Os micro-organismos que habitam o trato digestivo dos ruminantes têm uma profunda influência no desenvolvimento e funcionamento do animal hospedeiro. O rúmen abriga comunidades microbianas complexas dominadas por bactérias que participam de um processo eficiente de degradação dos materiais que compõem a parede celular vegetal. Por esta razão, o microbioma do rúmen representa uma fonte inexplorada de enzimas hidrolíticas com potencial aplicação na produção de combustíveis a partir da biomassa lignocelulósica. Nós usamos a plataforma Ion Torrent (PGM) para acessar o microbioma do rúmen de quatro animais da raça Santa Inês submetidos a uma dieta base. A fim de descrever a estrutura da comunidade microbiana no rúmen de ovinos e explorar o seu potencial como uma fonte de genes de degradação da biomassa, usamos a abordagem de sequenciamento do gene RNA ribossomal 16S (rRNA), utilizando Ion Tags, e a abordagem de sequenciamento metagenômico shotgun (DNA total), respectivamente. Além disso, medimos parâmetros químicos do ambiente do rúmen, relacionados a cada animal, incluindo pH, Degradabilidade da Matéria Orgânica (OMD), Produção total de Gás (GP) e Emissões de Metano (CH4), a fim de buscar correlações entre estas variáveis químicas e os grupos bacterianos. Em termos de estrutura da comunidade microbiana (bacteriana), encontramos Bacteroidetes como o filo dominante, seguido por Firmicutes, Proteobacteria e Actinobacteria. Alguns táxons foram correlacionados com os parâmetros químicos, como as famílias Corynebacteriaceae e Streptococcaceae, que foram positivamente correlacionadas com OMD; e a família Streptomycetaceae, negativamente correlacionada com GP e CH4. Algumas glicosil hidrolases conhecidas foram identificadas, como Endo-1,4-beta-glucanases, Beta-D-glicosídioglicohidrolases e outras foram designadas como putativas. Estas descobertas mostram interações ecológicas entre os grupos microbianos e funções importantes do rúmen, assim como o potencial do rúmen de ovinos para a descoberta de novas enzimas celulolíticas. / The microorganisms inhabiting the digestive tracts of ruminants have a profound influence on the host animal development and functioning. The rumen harbors complex microbial communities dominated by bacteria, which participate in an efficient process to digest plant cell wall materials. For this reason, the rumen microbiome represents an untapped source of hydrolytic enzymes with potential application for fuel production from lignocellulosic biomass. We used the Ion Torrent (PGM) platform to access the rumen microbiome of four animals of Santa Inês breed under a base diet. In order to describe the structure of the microbial community in the sheep rumen and explore its potential as a source of biomass-degrading genes, we used 16S ribosomal RNA (rRNA) Ion Tags sequencing approach and shotgun metagenomic sequencing (total DNA) approach, respectively. Furthermore, we measured rumen chemical environmental parameters related to each animal, including pH, Organic Matter Degradability (OMD), Total Gas Production (GP) and Methane emissions (CH4) in order to search for correlations between these chemical variables and bacterial groups. In terms of microbial (bacterial) community structure, we found Bacteroidetes as the dominant phylum in sheep rumen microbiome, followed by Proteobacteria, Firmicutes and Actinobacteria. Some taxa were correlated with the environmental parameters, like the Corynebacteriaceae and Streptococcaceae families, which was positively correlated with OMD, and the Streptomycetaceae family, negatively correlated with GP and CH4. Some known glycoside hydrolases were identified, such as Endo-1,4-betaglucanases, Beta-D-glucoside glucohydrolases and others were designated as putative ones. These findings show ecological interactions among microbial groups and important rumen functions, as well as the potential of the sheep rumen for the discovery of new cellulolytic enzymes.

La polarité apico-basale des cellules épithéliales est maintenue au niveau cellulaire par l’implication de plusieurs complexes protéiques, PAR, SCRIBBLE et CRUMBS, et par l’intégrité des jonctions serrées et adhérentes. La polarité apico-basale est essentielle au bon déroulement des processus de division, d’apoptose et de migration cellulaire, et de nombreuses études montrent que les perturbations de la polarité interviennent dans la progression tumorale. Notre travail a porté sur la recherche d’anomalies biologiques modifiant la polarité apico basale et l’adhésion cellulaire dans les carcinomes mammaires. Nous avons choisi d’étudier deux types spéciaux de carcinomes mammaires infiltrant, dont le phénotype suggère l’existence d’altérations spécifiques de ces processus biologiques: le type lobulaire (ILC) et le type micropapillaire (IMPC). Ces études ont été menées par des analyses combinées phénotypiques, transcriptomiques et génomiques comparatives, par rapport à un groupe de carcinomes mammaires sans autre spécificité (IC-NST).Les carcinomes lobulaires sont caractérisés par les altérations du complexe E cadhérine/ caténine et leur capacité de dissémination métastatique. Nous avons montré qu’ils présentent également une sous-expression de la protéine du complexe PAR, PAR-3, associée à des altérations des gènes impliqués dans l’adhésion cellulaire (ADAM12, LOXL2), l’interaction cellule-matrice extracellulaire (MMP11, COL11A1, etc…) et l’invasion (ACTR2, PAK1). Des défauts quantitatifs des constituants de la matrice extracellulaire ont également été mis en évidence. Nous avons, ainsi, pu établir une signature transcriptomique spécifique de cette entité tumorale, en accord avec les caractéristiques morphologiques observées. Les carcinomes micropapillaires présentent une polarité anormale caractérisée par des marqueurs apicaux positionnés vers la matrice extracellulaire ou absents, la perte de l’orientation de la protéine golgienne GM130, des anomalies des jonctions serrées (occludine) et des protéines du complexe PAR (CDC42 et aPKC). Au niveau génomique, nous avons mis en évidence des mutations somatiques de gènes impliqués dans la régulation de la polarité (DNAH9, FOXO3) et de la ciliogenèse (BBS9, BBS12, SEC63), l’organisation du cytosquelette (HSP90B1, UBR4, ZFYVE26) et la motilité (FMN2). Au niveau transcriptomique, une nette perturbation des gènes impliqués dans l’adhésion cellule cellule, cellule-matrice extracellulaire et l’angiogenèse est observée. Les IMPC présentent également une surexpression spécifique d’une protéine du complexe CRUMBS, LIN7A. Nous avons établi un modèle in vitro et avons montré que LIN7A est un puissant perturbateur de la polarité apico basale. Sa surexpression dans la lignée MCF10A cultivée en 3D induit la formation d’acini multilobés, à forte capacité proliférative et sans lumière centrale. Cette absence de lumière centrale est due à une inhibition de l’apoptose. Les cellules MCF10A-LIN7A présentent également une capacité accrue de croissance en suspension, témoignant d’une résistance à l’anoïkis. Cette résistance est due, entre autre, à une diminution de la phosphorylation de la protéine p38. Cette description approfondie des altérations biologiques de types spéciaux de carcinomes mammaires doit permettre à moyen terme de proposer une prise en charge plus spécifique des patientes, grâce à l’identification de nouvelles possibilités thérapeutiques ou à une stratégie de désescalade thérapeutique. / Apicobasal polarity is maintained by the combined action of several protein complexes – PAR, SCRIBBLE and CRUMBS – together with the structural organisation of adherent and tight junctions. Apicobasal polarity is important for the regulation of cell division, apoptosis and cell migration, and several studies show that disruption of cell polarity is involved in tumour progression. Our work focused on the biological mechanisms responsible for the altered apicobasal polarity and cell adhesion observed in breast cancers. To do so, we studied two types of breast carcinomas – invasive lobular carcinoma (ILC) and invasive micropapillary carcinoma (IMPC) – whose morphology suggests specific alterations of these cell processes. Our approach combined genomic, transcriptomic and phenotypical comparative analyses, using a group of invasive carcinomas not special type (IC-NST) as control.Lobular carcinomas are generally characterized by alterations of the E cadherin/ catenin complex and their ability to disseminate. We have shown that they also present a downregulation of PAR-3, a protein of the PAR complex, associated with deregulation of genes involved in cell adhesion (ADAM12, LOXL2), cell-extracellular matrix interactions (MMP11, COL11A1, etc…) and invasion (ACTR2, PAK1). Quantitative defects in components of the extracellular matrix were also observed. We have thus been able to establish a transcriptomic signature for this tumour entity, in agreement with the phenotypical observations.Micropapillary carcinomas show an abnormal polarity characterized by the absence of apical markers or their localization at the inverted apical pole, the loss of Golgi protein GM130 correct orientation and abnormal expression or localization of occludin (tight junctions), CDC42 and aPKC(PAR complex proteins). At the genomic level, we have identified somatic mutations in genes involved in polarity (DNAH9, FOXO3) and ciliogenesis regulation (BBS9, BBS12, SEC63), cytoskeleton organisation (HSP90B1, UBR4, ZFYVE26) and motility (FMN2). At the transcriptomic level, we observed deregulation of genes involved in cell-cell, cell-extracellular matrix adhesion and angiogenesis. IMPC also demonstrate the specific overexpression of a protein of the CRUMBS complex, LIN7A. We established an in vitro model and showed that LIN7A is an efficiently modifier of MCF10A’s polarity. Its overexpression in MCF10A cells cultured in 3-D conditions induces the formation of proliferating multi-lobar acini with no central lumen. This absence of a central lumen is due to an inhibition of apoptosis. MCF10A-LIN7A cells also show an increased ability to grow in suspension, indicating resistance to anoikis. This resistance seems to be linked to a decrease of p38protein’s phosphorylation.This detailed description of biological alterations in special types of breast carcinomas will contribute to more specific treatments provided to the patients, through the identification of new therapeutic targets or therapeutic strategies.

A terapia antiagregante é comumente indicada na prevenção e tratamento de doenças cardiovasculares. A dupla antiagregação com clopidrogrel e ácido acetilsalicílico (AAS) tem sido frequentemente adotada em pacientes com Doença Arterial Coronariana (DAC), mas apresenta ineficácia em uma parcela significativa da população com genótipo de respondedores. Essa falha terapêutica nos leva a questionar se outros mecanismos moleculares podem estar influenciando na resposta a esses fármacos. Recentes estudos sugerem que pequenas sequências de RNA não codificantes denominadas microRNAs (miRNAs) podem estar fortemente relacionadas com resposta ao tratamento fármaco-terapêutico, controlando as proteínas envolvidas na farmacocinética e farmacodinâmica. Entretanto, os principais miRNAs que atuam na dinâmica da resposta medicamentosa ainda não foram bem definidos. O objetivo deste estudo foi avaliar o perfil de miRNAs no sangue total periférico, procurando melhor esclarecer os mecanismos envolvidos na resposta aos antiagregantes plaquetários AAS e clopidogrel. Para isso, selecionou-se pacientes com DAC, os quais apresentavam diferentes respostas à dupla terapia de antiagregação determinadas pelo teste de agregação plaquetária. Baseados nos fenótipos, os perfis de expressão de miRNAs foram comparados entre os valores da taxa de agregação categorizados em tercis (T) de resposta. O grupo T1 foi constituído de pacientes respondedores, o T2 de respondedores intermediários e o T3 de não respondedores. Os perfis de miRNAs foram obtidos após sequenciamento de última geração e os dados obtidos foram analisados pelo pacote Deseq2. Os resultados mostraram 18 miRNAs diferentemente expressos entre os dois tercis extremos. Dentre esses miRNAs, 10 deles apresentaram importantes alvos relacionados com vias de ativação e agregação plaquetária quando analisados pelo software Ingenuity®. Dos 10 miRNAs, 4 deles, os quais apresentaram-se menos expressos no sequenciamento, demonstraram os mesmos perfis de expressão quando analisados pela reação em cadeia pela polimerase quantitativa (qPCR): hsa-miR-423-3p, hsa-miR-744-5p, hsa-miR- 30a-5p e hsa-let-7g-5p. A partir das análises de predição de alvos, pôde-se observar que os quatro miRNAs, quando menos expressos simultaneamente, predizem ativação da agregação plaquetária. Além disso, os miRNAs hsa-miR- 423-5p, hsa-miR-744-5p e hsa-let-7g-5p mostraram correlação com o perfil lipídico dos pacientes que, por sua vez, apresentou influência nos valores de agregação compreendidos no T3 de resposta a ambos os medicamentos. Sendo assim, conclui-se que maiores taxas de agregação plaquetária podem estar indiretamente relacionadas com os padrões de expressão de hsa-miR- 423-3p, hsa-miR-744-5p e hsa-let-7g-5p. Sugere-se que a avaliação do perfil de expressão destes 3 miRNAs no sangue periférico de pacientes com DAC possa predizer resposta terapêutica inadequada ao AAS e ao clopidogrel / The antiplatelet therapy is often indicated for the prevention and treatment of cardiovascular diseases. Dual antiplatelet therapy with clopidogrel and acetylsalicylic acid (ASA) has often been adopted in patients with coronary artery disease (CAD), but it has been ineffective in a significant portion of the population with genotype of responders. This fact leads us to question whether other molecular mechanisms may be influencing the response to these drugs. Recent studies suggest that small non-coding RNA sequences known as microRNAs (miRNAs) may be closely related to response to drug-therapeutic treatment, controlling proteins involved in pharmacokinetics and pharmacodynamics. The aim of this study was to evaluate the profile of miRNAs in whole blood, looking to better clarify mechanisms involved in ASA and clopidogrel response. For this purpose, we selected CAD patients who showed different responses to dual antiplatelet therapy determined by aggregation test. Based on the phenotypes, the miRNA expression profiles were compared between the platelet aggregation values categorized into tertiles (T) of response. The T1 group consisted of responding patients, the T2 consisted of intermediate responders and the T3 consisted of non-responders. The miRNA profiles were obtained after next-generation sequencing and data were analyzed by Deseq2 package. Results showed that 18 miRNAs were differentially expressed between the two extreme tertiles. By Ingenuity® software prediction analysis, 10 miRNAs showed important targets related with activation and aggregation of blood platelets. Of the 10 miRNAs, 4 of them, which were down-expressed on sequencing, showed the same fold-regulation when expression profiles were analyzed by quantitative polymerase chain reaction (qPCR): hsa-miR-423-3p, hsa-miR-744-5p, hsa-miR-30a-5p and has-hsa- let-7g-5p. By target prediction analysis, it was observed that, when the four miRNAs are simultaneously down-expressed, they predict activation of platelet aggregation. Furthermore, hsa-miR-423-5p, hsa-miR-744-5p, and hsa-let-7g-5p showed correlation with the lipid profile of patients which, in turn, demonstrated influence in aggregation values reaching T3 of response to both drugs. Therefore, we concluded that increased platelet aggregation rates may be indirectly related to the expression profiles of hsa-miR-423-3p, hsa-miR-744-5p and hsa-let-7g-5p. It is suggested that the evaluation of the expression profile of these three miRNAs in the peripheral blood of patients with CAD may predict inadequate therapeutic response to aspirin and clopidogrel.

Orientador: Fausto Foresti / Resumo: Characidium é um grupo de peixes amplamente distribuídos pela região Neotropical, embora seja considerado o mais especioso dentro de Crenuchidae, do ponto de vista citogenético o número de espécies investigadas ainda é baixo, o que dificulta a caracterização quanto a organização cromossômica do gênero. Em relação ao número diploide, as espécies de Characidium conservaram um cariótipo com 2n = 50 cromossomos, do tipo metacêntricos e submetacêntricos (com exceções), o que resulta em uma macroestrutura homogênea para o grupo. Porém, investigações utilizando sequências repetitivas têm contribuído para ilustrar que a organização microestrutural cromossômica pode diferir entre as espécies, refletindo o hábito destes peixes constituírem populações pequenas e isoladas em cabeceiras de riachos. Adicionalmente, algumas espécies de Characidium também foram descritas portando cromossomos B em seus cariótipos, e a utilização de ferramentas citomoleculares têm contribuído para explorar quanto a origem e evolução destes componentes cariotípicos. Neste sentido, o objetivo do presente estudo foi agregar técnicas citomoleculares com resultados de sequenciamento massivo, para tentar compreender a ocorrência de cromossomos B no genoma de Characidium sp. aff. C. vidali. Os resultados obtidos mostraram que i) o mapeamento físico de diferentes sondas de DNA repetitivo contribuíram não apenas para caracterizar o cariótipo da espécie em estudo, como também adicionaram mais informações quanto a organi... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Characidium is a group of fish widely distributed in the Neotropical region, although it is considered the most specious within Crenuchidae, from the cytogenetic point of view the number of species investigated is still low, which makes it difficult to characterize the chromosomal organization of the genus. In relation to the diploid number, Characidium species retained a karyotype with 2n = 50 chromosomes, metacentric and submetacentric (with exceptions), resulting in a homogeneous macrostructure for the group. However, investigations using repetitive sequences have contributed to illustrate that the chromosomal microstructural organization may differ between species, reflecting the habit of these fish constituting small and isolated populations in headwaters of streams. In addition, some species of Characidium have also been described carrying B chromosomes in their karyotypes, and the use of cyto-molecular tools has contributed to explore the origin and evolution of these karyotype components. In this sense, the objective of the present study was to aggregate cyto-molecular techniques with massive sequencing results to try to understand the occurrence of B chromosomes in the genome of Characidium sp. aff. C. vidali. The results showed that i) the physical mapping of different repetitive DNA probes contributed not only to characterize the karyotype of the species under study, but also added more information about the organization and evolution of the chromosomal microstruct... (Complete abstract click electronic access below) / Doutor

Saccharomyces yeasts are ideal candidates for genomic and evolutionary studies in eukaryotes due to their small genome, short generation time and availability of genomic data. Species freely hybridize producing viable but largely sterile cells. A hybridization event can be a swift mechanism for evolutionary innovation that if successful, may produce individuals fitter than either parents. It is largely unclear which mechanisms contribute to such hybrid vigour. This thesis investigated three mechanisms by which a natural hybrid may utilise one or both subgenomes to its advantage: recombination, the formation of chimeric protein complexes and the inheritance of mitochondrial DNA. Three strains of Saccharomyces pastorianus, a natural hybrid of Saccharomyces cerevisiae and Saccharomyces eubayanus, used in the lager fermentation process were sequenced using a NGS SOLiD platform. An analysis of recombination between each subgenome revealed the presence of 30 breakpoints, 28 of which are found within coding regions. Two breakpoints, present within the genes XRN1 and HSP82 have been reused in all three strains of S. pastorianus. This thesis investigated the formation of chimeric protein complexes in S. pastorianus by determining the configuration of protein complex-forming gene pairs to see whether they were mainly uni-specific, with all members belonging to the same parent, or chimeric, comprising one member from each parental species. A total of 21 pairwise protein complexes were found to be obligatorily chimeric in three strains of S. pastorianus. We used PCR-mediated gene deletion to recreate chimeric protein complexes in laboratory hybrids of S. cerevisiae and S. uvarum. The allelic configuration of one protein-complex forming gene pair, MLP2 and SPC110, impacted the growth of hybrid strains in a temperature-dependent manner. Finally, we looked at the mitochondrial inheritance in hybrids. Yeast hybrids can initially inherit mitochondrial DNA (mtDNA) from both parents, but rapidly become homoplasmic. To investigate the mechanisms influencing mtDNA inheritance, strains of Saccharomyces cerevisiae and Saccharomyces uvarum were crossed under different environmental conditions. The majority of hybrids inherited S. cerevisiae mtDNA when crossed in glycerol, a carbon source that can only be respired by yeast, in a range of temperatures. Those crossed in glucose, a fermentable source, did not show a preference for the inheritance of mtDNA at 30°, but at 10°C preferentially inherited S. uvarum mtDNA. In subsequent growth assays, hybrids with S. cerevisiae mtDNA grew better than those with S. uvarum mtDNA at 30°C and 20°C. However, at 10°C, the reverse was true: hybrids with S. uvarum mtDNA grew better that those with S. cerevisiae mtDNA, although only in glycerol. Overall this works sheds light on the molecular mechanisms contributing to fitness and evolutionary vigour in yeast hybrids.

A Síndrome Ablefaria Macrostomia (SAM) é uma condição rara, onde os pacientes apresentam características clínicas marcantes como o encurtamento ou ausência das pálpebras superiores e inferiores, ausência de sobrancelhas e cílios, macrostomia por defeitos na fusão dos lábios, entre outros. O padrão de herança da síndrome não está elucidado, tendo a herança autossômica dominante com expressividade variável sido sugerida. SAM possui sobreposição fenotípica com a Síndrome de Barber-Say e com a Síndrome de Fraser, porém nenhum gene já descrito apresentou mutação nos pacientes portadores da SAM. A abordagem genômica no estudo de doenças raras tem sido amplamente utilizada, devido principalmente ao surgimento da Nova Geração de Sequenciamento, que possui alto poder de descriminar as seqüencias nucleotídicas com grande cobertura, em um curto período de tempo. No presente estudo o sequenciamento completo do exoma foi realizado, com cinco indivíduos de uma mesma família, três membros afetados e dois não, e permitiu a análise das regiões codificantes nestes indivíduos. A base molecular da Síndrome Ablefaria Macrostomia é aqui sugerida como autossômica dominante, e decorrente da mutação nova não sinônima c.223G>A (p.E75K) no gene TWIST2. Essa mutação patogênica ocasiona a troca de um aminoácido pequeno de carga negativa, o ácido glutâmico, para um aminoácido de cadeia maior carregado positivamente, a lisina. A modelagem in silico da proteína Twist2 mostrou que a estrutura geral tridimensional da proteína não foi alterada, mas a troca do aminoácido ocorre na posição 75 dentro do domínio básico HLH, e pode impedir a formação de dímeros, ou a própria ligação ao DNA. Sugere-se ainda que a heterogeneidade de fenótipos associados a mutações no gene TWIST2, pode ser atribuída às interações que essa proteína é capaz de formar, e a ampla ação regulatória que ela desempenha em diversos genes do desenvolvimento. / Ablepharon-Macrostomia Syndrome (AMS) is a rare condition characterised by absent or hypoplastic eyelids, absent eyebrows and eyelashes, macrostomia caused by fusion defects of the mouth with unfused lateral commissures, as well as other clinical features. The inheritance pattern has not been confirmed and while autosomal dominant inheritance with variable expressivity has been suggested, recessive inheritance has not been ruled out. The phenotype of AMS overlaps that of Barber-Say and Fraser Syndrome, but any reported gene for these syndromes is mutated on AMS patients. The genomic approach for rare disease studies has been widely used mainly due to the emergence of Next Generation Sequencing, which is very effective at determining nucleotide sequences with large coverage in a short period of time. The whole exome sequencing of five family members was undertaken, with three affected and two unaffected, and the coding regions of the individuals were subsequently analysed. The molecular basis of AMS is suggested here as autosomal dominant, and due to a novel non-synonymous mutation c.223G>A (p.E75K), in TWIST2 gene. This pathogenic mutation causes glutamic acid, a small negatively charged amino acid, to be substituted for a larger and positively charged lysine. The in silico protein modeling of Twist2 shows that the general 3D-structure of the protein is not affected, but the amino acid change is located inside the basic Helix-Loop-Helix domain which could disrupt dimerization and DNA binding. It has also been suggested that the phenotype heterogeneity associated with mutations on TWIST2 gene can be attributed to the interactions that this protein is capable of, and the role that it plays in the regulation of several developmental genes.

Submitted by Ana Damasceno (ana.damasceno@unipampa.edu.br) on 2016-11-07T16:30:36Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação - Evelise Carvalho.pdf: 2321912 bytes, checksum: 3957e800afc8b54ee2d2bc427c9dffd6 (MD5) / Made available in DSpace on 2016-11-07T16:30:36Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação - Evelise Carvalho.pdf: 2321912 bytes, checksum: 3957e800afc8b54ee2d2bc427c9dffd6 (MD5) Previous issue date: 2015-05-19 / Algas verdes da classe Trebouxiphyceae estão entre os organismos presentes no continente Antártico, onde a espécie mais relatada é a macroalga verde Prasiola crispa (Lightfoot) Kützing. Considerada um organismo extremófilo, pois se desenvolve com muito sucesso no habitat extremo da Antártica, ainda são raros na literatura dados moleculares sobre esta espécie, o que impede uma avaliação sobre sua taxonomia e posição filogenética. Com o advento das tecnologias de sequenciamento de nova geração, os genomas de organelas tornaram-se uma grande ferramenta para estudos de filogenia, pois fornecem inúmeros dados filogenéticos, sequências de proteínas e nucleotídeos e também informações sobre conteúdo gênico e arquitetura. Neste trabalho, foi determinada a sequência dos genomas do cloroplasto (cpDNA) e mitocondrial (mtDNA) de P. crispa, com o intuito de inferir as relações evolutivas deste organismos com outras espécies de plantas verdes, bem como uma análise estrutural. Os genomas plastidial e mitocondrial foram sequenciados por Macrogen Service (SolexaIllumina Hi-Seq 2500). A montagem, anotação, alinhamento, construção da filogenia e análise sintênica foram realizados in silico com softwares específicos. O cpDNA e mtDNA P. crispa apresentam 196.502 pb e 89.819 pb, respectivamente. Estes genomas acessórios apresentam 21 genes putativos relacionados com a fotossíntese e 18 genes relacionados com o metabolismo oxidativo. A análise filogenômica baseada no cpDNA demonstrou que P. crispa agrupou com alga trebouxiophyceae Prasiolopsis sp. formando o clado Prasiola juntamente com Stichococcus bacilaris. Nossos resultados para filogenômica embasada no mtDNA revelam que P. crispa agrupa com as outras espécies da classe Trebouxiphyceae. A análise de sintenia do cpDNA e mtDNA de P. crispa com a espécies de plantas verdes relacionadas evolutivamente demonstram que estes organismos apresentam poucos blocos gênicos sintênicos. Este trabalho pioneiro com a alga P. crispa, demonstra que os genomas acessórios suprem uma gama de dados moleculares que podem ser utilizados para estudos filogenômicos. Além disto, as informações geradas a partir do sequenciamento do cpDNA e mtDNA de P. crispa fornecem um aporte para estudos futuros mais aprofundados / Green algae from Trebouxiophyceae class are among the organisms in the Antarctic continent, where the most reported species is the green macroalga Prasiola crispa (Lightfoot) Kützing. This algae is considered an extremophile organism because develops successfully in the harsh Antarctic habitat, however studies reporting molecular data of this species are still lacking in the literature, which prevents an assessment of their correct taxonomy and phylogenetic position. With the advent of next generation sequencing technologies, it because easier to obtain molecular information as for example from organelle genomes making them a great tool for taxonomic studies because they provide a great number of, phylogenetic data, nucleotides, protein sequences, gene content and architecture information. In this study, we determined the sequence of the chloroplast (cpDNA) and mitochondrial (mtDNA) genome of P. crispa in order to infer the evolutionary relationships of the organisms with other species of green plants, as well as a structural analysis. Plastid and mitochondrial genome was sequenced by Macrogen Service (Illumina Solexa Hi-Seq 2500). The genome assembly, annotation, sequences alignment, phylogeny construction, and structural analyses were performed in silico with specific softwares. Plastid and Mitochondrial genomes have a total length of 196,502 bp and 89,819 bp, respectively. These genomes presented 21 putative photosynthesis related genes and 18 oxidative metabolism related genes, respectively. Phylogenetic analysis based on the cpDNA demonstrated that P. crispa grouped with Trebouxiophyceae algae Prasiolopsis sp. forming the Prasiola clade along with Stichococcus bacilaris. Our results for phylogenetic analysis grounded in mtDNA show that P. crispa groups with other species of Trebouxiphyceaen alga. Synteny analysis of P. crispa cpDNA and mtDNA with evolutionarily related species of green plants shows that these organisms have few syntenic gene blocks. This pioneering work with P. crispa provided the accessories genomes which suppled a range of molecular data that can be employed to taxonomic studies. In addition, the information generated from the sequencing of cpDNA and mtDNA of P. crispa provide a contribution for further studies.

A espécie de cianobactéria Cylindrospermopsis raciborskii pertence a ordem Nostocales e tem a capacidade de realizar a fixação biológica do N2 atmosférico, sintetizar cianotoxinas e também de proliferar em águas doces formando florações de cianobactérias nocivas. A população de C. raciborskii encontrada no Brasil é conhecida por produzir derivados de saxitoxina (STX), enquanto cilindrospermopsinas (CYN), as quais são detectadas em isolados de outros países, não têm sido detectada em isolados brasileiros. Neste estudo, a produção de CYN por dois isolados brasileiros de C. raciborskii, CENA302 e CENA303, submetidos a diferentes condições nutricionais, foi avaliada usando o teste imunológico ELISA específico para detecção de CYN. Em seguida, genes evolvidos na biossíntese de CYN foram buscados no genoma dessas duas linhagens usando amplificações por PCR com iniciadores específicos e posterior sequenciamento Sanger, bem como sequenciamento genômico na plataforma HiScan SQ. Além disso, foram realizadas buscas nos dois genomas obtidos pela plataforma HiScan SQ de genes envolvidos com a síntese de algumas outras moléculas já descritas em cianobactérias, bem como aqueles relacionados com a fixação biológica de N2 e diferenciação do heterócito. Apesar da utilização de diferentes condições nutricionais, não foi detectada a produção de CYN no ensaio de ELISA em ambos os isolados de cianobactérias. No sequenciamento Sanger, dos nove genes sintetases de CYN buscados, quatro foram amplificados e sequenciados na linhagem C. raciborskii CENA302 e três na linhagem CENA303. Estas sequências de nucletídeos foram traduzidas em aminoácidos, e as funções das proteínas e os domínios preditos confirmaram a sua identidade como genes sintetase de CYN. O sequenciamento dos genomas completos das duas linhagens apresentou altos valores de qualidade de bases e elevada cobertura genômica. A busca por genes sintetase de CYN nos dois genomas foi realizada pelo mapeamento por referência das leituras, mostrando que algumas porções do agrupamento estavam contempladas no genoma, representando aproximadamente 10% do agrupamento de CYN. A anotação das regiões entre os genes descritos como flanqueadores do agrupamento CYN mostrou uma inversão gênica indicando a ocorrência de eventos que podem ter levado a perda ou dispersão no genoma deste agrupamento. Os genes anotados relacionados com fixação biológica de N2 e diferenciação do heterócito nas linhagens CENA302 e CENA303 apresentaram altas identidades com genes homólogos descritos em C. raciborskii. A ausência de alguns nucleotídeos no gene hetP envolvido na formação do heterócito possivelmente levou a perda de sua função na C. raciborskii CENA303, impedindo a diferenciação das células vegetativas em heterócitos, uma vez que não foi observado a presença dessa célula diferenciada em nenhuma das condições nutricionais avaliadas. Todos os genes de moléculas bioativas conhecidas de cianobactérias buscados pelo mapeamento por referência das leituras não foram encontrados nos genomas das linhagens C. raciborskii CENA302 e CENA303. Os resultados obtidos neste estudo revelaram a possibilidade de estar ocorrendo eventos evolutivos que estão causando a perda dos genes responsáveis pela síntese de CYN nos isolados brasileiros de C. raciborskii, demonstrando divergência alopátrica. / The cyanobacterial species Cylindrospermopsis raciborskii belongs to the order Nostocales and has the ability to perform biological nitrogen fixation, synthesize cyanotoxins and also to proliferate in freshwaters to form harmful cyanobacterial blooms. The C. raciborskii population from Brazil is known to produces saxitoxin derivatives (STX), while cylindrospermopsin (CYN), which is commonly detected in isolates from other countries, has thus far not been detected in Brazilian strains. In this study, the production of CYN by two Brazilian isolates of C. raciborskii CENA302 and CENA303, under different nutritional conditions, was evaluated using ELISA immunoassay specific for the detection of CYN. Then, genes involved in the biosynthesis of CYN were searched in the genome of these two strains using PCR amplification with specific primers and subsequent Sanger sequencing, as well as genomic sequencing on the platform HiScan SQ. Furthermore, searches were performed in the two genomes obtained by the platform HiScan SQ of genes involved in the synthesis of several other molecules already described in cyanobacteria, as well as those related to the biological N2 fixation and heterocyte differentiation. Despite the use of different nutritional conditions, no CYN production was detected by the ELISA assay for both cyanobacterial isolates. In the Sanger sequencing, of the nine CYN synthetase gene searched, four were amplified and sequenced in the C. raciborskii strain CENA302 and three in the strain CENA303. These nucleotide sequences were translated into amino acids, and the predicted protein functions and domains confirmed their identity as CYN synthetase genes. The complete genome sequencing of the two strains showed high values of bases quality and high genomic coverage. The search for CYN synthetase genes in the two genomes was performed by mapping reads to reference, showing that some portions of the cluster were contemplated in the genome, representing approximately 10% of CYN cluster. The annotation of regions between the genes described as flanking the CYN cluster showed a gene inversion indicating the occurrence of events that may have caused the loss or dispersion in the genome of this cluster. The annotated genes related to biological N2 fixation and heterocyte differentiation in the CENA302 and CENA303 strains showed high identities with homologous genes from other strains of C. raciborskii. The absence of a few nucleotides in the hetP gene involved in the heterocyte formation perhaps led to the loss of its function in the C. raciborskii CENA303, preventing the differentiation of vegetative cells into heterocyte, since it was not observed the presence of this differentiated cell in any of the nutritional conditions evaluated. All genes of known cyanobacterial molecules searched by mapping reads to reference were not found in the genomes of the C. raciborskii strains CENA302 and CENA303. The results of this study revealed the possibility of being occurring evolutionary events that are leading to loss of the genes responsible for the synthesis of CYN in the C. raciborskii Brazilian isolates, demonstrating allopatric divergence.

Os micro-organismos que habitam o trato digestivo dos ruminantes têm uma profunda influência no desenvolvimento e funcionamento do animal hospedeiro. O rúmen abriga comunidades microbianas complexas dominadas por bactérias que participam de um processo eficiente de degradação dos materiais que compõem a parede celular vegetal. Por esta razão, o microbioma do rúmen representa uma fonte inexplorada de enzimas hidrolíticas com potencial aplicação na produção de combustíveis a partir da biomassa lignocelulósica. Nós usamos a plataforma Ion Torrent (PGM) para acessar o microbioma do rúmen de quatro animais da raça Santa Inês submetidos a uma dieta base. A fim de descrever a estrutura da comunidade microbiana no rúmen de ovinos e explorar o seu potencial como uma fonte de genes de degradação da biomassa, usamos a abordagem de sequenciamento do gene RNA ribossomal 16S (rRNA), utilizando Ion Tags, e a abordagem de sequenciamento metagenômico shotgun (DNA total), respectivamente. Além disso, medimos parâmetros químicos do ambiente do rúmen, relacionados a cada animal, incluindo pH, Degradabilidade da Matéria Orgânica (OMD), Produção total de Gás (GP) e Emissões de Metano (CH4), a fim de buscar correlações entre estas variáveis químicas e os grupos bacterianos. Em termos de estrutura da comunidade microbiana (bacteriana), encontramos Bacteroidetes como o filo dominante, seguido por Firmicutes, Proteobacteria e Actinobacteria. Alguns táxons foram correlacionados com os parâmetros químicos, como as famílias Corynebacteriaceae e Streptococcaceae, que foram positivamente correlacionadas com OMD; e a família Streptomycetaceae, negativamente correlacionada com GP e CH4. Algumas glicosil hidrolases conhecidas foram identificadas, como Endo-1,4-beta-glucanases, Beta-D-glicosídioglicohidrolases e outras foram designadas como putativas. Estas descobertas mostram interações ecológicas entre os grupos microbianos e funções importantes do rúmen, assim como o potencial do rúmen de ovinos para a descoberta de novas enzimas celulolíticas. / The microorganisms inhabiting the digestive tracts of ruminants have a profound influence on the host animal development and functioning. The rumen harbors complex microbial communities dominated by bacteria, which participate in an efficient process to digest plant cell wall materials. For this reason, the rumen microbiome represents an untapped source of hydrolytic enzymes with potential application for fuel production from lignocellulosic biomass. We used the Ion Torrent (PGM) platform to access the rumen microbiome of four animals of Santa Inês breed under a base diet. In order to describe the structure of the microbial community in the sheep rumen and explore its potential as a source of biomass-degrading genes, we used 16S ribosomal RNA (rRNA) Ion Tags sequencing approach and shotgun metagenomic sequencing (total DNA) approach, respectively. Furthermore, we measured rumen chemical environmental parameters related to each animal, including pH, Organic Matter Degradability (OMD), Total Gas Production (GP) and Methane emissions (CH4) in order to search for correlations between these chemical variables and bacterial groups. In terms of microbial (bacterial) community structure, we found Bacteroidetes as the dominant phylum in sheep rumen microbiome, followed by Proteobacteria, Firmicutes and Actinobacteria. Some taxa were correlated with the environmental parameters, like the Corynebacteriaceae and Streptococcaceae families, which was positively correlated with OMD, and the Streptomycetaceae family, negatively correlated with GP and CH4. Some known glycoside hydrolases were identified, such as Endo-1,4-betaglucanases, Beta-D-glucoside glucohydrolases and others were designated as putative ones. These findings show ecological interactions among microbial groups and important rumen functions, as well as the potential of the sheep rumen for the discovery of new cellulolytic enzymes.

The future of Lake Baikal’s biodiversity is uncertain in response to climate change. Unlike its diverse benthos, Lake Baikal’s zooplankton is species poor, with up to 96% of its biomass being composed of a single Calanoid copepod species, Epischura baikalensis. This study characterizes the genetic differentiation and differential gene expression of E. baikalensis. Using partial-transcriptome sequences obtained by 454 Rosche and Illumina sequencing technologies, the genetic differentiation at inferred single nucleotide polymorphism (SNP) sites and differential gene expression in populations sampled from various parts of the lake were analyzed. The functional genomics of genes showed significant differential expression among the lake’s regions with some genes being highly up-or down-regulated. High genetic differentiation among regions suggests isolated subpopulations. Moreover, significantly differentially expressed transcripts were significantly more genetically differentiated than transcripts exhibiting no differential expression. These results suggest high potential phenotypic plasticity and adaptability in response to climate change, e.g., temperature.