Transcription of the ribosomal genes accounts for the majority of transcription in the cell due to the constant high demand for ribosomes. The number of proteins synthesized correlates with an effective ribosomal biogenesis, which is regulated by cell growth and proliferation. In the work presented in this thesis, we have investigated the ribosomal RNA genes 45S and 5S rRNA, which are transcribed by RNA Pol I and RNA Pol III, respectively. The focus of this work is the chromatin remodelling complex B-WICH, which is composed of WSTF, the ATPase SNF2h and NM1. We have studied in particular its role in ribosomal gene transcription. We showed in Study I that B-WICH is required to set the stage at rRNA gene promoters by remodelling the chromatin into an open, transcriptionally active configuration. This results in the binding of histone acetyl transferases to the genes and subsequent histone acetylation, which is needed for ribosomal gene activation. Study II investigated the role of B-WICH in transcription mediated by RNA polymerase III. We showed that B-WICH is essential to create an accessible chromatin atmosphere at 5S rRNA genes, which is compatible with the results obtained in Study 1. In this case, however, B-WICH operates as a licensing factor for c-Myc and the Myc/Max/Mxd network. Study III confirmed the importance and the function of the B-WICH complex as an activator of ribosomal genes. We demonstrated that B-WICH is important for the remodelling of the rDNA chromatin into an active, competent state in response to extracellular stimuli, and that the association of the B-WICH complex to the rRNA gene promoter is regulated by proliferative and metabolic changes in cells. The work presented in this thesis has confirmed that the B-WICH complex is an important regulator and activator of Pol I and Pol III transcription. We conclude that B-WICH is essential for remodelling the rDNA chromatin into a transcriptionally active state, as required for efficient ribosomal gene transcription. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: Manuscript.</p><p> </p>

Anuros há muito tempo são conhecidos como portadores de tripanossomas em todo o mundo. Esses tripanossomas são transmitidos por sanguessugas e insetos hematófagos. Nenhum levantamento foi feito na América do Sul. A taxonomia tradicional resulta em identificações não confiáveis desses tripanossomas, a diversidade genética foi pouco investigada e o a filogenético é pouco conhecida. Neste estudo mostramos grande prevalência e marcada diversidade molecular e morfológica entre tripanossomas de várias espécies de anuros da Amazônia, Floresta Atlântica e Pantanal. O relacionamento filogenético inferido entre tripanossomas do Brasil, América do Norte, Europa e África revelou 5 clados de isolados de anuros. Um deles contem isolados de anuros e flebotomíneos, sugerindo que esses insetos possam ser vetores de tripanossomas de anuros. Em geral, os dados mostraram certo grau de associação entre clados de isolados e filogeografia de anuros e sugeriram que trocas de hospedeiros e adaptação ecológica desempenharam um papel importante na história evolutiva desses tripanossomas. / Amphibians of the order Anura have long been known to be infected with trypanosomes worldwide, infecting frogs and toads and transmitted by leeches and insects. No surveys were carried out in South American anurans. Traditional taxonomy renders unreliable identification of these trypanosomes, genetic diversity was poorly investigated and phylogenetic relationships are far from be understood. In this study, we showed high prevalence and marked morphological and molecular diversity among trypanosomes from several species from Amazonia, Atlantic Forest and Pantanal. Phylogenetic relationships inferred among trypanosomes from Brazil, North America, Europe and Africa disclosed 5 major clades of anuran isolates. One clade contains anuran and sand fly isolates, suggesting that these insects can be vectors of anuran trypanosomes. In general, data showed a certain degree of association between clades of isolates and anuran phylogeography and suggested that host switching and ecological fitting also play a role in the evolutionary history of these trypanosomes.

Made available in DSpace on 2017-07-21T19:59:46Z (GMT). No. of bitstreams: 1 Thais Dulz.pdf: 2466351 bytes, checksum: 208f40d4e86e959ef9f685c1e5a3cac5 (MD5) Previous issue date: 2015-02-27 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The Characidae is considered the largest family and most complex among the Characiforms. The Astyanax genus is one of the most specious between the characids, comprising 142 valid species distributed in the Neotropical Region. Many of these species are restricted to certain river basins. The fish fauna of the Iguaçu River basin is characterized by having a high endemism, possibly because its isolation of the Paraná River basin, occurred about 22 million years, caused by the emergence of the Iguaçu Falls. Were recently described new species of this genus in the Rio Iguaçu, however its systematic position opposite the other Astyanax is still unknown, reinforcing necessary studies aimed at understanding their diversity and their evolutionary relationships. Therefore, this research aimed was to perform the karyotype characterization of the Astyanax species from the middle Rio Iguaçu: Astyanax altiparanae, A. bifasciatus, A. dissimilis, A. minor, A. ribeirae and A. serratus, using cytogenetics as a tool, in order to contribute to a more consistent scenario about the evolution of this group. The analysis were based on conventional Giemsa staining techniques, detection of nucleolar organizing regions by silver nitrate (Ag-NORs), C-banding and fluorescence in situ hybridization (FISH) to ribosomal genes 18S e 5S probes. All species presented 2n = 50 chromosomes, confirming the conservatism to the genus. The karyotypes comprise all chromosomal types and prevalence of chromosomes with two arms, mainly on the metacentric type except for A. ribeirae with the karyotype formula containing high number of acrocentrics. Despite the diploid number remain constant in these species; there were variations in karyotype formula and consequently the fundamental number, ranging from 66 to 92. The C-banding showed differences in the distribution of heterochromatic blocks between species, occurring polymorphism in A. serratus, which was also observed in other studies involving Astyanax sp. D (A. serratus). The six species showed the presence of NORs located in the telomeric region of the short arm of a chromosome pair of the type subtelocentric, characteristic conserved for the genus. Was identified single NORs in A. altiparanae and A. dissimilis, although the most common being multiple NORs, in almost all cases confirmed by 18S rDNA. All species showed 5S rDNA located in the interstitial segments of the chromosomes. Have been observed simple rDNA 5S in A. altiparanae, A. minor and A. serratus, and multiple in A. bifasciatus, A. dissimilis and A. ribeirae. Highlighted A. ribeirae by presenting a high number of rDNA 18S and 5S sites, which proved to be sintenics and co-located. In addition, this species is considered endemic for Ribeira de Iguape basin; however, was first found in the Iguaçu basin, strengthening fauna sharing hypothesis between rivers of the plateau and coastal regions in southern Brazil. The karyotype diversity evident for all species is mainly due to non-Robertsonian rearrangements. This study presented the first cytogenetic data for the genus Astyanax occurring in the Middle Rio Iguaçu region. / A família Characidae é considerada a maior e mais complexa entre os Characiformes. O gênero Astyanax é um dos mais especiosos entre os caracídeos, compreendendo 142 espécies válidas distribuídas na região Neotropical. Muitas destas espécies encontram-se restritas a determinadas bacias hidrográficas. A ictiofauna da bacia do Rio Iguaçu caracteriza-se por apresentar um elevado grau de endemismo, possivelmente devido o seu isolamento da bacia do Rio Paraná, ocorrido há cerca de 22 milhões de anos, causado pelo surgimento das Cataratas do Iguaçu. Recentemente, foram descritas novas espécies deste gênero no Rio Iguaçu, entretanto seu posicionamento sistemático frente aos demais Astyanax ainda é desconhecido, fazendo-se necessários estudos que visem à compreensão de sua diversidade e de suas relações evolutivas. Sendo assim, objetivou-se realizar a caracterização cariotípica de espécies do gênero Astyanax provenientes do médio Rio Iguaçu: Astyanax altiparanae, A. bifasciatus, A. dissimilis, A. minor, A. ribeirae e A. serratus, utilizando como ferramenta a citogenética, a fim de contribuir com um cenário mais consistente acerca da evolução deste grupo. Utilizou-se técnicas baseadas na coloração convencional por Giemsa, detecção de regiões organizadoras de nucléolo por nitrato de prata (Ag-RONs), bandamento C e hibridação in situ fluorescência (FISH) com sondas de genes ribossomais 18S e 5S. Todas as espécies estudadas apresentaram 2n = 50 cromossomos, confirmando o conservadorismo para o gênero. Observaram-se cariótipos compostos por todos os tipos cromossômicos e predominância de cromossomos com dois braços, principalmente do tipo metacêntrico, exceto para A. ribeirae com fórmula cariotípica composta por elevado número de acrocêntricos. Apesar do número diploide se manter constante nestas espécies, houve variações na fórmula cariotípica e, consequentemente no número fundamental, variando de 66 até 92. O bandamento C evidenciou variações na distribuição dos blocos heterocromáticos entre as espécies, com ocorrência de polimorfismo em A. serratus, o que também foi observado em outros estudos envolvendo Astyanax sp. D (A. serratus). As seis espécies estudadas mostraram a presença de RONs localizadas na região telomérica do braço curto de um par cromossômico do tipo subtelocêntrico, característica conservada para o gênero. Ocorreram RONs simples em A. altiparanae e A. dissimilis, as demais espécies possuem RONs múltiplas, em quase todos os casos confirmadas pela FISH com rDNA 18S. Todas as espécies evidenciaram rDNA 5S localizados nos segmentos intersticiais dos cromossomos. Foram observados rDNA 5S em um único par em A. altiparanae, A. minor e A. serratus, e múltiplos em A. bifasciatus, A. dissimilis e A. ribeirae. Destaca-se A. ribeirae por apresentar um elevado número de sítios rDNA 18S e 5S, os quais mostraram-se sintênicos e co-localizados. Além disso, esta espécie é considerada endêmica para bacia do Ribeira de Iguape; no entanto, foi encontrada pela primeira vez na bacia do Iguaçu, reforçando a hipótese de compartilhamento de fauna entre rios do planalto cristalino e regiões costeiras no sul do Brasil. A diversidade cariotípica evidenciada para todas as espécies devem-se principalmente a rearranjos não Robertsoniano. Neste estudo foram apresentados os primeiros dados citogenéticos para o gênero Astyanax ocorrentes na região do médio Rio Iguaçu.

Cell growth and proliferation are processes in the cell that must be tightly regulated. Transcription of ribosomal RNA and ribosomal biogenesis are directly linked to cell growth and proliferation, since the ribosomal RNA encodes for the majority of transcription in a cell and ribosomal biogenesis influences directly the number of proteins that are synthesized. In the work presented in this thesis, we have investigated the ribosomal RNA genes, namely the ribosomal DNA genes and the 5S rRNA genes, and their transcriptional regulation. One protein complex that is involved in RNA polymerase I and III transcription is the chromatin remodelling complex B‑WICH (WSTF, SNF2h, NM1). RNA polymerase I transcribes the rDNA gene, while RNA polymerase III transcribes the 5S rRNA gene, among others. In Study I we determined the mechanism by which B‑WICH is involved in regulating RNA polymerase I transcription. B‑WICH is associated with the rDNA gene and was able to create a more open chromatin structure, thereby facilitating the binding of HATs and the subsequent histone acetylation. This resulted in a more active transcription of the ribosomal DNA gene. In Study II we wanted to specify the role of NM1 in RNA polymerase I transcription. We found that NM1 is not capable of remodelling chromatin in the same way as B‑WICH, but we demonstrated also that NM1 is needed for active RNA polymerase I transcription and is able to attract the HAT PCAF. In Study III we investigated the intergenic part of the ribosomal DNA gene. We detected non-coding RNAs transcribed from the intergenic region that are transcribed by different RNA polymerases and that are regulated differently in different stress situations. Furthermore, these ncRNAs are distributed at different locations in the cell, suggesting that they have different functions. In Study IV we showed the involvement of B‑WICH in RNA Pol III transcription and, as we previously had shown in Study I, that B‑WICH is able to create a more open chromatin structure, in this case by acting as a licensing factor for c-Myc and the Myc/Max/Mxd network. Taken together, we have revealed the mechanism by which the B‑WICH complex is able to regulate RNA Pol I and Pol III transcription and we have determined the role of NM1 in the B‑WICH complex. We conclude that B‑WICH is an important factor in the regulation of cell growth and proliferation. Furthermore, we found that the intergenic spacer of the rDNA gene is actively transcribed, producing ncRNAs. Different cellular locations suggest that the ncRNAs have different functions. / <p>At the time of the doctoral defence the following papers were unpublished and had a status as follows: Paper 2: Manuscript; Paper 3: Manuscript</p>

Anuros há muito tempo são conhecidos como portadores de tripanossomas em todo o mundo. Esses tripanossomas são transmitidos por sanguessugas e insetos hematófagos. Nenhum levantamento foi feito na América do Sul. A taxonomia tradicional resulta em identificações não confiáveis desses tripanossomas, a diversidade genética foi pouco investigada e o a filogenético é pouco conhecida. Neste estudo mostramos grande prevalência e marcada diversidade molecular e morfológica entre tripanossomas de várias espécies de anuros da Amazônia, Floresta Atlântica e Pantanal. O relacionamento filogenético inferido entre tripanossomas do Brasil, América do Norte, Europa e África revelou 5 clados de isolados de anuros. Um deles contem isolados de anuros e flebotomíneos, sugerindo que esses insetos possam ser vetores de tripanossomas de anuros. Em geral, os dados mostraram certo grau de associação entre clados de isolados e filogeografia de anuros e sugeriram que trocas de hospedeiros e adaptação ecológica desempenharam um papel importante na história evolutiva desses tripanossomas. / Amphibians of the order Anura have long been known to be infected with trypanosomes worldwide, infecting frogs and toads and transmitted by leeches and insects. No surveys were carried out in South American anurans. Traditional taxonomy renders unreliable identification of these trypanosomes, genetic diversity was poorly investigated and phylogenetic relationships are far from be understood. In this study, we showed high prevalence and marked morphological and molecular diversity among trypanosomes from several species from Amazonia, Atlantic Forest and Pantanal. Phylogenetic relationships inferred among trypanosomes from Brazil, North America, Europe and Africa disclosed 5 major clades of anuran isolates. One clade contains anuran and sand fly isolates, suggesting that these insects can be vectors of anuran trypanosomes. In general, data showed a certain degree of association between clades of isolates and anuran phylogeography and suggested that host switching and ecological fitting also play a role in the evolutionary history of these trypanosomes.

A cana-de-açúcar é uma cultura importante no contexto agrário brasileiro, sobretudo com relação a manutenção e sustentabilidade dos agroecossistemas e da biodiversidade do solo. As comunidades microbianas associadas à canade- açúcar são participantes da manutenção dos ciclos biogeoquímicos, podendo ter sua estrutura e diversidade alteradas por mudanças no manejo da cultura e nas condições climáticas. Esse estudo teve como objetivo avaliar a diversidade microbiana associada à rizosfera de diferentes genótipos de cana-de-açúcar e empregar a metodologia de Stable Isotope Probing (DNA-SIP) para se avaliar a estrutura dos grupos responsivos a este ambiente. Para tanto, variedades de cana-de-açúcar foram selecionadas, extraindo-se o DNA total rizosférico e do bulk soil para análise por PCR-DGGE das regiões do gene 16S rDNA de bactérias, selecionando-se amostras representativas para o sequenciamento da região V6 do gene 16S rDNA através da plataforma Ion Torrent(TM). Os resultados demonstraram diferenças entre a diversidade das comunidades microbianas da rizosfera e do bulk soil, havendo a predominância dos grupos Actinobacteria, Proteobacteria e Acidobateria. Para o estudo da estrutura dos grupos responsivos na rizosfera, plantas da variedade RB86-7515 foram cultivadas sob duas concentrações de CO2 (350 e 700 ppm), realizando-se o enriquecimento com 13CO2, e posteriormente realizando a extração do DNA rizosférico para aplicação na técnica de DNA-SIP. A eficiência desta técnica foi avaliada por meio da técnica de PCR-DGGE para as regiões 16S rDNA de bactérias e ITS de fungos, onde foi verificado que após 48 horas já ocorre a incorporação de 13C pelas comunidades microbianas, havendo diferença entre os grupos que incorporaram o 13C. Diferenças foram também observadas para as distintas concentrações de CO2, indicando o DNA-SIP como uma poderosa ferramenta de estudos da ecologia das comunidades microbianas na rizosfera de cana-deaçúcar. / The sugarcane is an important crop in Brazilian agrarian context, especially in respect to maintenance and sustainability of agroecosystems and soil biodiversity. The microbial communities associated to sugarcane are involved biogeochemical cycles processes and it may have their structure and diversity changed due to crop management and climatic conditions. The aim of this study was to evaluate the microbial diversity associated to the rhizosphere of different sugarcane\'s genotypes and employ the Stable Isotope Probing tecnique (DNASIP) to evaluate the structure of the groups that are responding to this environment attributes. Therefore, some sugarcane varieties were selected and the total DNA in bulksoil and rhizosphere for analysis by PCR-DGGE of 16S rDNA gene regions of bacteria was extracted, selecting representative samples for sequencing the 16S rDNA gene of V6 region by Ion Torrent (TM) platform. The results showed differences between the diversity of microbial communities in the rhizosphere and bulk soil, with the predominance of Actinobacteria, Proteobacteria and Acidobateria groups. To study the structure of the responsive rhizosphere groups, the genotype RB86-7515 were grown under two CO2 concentrations (350 and 700 ppm), performing the 13CO2 enrichment. Afterwards, was performed the extraction of DNA for application of the SIP-rhizosphere DNA technique. The efficiency of this technique was assessed by PCR-DGGE over the regions of bacteria 16S rDNA and fungi ITS, which of these showed that occurs after 48 hours the incorporation of 13C by microbial communities, and it elucidate differences between the groups that incorporate the 13C. These differences were also observed for those different CO2 concentrations, indicating that the DNA-SIP is a powerful tool for studies of the ecology of microbial communities in the rhizosphere of sugarcane.

A cana-de-açúcar é uma cultura importante no contexto agrário brasileiro, sobretudo com relação a manutenção e sustentabilidade dos agroecossistemas e da biodiversidade do solo. As comunidades microbianas associadas à canade- açúcar são participantes da manutenção dos ciclos biogeoquímicos, podendo ter sua estrutura e diversidade alteradas por mudanças no manejo da cultura e nas condições climáticas. Esse estudo teve como objetivo avaliar a diversidade microbiana associada à rizosfera de diferentes genótipos de cana-de-açúcar e empregar a metodologia de Stable Isotope Probing (DNA-SIP) para se avaliar a estrutura dos grupos responsivos a este ambiente. Para tanto, variedades de cana-de-açúcar foram selecionadas, extraindo-se o DNA total rizosférico e do bulk soil para análise por PCR-DGGE das regiões do gene 16S rDNA de bactérias, selecionando-se amostras representativas para o sequenciamento da região V6 do gene 16S rDNA através da plataforma Ion Torrent(TM). Os resultados demonstraram diferenças entre a diversidade das comunidades microbianas da rizosfera e do bulk soil, havendo a predominância dos grupos Actinobacteria, Proteobacteria e Acidobateria. Para o estudo da estrutura dos grupos responsivos na rizosfera, plantas da variedade RB86-7515 foram cultivadas sob duas concentrações de CO2 (350 e 700 ppm), realizando-se o enriquecimento com 13CO2, e posteriormente realizando a extração do DNA rizosférico para aplicação na técnica de DNA-SIP. A eficiência desta técnica foi avaliada por meio da técnica de PCR-DGGE para as regiões 16S rDNA de bactérias e ITS de fungos, onde foi verificado que após 48 horas já ocorre a incorporação de 13C pelas comunidades microbianas, havendo diferença entre os grupos que incorporaram o 13C. Diferenças foram também observadas para as distintas concentrações de CO2, indicando o DNA-SIP como uma poderosa ferramenta de estudos da ecologia das comunidades microbianas na rizosfera de cana-deaçúcar. / The sugarcane is an important crop in Brazilian agrarian context, especially in respect to maintenance and sustainability of agroecosystems and soil biodiversity. The microbial communities associated to sugarcane are involved biogeochemical cycles processes and it may have their structure and diversity changed due to crop management and climatic conditions. The aim of this study was to evaluate the microbial diversity associated to the rhizosphere of different sugarcane\'s genotypes and employ the Stable Isotope Probing tecnique (DNASIP) to evaluate the structure of the groups that are responding to this environment attributes. Therefore, some sugarcane varieties were selected and the total DNA in bulksoil and rhizosphere for analysis by PCR-DGGE of 16S rDNA gene regions of bacteria was extracted, selecting representative samples for sequencing the 16S rDNA gene of V6 region by Ion Torrent (TM) platform. The results showed differences between the diversity of microbial communities in the rhizosphere and bulk soil, with the predominance of Actinobacteria, Proteobacteria and Acidobateria groups. To study the structure of the responsive rhizosphere groups, the genotype RB86-7515 were grown under two CO2 concentrations (350 and 700 ppm), performing the 13CO2 enrichment. Afterwards, was performed the extraction of DNA for application of the SIP-rhizosphere DNA technique. The efficiency of this technique was assessed by PCR-DGGE over the regions of bacteria 16S rDNA and fungi ITS, which of these showed that occurs after 48 hours the incorporation of 13C by microbial communities, and it elucidate differences between the groups that incorporate the 13C. These differences were also observed for those different CO2 concentrations, indicating that the DNA-SIP is a powerful tool for studies of the ecology of microbial communities in the rhizosphere of sugarcane.