In Saccharomyces cerevisiae, the telomeres and rDNA repeats are repetitive silent chromatin domains that are tightly regulated to maintain silencing and genome stability. Disruption of the Cohibin complex, which maintains rDNA silencing and stability, also abrogates telomere localization and silencing. Cohibin-deficient cells have decreased Sir2 localization at telomeres, and restoring telomeric Sir2 concentrations rescues the telomeric defects observed in Cohibin-deficient cells. Genetic and molecular interactions suggest that Cohibin clusters telomeres to the nuclear envelope by binding inner nuclear membrane proteins. Futhermore, telomeric and rDNA sequences can form G-quadruplex structures. G-quadruplexes are non-canonical DNA structures that have been linked to processes affecting chromosome stability. Disruption of the G-quadruplex stabilizing protein Stm1, which also interacts with Cohibin, increases rDNA stability without affecting silent chromatin formation. In all, our findings have led to the discovery of new processes involved in the maintenance of repetitive silent chromatin domains that may be conserved across eukaryotes.

As dermatofitoses apresentam alta prevalência na população em geral, sendo Trichophyton interdigitale (Trichophyton mentagrophytes) a segunda espécie mais frequentemente relatada como causadora de infecção em humanos. O objetivo do trabalho foi determinar a identidade genética, o perfil enzimático e de assimilação de açúcares e a suscetibilidade a antifúngicos de isolados clínicos do gênero Trichopyton. Os isolados foram avaliados por de ensaios enzimáticos, assimilação de fontes de carbono e da atividade antifúngica in vitro. A caracterização genotípica foi realizada através da amplificação e do sequenciamento da região do gene que codifica a região interna do gene DNA ribossomal dos isolados testados. No estudo, todos os isolados secretaram DNase, mas nenhum deles foi capaz de secretar fosfolipase e proteinase. Por outro lado, urease e lipase foram produzidas pela maioria dos isolados testados. Entre os antifúngicos avaliados, a terbinafina e o itraconazol demonstraram melhores resultados de sensibilidade, enquanto o fluconazol apresentou baixa atividade para as amostras. A anfotericina B, apesar de menos eficaz que a terbinafina e o itraconazol, também demonstrou resultados satisfatórios. A combinação entre os antifúngicos tioconazol e terbinafina demonstrou ter atividade antagônica em todos os isolados. Todos os dermatófitos testados assimilaram inulina, sorbitol, manose, glicose e trealose. Os demais carboidratos tiveram assimilação variável entre os isolados. Genotipicamente, todos os isolados do estudo foram identificados como pertencentes à espécie T. interdigitale. Os ensaios enzimáticos e de assimilação de carboidratos apresentaram resultados variáveis entre os isolados testados, inferindo variação intraespecífica entre as amostras de T. interdigitale. O perfil de sensibilidade aos antimicóticos testados seguiu o padrão de resultados observado em estudos anteriores. A identificação genotípica através da amplificação e do sequenciamento da região ITS permitiu garantir a identidade entre os isolados testados. / The prevalence of dermatophytosis among population is high, and Trichophyton interdigitale (Trichophyton mentagrophytes) is the second most frequently reported species to cause infection in humans. This study objective was to evaluate the genetic identity, the enzymatic profile and the assimilation of carbon sources, as well as the antifungal susceptibility of clinical isolates of Trichophyton sp. The isolates were analyzed phenotypically by enzymatic assays, assimilation of carbon sources and antifungal activity in vitro. The genotypic characterization was performed by amplification and sequencing the gene region encoding the internal region of the DNA ribosomal gene of the isolates tested. In our study, all isolates secreted DNase, while none of them was capable of secreting phospholipase, keratinase and proteinase. On the other hand, lipase and urease were secreted by most of the isolates. Of the antifungal agents tested, the best results in terms of sensitivity were found with terbinafine and itraconazole, while the antifungal activity of fluconazole was found to be weak. Amphotericin B, although less effective than terbinafine and itraconazole, also yielded satisfactory results. Evaluation of the drug combination of tioconazole and terbinafine revealed an antagonistic effect for all tested isolates. All isolates assimilated inulin, sorbitol, glucose, trehalose and mannose. Arabinose, dulcitol, galactose and xylose were assimilated by some of the isolates, whereas the most isolates assimilated cellobiose, dulcitol, erytritol and mannitol. Genotypically, all isolates in the study were identified as belonging to Trichophyton interdigitale species. In general, enzyme assays and assimilation of carbohydrates showed variable results among the isolates tested, inferring intraspecific variation between Trichophyton interdigitale samples. The sensitivity profile of the antifungal agents tested in this study was similar to results obtained in previous studies. The genotypic identification, by amplification and sequencing of the ITS region, has ensured the identity of the isolates tested.

As dermatofitoses apresentam alta prevalência na população em geral, sendo Trichophyton interdigitale (Trichophyton mentagrophytes) a segunda espécie mais frequentemente relatada como causadora de infecção em humanos. O objetivo do trabalho foi determinar a identidade genética, o perfil enzimático e de assimilação de açúcares e a suscetibilidade a antifúngicos de isolados clínicos do gênero Trichopyton. Os isolados foram avaliados por de ensaios enzimáticos, assimilação de fontes de carbono e da atividade antifúngica in vitro. A caracterização genotípica foi realizada através da amplificação e do sequenciamento da região do gene que codifica a região interna do gene DNA ribossomal dos isolados testados. No estudo, todos os isolados secretaram DNase, mas nenhum deles foi capaz de secretar fosfolipase e proteinase. Por outro lado, urease e lipase foram produzidas pela maioria dos isolados testados. Entre os antifúngicos avaliados, a terbinafina e o itraconazol demonstraram melhores resultados de sensibilidade, enquanto o fluconazol apresentou baixa atividade para as amostras. A anfotericina B, apesar de menos eficaz que a terbinafina e o itraconazol, também demonstrou resultados satisfatórios. A combinação entre os antifúngicos tioconazol e terbinafina demonstrou ter atividade antagônica em todos os isolados. Todos os dermatófitos testados assimilaram inulina, sorbitol, manose, glicose e trealose. Os demais carboidratos tiveram assimilação variável entre os isolados. Genotipicamente, todos os isolados do estudo foram identificados como pertencentes à espécie T. interdigitale. Os ensaios enzimáticos e de assimilação de carboidratos apresentaram resultados variáveis entre os isolados testados, inferindo variação intraespecífica entre as amostras de T. interdigitale. O perfil de sensibilidade aos antimicóticos testados seguiu o padrão de resultados observado em estudos anteriores. A identificação genotípica através da amplificação e do sequenciamento da região ITS permitiu garantir a identidade entre os isolados testados. / The prevalence of dermatophytosis among population is high, and Trichophyton interdigitale (Trichophyton mentagrophytes) is the second most frequently reported species to cause infection in humans. This study objective was to evaluate the genetic identity, the enzymatic profile and the assimilation of carbon sources, as well as the antifungal susceptibility of clinical isolates of Trichophyton sp. The isolates were analyzed phenotypically by enzymatic assays, assimilation of carbon sources and antifungal activity in vitro. The genotypic characterization was performed by amplification and sequencing the gene region encoding the internal region of the DNA ribosomal gene of the isolates tested. In our study, all isolates secreted DNase, while none of them was capable of secreting phospholipase, keratinase and proteinase. On the other hand, lipase and urease were secreted by most of the isolates. Of the antifungal agents tested, the best results in terms of sensitivity were found with terbinafine and itraconazole, while the antifungal activity of fluconazole was found to be weak. Amphotericin B, although less effective than terbinafine and itraconazole, also yielded satisfactory results. Evaluation of the drug combination of tioconazole and terbinafine revealed an antagonistic effect for all tested isolates. All isolates assimilated inulin, sorbitol, glucose, trehalose and mannose. Arabinose, dulcitol, galactose and xylose were assimilated by some of the isolates, whereas the most isolates assimilated cellobiose, dulcitol, erytritol and mannitol. Genotypically, all isolates in the study were identified as belonging to Trichophyton interdigitale species. In general, enzyme assays and assimilation of carbohydrates showed variable results among the isolates tested, inferring intraspecific variation between Trichophyton interdigitale samples. The sensitivity profile of the antifungal agents tested in this study was similar to results obtained in previous studies. The genotypic identification, by amplification and sequencing of the ITS region, has ensured the identity of the isolates tested.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / A pesquisa foi desenvolvida no perÃodo de janeiro de 2003 a marÃo de 2006, nos Departamentos de Zootecnia, e Biologia da Universidade Federal do CearÃ, localizada no municÃpio de Fortaleza, estado do CearÃ. As amostras de abelhas foram coletadas nos estados do CearÃ, Piauà e GoiÃs. O objetivo desta tese foi estudar a diversidade genÃtica de populaÃÃes da abelha sem ferrÃo Melipona quinquefasciata, ocorrendo naturalmente na Chapada do Araripe (Sul do CearÃ), na Chapada da Ibiapaba (Oeste do CearÃ), na cidade de Canto do BuritÃ-PI, e na cidade de LuziÃnia-GO (Centro-Oeste do Brasil), visando contribuir para a correta classificaÃÃo taxonÃmica da populaÃÃo nordestina e dar subsÃdios iniciais para trabalhos de criaÃÃo racional desta espÃcie, em colmÃias para produÃÃo de mel, motivando entÃo que a atual aÃÃo extrativista e predatÃria, reverta-se numa atividade zootÃcnica produtiva e ecologicamente sustentÃvel. As regiÃes do DNA ribossÃmico nuclear 18S e ITS-1 parcial foram extraÃdas e seqÃenciadas, podendo-se verificar os seguintes aspectos: ComposiÃÃo nucleotÃdica, matrizes de distÃncia genÃtica, mÃltiplos alinhamentos e cladogramas. Os resultados mostraram alto grau de similaridade entre as amostras, 0,008 para regiÃo 18S e 0,015 para o ITS-1 parcial. O tamanho das seqÃÃncias correspondente à regiÃo 18S, foram de 1823 a 1869, do ITS-1 491 a 572. O cladograma gerado para o 18S, apresentou um Ãnico clado, porÃm, o ITS-1 quando acrescido de amostras externas (M. quadrifasciata, M subnitida, M scutellaris, M. mandaÃaia), derivou-se em trÃs grupos, refletindo os subgÃneros descritos pela taxonomia morfolÃgica. A distÃncia entre as Ãreas nÃo se correlacionou significativamente com a dissimilaridade das abelhas para o 18S, porÃm houve correlaÃÃo com o ITS-1 parcial, que agregou a amostra oriunda do estado do PiauÃ. Conclui-se (i) que as abelhas amostradas nos estados do Cearà e Piauà sÃo indistinguÃveis em termos moleculares das abelhas do estado de GoiÃs, sugerindo tratar-se da mesma espÃcie, embora apresentando algum nÃvel de variabilidade entre as populaÃÃes; (ii) os resultados encontrados refletem a taxonomia por dados morfolÃgicos, para a espÃcie Melipona quinquefasciata; (iii) o distanciamento geogrÃfico sugeriu algum grau de alteraÃÃo no genoma das abelhas que nidificam nos trÃs estados estudados; (iv) a regiÃo ITS-1 parcial do DNA ribossÃmico nuclear, mesmo em pequenas amostragens de abelhas, pode ajudar a resolver dÃvidas taxonÃmicas ao nÃvel de subgÃneros, em Melipona / The research was carried out from January 2003 to March 2006, at the Department of Animal Science and Department of Biology in the Federal University of CearÃ, in Fortaleza, CearÃ. Bee samples were collected in the states of CearÃ, Piauà and GoiÃs, Brazil. The objective of this work was to study the genetic variability of populations of the stingless bee Melipona quinquefasciata, which occurs naturally in the plateau of Araripe (South of CearÃ), in the plateau of Ibiapaba (West of CearÃ), in Canto do BuritÃ, Piauà and LuziÃnia-GoiÃs (Center-Western, Brazil). Our aims were to contribute to the correct taxonomic classification of the Northeastern population and to give initial support to future work on the rational breeding of this species. Meliponiculture in rational hives for honey production will stimulate the change of the present predatory actions into a productive bee rearing activity which is ecologically sustainable. The regions of nuclear ribosomal DNA 18S and partial ITS-1 were extracted and sequenced and the following aspects were determined: nucleotid composition, matrixes of genetic distances, multiple alignments and cladograms. Results showed a high degree of similarity among the samples: 0,008 to region 18S, and 0,015 to partial ITS-1. The sequencesâ size found to region 18S varied: from 1823 to 1869, and to ITS-1 from 491 to 572. The cladogram made to 18S presented a single clade. However, when external samples (M. quadrifasciata, M. subnitida, M. scutellaris, M. mandaÃaia), were added to ITS-1, three groups were formed reflecting the described subgenus by the morphological taxonomy. Distance among the localities where samples were colleted was not significantly correlated to the dissimilarity of the bees to 17 18S. Nevertheless, there was a correlation with partial ITS-1, which contained the Piauà sample. Our conclusions are: (i) the bee samples from Cearà and Piauà cannot be distinguished, in molecular terms, from the bee samples of GoiÃs, suggesting they are the same species, although presenting some level of variability among the populations; (ii) the results reflect the taxonomy based in morphological aspects for Melipona quinquefasciata; (iii) the geographical distance suggested some level of alteration in the genoma of bees which inhabit in the three studied regions; (iv) the region partial ITS-1 of the nuclear ribosomal DNA, even in small bee samples, can help to solve taxonomic doubts at the subgenus level, in Melipona

>Magister Scientiae - MSc / The class Actinobacteria consists of a heterogeneous group of filamentous, Gram-positive bacteria that colonise most terrestrial and aquatic environments. The industrial and biotechnological importance of the secondary metabolites produced by members of this class has propelled it into the forefront of metagenomics studies. The Ethiopian Rift Valley lakes are characterized by several physical extremes, making it a polyextremophilic environment and a possible untapped source of novel actinobacterial species. The aims of the current study were to identify and compare the eubacterial diversity between three geographically divided soda lakes within the ERV focusing on the actinobacterial subpopulation. This was done by means of a culture-dependent (classical culturing) and culture-independent (DGGE and ARDRA) approach. The results indicate that the eubacterial 16S rRNA gene libraries were similar in composition with a predominance of α-Proteobacteria and Firmicutes in all three lakes. Conversely, the actinobacterial 16S rRNA gene libraries were significantly different and could be used to distinguish between sites. The actinobacterial OTUs detected belonged to both the Rubrobacterales and Actinomycetales orders with members of the genus Arthrobacter being found in all three lakes. Geochemical properties were significantly different between the lakes, although more than one property attributed to the variance between community compositions. The diversity detected in the culture-based study differed significantly and all isolates belonged to the genus Streptomyces. Two novel strains were characterized by means of phylogenetic (16S rRNA gene sequence), physiological, morphological and biochemical analyses. Both novel isolates were capable of growing under "extreme" conditions- pH 12, 10% NaCl and 45°C. Partial enzyme characterization revealed that both strains produced xylanase enzymes that were active at pH 6.5 and 8.5 with an increase in activity up to 45°C. The results obtained revealed a previously undetected diversity of actinobacteria in the Ethiopian Rift Valley with a potentially novel subpopulation adapted to haloalkaline conditions. The low 16S rRNA sequence similarity of a substantial proportion of the libraries suggests that culture-based isolation may play a vital role in deciphering the community fingerprint. / The National Research Foundation and the Norwegian Research Council

Os peixes apresentam uma grande diversidade quanto a sua morfologia, seus habitats e também sua biologia. São encontrados em lagos, córregos, estuários e oceanos, constituindo assim mais de 50% do número total das espécies de vertebrados conhecidas atualmente. Essa fauna tem sido objeto de um número expressivo de estudos citogenéticos e moleculares, tendo-se já conhecimento não só das relações cromossômicas, mas também da sistemática de vários grupos. Essas pesquisas têm investigado não somente o número e fórmula cromossômica, mas também a presença de cromossomos sexuais diferenciados, presença de cromossomos supranumerários, padrões de distribuição da heterocromatina, localização das regiões organizadoras de nucléolo, padrões de bandamento de restrição e replicação, permitindo a localização de diferentes classes de DNAs repetitivos, bem como a identificação de homeologias cromossômicas que auxiliam a compreensão da evolução cariotípica dos grupos. Os estudos moleculares, por sua vez, têm se tornado cada vez mais importantes nesse grupo e têm fornecido dados fundamentais não só no que diz respeito à filogenia dos grupos, como também em relação a regiões repetitivas do DNA e sua importância no genoma. A união dessa área com a Citogenética tem permitido uma maior e melhor compreensão sobre os processos evolutivos associados às alterações de seqüências específicas do genoma visíveis tanto a níveis cromossômicos, quanto moleculares. O gênero Gymnotus (Teleostei: Gymnotiformes) inclui representantes com características biológicas peculiares, o que os torna objeto de estudo de diversas áreas da Biologia. Estudos sobre o gênero incluem sua caracterização cariotípica, estudo das regiões organizadoras de nucléolo (RONs) polimórficas, bem como estudos envolvendo marcadores moleculares, os quais conjuntamente com a Citogenética permitiram a análise de filogenética molecular, com inferência na evolução cromossômica, permitindo uma melhor compreensão das relações dentro do gênero. No presente trabalho foram levados a efeito estudos sobre as regiões heterocromáticas e os DNAs repetitivos desse grupo, para uma melhor compreensão da organização e localização dessas seqüências no genoma e a identificação de possíveis marcadores moleculares. Foram efetuados ainda, estudos envolvendo a evolução cariotípica das espécies G. carapo e G. sylvius, localização de genes ribossômicos e análise molecular do gene ribossômico 5S juntamente com seu espaçador não transcrito, propiciando uma melhor compreensão da evolução dessa família gênica em Gymnotus. / Fishes present a great diversity in relation to their morphology, habitat and biology. They are found in lakes, rivers, estuaries and oceans, comprising more than 50% of the total number of known vertebrates. Cytogenetic and molecular aspects of the fish fauna have been extensively studied, providing information about their chromosomal relationships and also about the systematic status of several groups. These researches have focused on the description of both chromosomal number and formula as well as the presence of differentiated sex chromosomes, occurrence of B-chromosomes, patterns of heterochromatin distribution, localization of nucleolar organizer regions, restriction or replication banding profiles allowing to locate distinct classes of repetitive DNAs and to identify chromosomal homeologies in order to understand the karyotypic evolution in distinct groups. On the other hand, molecular studies have become of utmost importance in this group, providing essential data about phylogeny of many groups and about repetitive DNA regions and their role in the genome. The union between this approach and cytogenetics has favored a better comprehension about the evolutionary processes associated with visible alterations in specific sequences within the genome at both chromosomal and molecular levels. The genus Gymnotus is composed of representatives with peculiar biological features, which turn them suitable for studies in a variety of biology approaches. Genetic studies in this genus comprise karyotype characterization, analysis of polymorphic NORs, besides studies of molecular markers that, coupled with cytogenetics, have fostered molecular phylogenetic analyzes with inferences on their chromosomal evolution, which have led to a better understanding about the interrelationships in the group. In the present work, we carried out studies about the heterochromatic regions and the repetitive DNAs in this group for a better comprehension about the organization and localization of these sequences in the genome and identification of potential molecular markers. Furthermore, studies related to the karyotype evolution in the species G. carapo and G. sylvius, location of ribosomal genes and molecular analysis of both 5S ribosomal gene and its non-transcribed spacer were performed to provide a better comprehension about the evolution of this gene family in Gymnotus.

Discoveries of new natural antibiotics are now relatively rare, therefore the construction of strains producing hybrid substances seems to be a very promising opportunity to gain new interesting biologically active compounds. This work is part of a larger project focused on the preparation of new biologically active substances derived from the antibiotic lincomycin. Lincomycin is composed of saccharide (MTL) and amino acid (propylhygric acid) moieties condensed by amide bond. Various modifications of amino acid moiety, especially of the side alkyl chain, are known to improve the antibiotic properties of final molecule. The bottleneck of biosynthesis of such modified compounds is the condensing enzyme NDL-synthetase, and especially its A-domain, which, similarly to nonribosomal peptide synthetases (NRPS), specifically recognizes and activates the amino acid precursor. In this work a set of degenerate primers for PCR searching of NRPS A-domains was proposed and the conditions of PCR reaction were optimized. In the first step a collection approximately 800 isolates of soil actinomycetes will serve as a source of genetic information for search of interesting NRPS A-domains, applicable for the construction of hybrid biosynthetic clusters. The isolates of this collection have been also characterized taxonomically...

Yes / Spinal muscular atrophy, the leading genetic cause of infant mortality, is a motor neuron disease caused by low levels of survival motor neuron (SMN) protein. SMN is a multifunctional protein that is implicated in numerous cytoplasmic and nuclear processes. Recently, increasing attention is being paid to the role of SMN in the maintenance of DNA integrity. DNA damage and genome instability have been linked to a range of neurodegenerative diseases. The ribosomal DNA (rDNA) represents a particularly unstable locus undergoing frequent breakage. Instability in rDNA has been associated with cancer, premature ageing syndromes, and a number of neurodegenerative disorders. Here, we report that SMN-deficient cells exhibit increased rDNA damage leading to impaired ribosomal RNA synthesis and translation. We also unravel an interaction between SMN and RNA polymerase I. Moreover, we uncover an spinal muscular atrophy motor neuron-specific deficiency of DDX21 protein, which is required for resolving R-loops in the nucleolus. Taken together, our findings suggest a new role of SMN in rDNA integrity.

No description available.

The following work demonstrates that paternal diet controls medically important metabolic phenotypes in offspring. We observe transmission of dietary information to the zygote via sperm, and this information evades reprogramming that typically occurs after fertilization. Cytosine methylation is implicated as a major contributor to meiotic epigenetic inheritance in several transgenerational phenomena. Our extensive characterization of the sperm methylome reveals that diet does not significantly affect methylation patterns. However, we find that extensive epivariability in the sperm epigenome makes important contributions to offspring variation. Importantly, coordinate cytosine methylation and copy number changes over the ribosomal DNA locus contributes to variation in offspring metabolism. Thus, rDNA variability acts independently of postadolescent paternal diet to influence offspring metabolism. Therefore, at least two mechanisms exist for epigenetically controlling offspring metabolism: stochastic epivariation and diet acting by an unknown mechanism to further modulate metabolism. This work argues that an offspring's phenotype can no longer be viewed solely as the result of genetic interactions with the developmental environment - the additional influences of paternal environment and inherited epigenetic variability must also be considered. These findings reveal novel contributions to metabolism that could revolutionize how we think about the risk factors for human health and disease.