s. and A. konderi developed oocysts in midguts, however, the percentage of oocyst-positives in A. oswaldoi s.s. (13.8%) was higher than in A. konderi (3.3%), and only A. oswaldoi s.s. developed salivary infection with sporozoites (6.9% of positivity). Infection rates in A. darlingi ranged from 22.5% to 30.0% for both oocysts and sporozoites. These results indicate that A. oswaldoi s.s. can transmit P. vivax and suggest that it is more susceptible than A. konderi. Although A. oswaldoi s.s. is an exophilic and zoophilic species, it may be involved in human malaria transmission as it seems to be occuring in the State of Acre.

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