With an adoption of modern methods of polyphasic approach to the study of cyanobacteria, an increased demand for the revision of the traditional taxonomy has emerged. This thesis is devoted to the systematic revisions of selected terrestrial cyanobacteria at several taxonomic levels. The methodology included thorough morphological characterization of cultured cyanobacterial strains using light and electron microscopy complemented with analyses of the molecular data: DNA sequencing, phylogenetic analyses based on 16S rRNA gene and the adjacent 16S-23S ITS region, and comparison of the predicted secondary structures of this region. Descriptions of new species, genera, families and an in-depth characterization of a previously poorly known family were achieved.

The study of the gut microbiota of fish began in the 1930’s and since that time a considerable amount of information has been collated on its composition and diversity. These studies have revealed that the microbial communities of the fish gastrointestinal tract are generally difficult to culture on bacteriological media and mainly consist of bacteria, archaea, viruses, yeasts and protists. The bacteria appear to be the most abundant of these microbial groups and their activity may have major implications for host health, development, immunity and nutrition. Therefore, much of the most recent published research has focused on developing improved methods of identifying the extent of the bacterial diversity within the fish gut and unravelling the potential influence of these microorganisms on the health of farmed fish species. However, whilst such studies have improved our knowledge of the dominant bacterial groups present in the rainbow trout gastrointestinal tract, the limited resolution capacity of many of the methods used has meant that our understanding of their baseline composition in healthy fish remains poorly understood. In this study, the bacterial communities that inhabit the intestine, now commonly referred to as the ‘microbiome’, of farmed Rainbow trout (Oncorhynchus mykiss) were characterized using a culture independent high-throughput molecular sequencing method. The microbiome of the intestinal lumen and mucosa was investigated to ascertain the true extent of the bacterial diversity present in this fish species prior to further experiments. It was found that the diversity of the intestinal microbiome was greater than previous studies had reported with a total of 90 and 159 bacterial genera being identified in both the lumen and mucosal regions respectively. The dominant bacterial phyla identified in both of the regions investigated were Proteobacteria, Firmicutes, Fusobacteria, Bacteroidetes and Actinobacteria. Furthermore, the data collected suggested that the intestinal microbiome may be similar in structure between individual fish, and illustrate the utility of next generation molecular methods in the investigation of the fish gut microbiome. A study was conducted to examine the effect of diet on the composition of the intestinal microbiome of rainbow trout. Two diets, one control and one treatment, were prepared which were identical apart from that the treatment diet contained a microalgal component at 5% of the total formulation. These diets were fed to rainbow trout for a total of 15 weeks. At the end of the trial period a total of 12 fish, three from each of four tanks, were sacrificed from each of the control and treatment groups and their intestinal tissue was sampled in order to compare the composition of the microbiome of both groups. The results revealed that both groups of fish shared similar microbiome compositions, with the Tenericutes being by far the most dominant phylum observed. The structure of the intestinal microbiome was not significantly different between both populations of trout tested. An increased level of bacterial diversity was noted in the treatment fish, however, this was not found to be statistically significant. A limited number of bacterial taxa were discriminatory between diets and were significantly elevated in the treatment group. These taxa were predominantly lactic acid bacteria of the genera Streptococcus, Leuconstoc, Lactobacillus, Lactococcus and Weissella. The results of this study suggested that the minor difference in the diets fed resulted in a correspondingly minor alteration in the intestinal microbiome of the tested rainbow trout. This may indicate that diet composition can modify the composition of the intestinal microbiome of these fish. A further study was conducted to investigate the structure of the intestinal microbiome from groups of fish reared in both freshwater cages and aquarium systems, in order to assess whether or not fish raised in different environments share similar microbiomes. This study also employed a novel computational tool, PICRUSt, to analyse the predicted functional capacity of the microbial communities of individual fish sampled from both environments. The data collected suggested that the structure of the intestinal microbiome was similar regardless of where the fish were raised, with the Tenericutes, Firmicutes, Proteobacteria, Spirochaetae and Bacteroidetes representing the dominant bacterial phyla recorded in the rainbow trout intestine. This suggests that the host may regulate the formation of the intestinal microbiome. A significant difference was however noted in community membership between the fish populations tested, which may point to an environmental influence on the intestinal microbiome. These data suggest that both deterministic host factors and stochastic environmental influences play important roles in shaping the composition of the bacterial communities in the intestine of these fish. The PICRUSt analysis revealed that gene pathways relating to metabolism, transport and cellular processes were enhanced in all of the fish studied, which may signal an involvement of these communities in the digestive processes of rainbow trout. In conclusion, this study used high-throughput sequencing methods in order to improve our understanding of the intestinal microbiome of farmed rainbow trout, and the effect of dietary and environmental factors on its composition. This research has generated scientific information relating to baseline bacterial community compositions in healthy fish, which may be used in future experiments including screening these baselines against the effects of novel aquafeed formulations, environmental perturbations or pathogenic challenges.

abstract: Obesity is a worldwide epidemic accompanied by multiple comorbidities. Bariatric surgery is currently the most efficient treatment for morbid obesity and its comorbidities. The etiology of obesity is unknown, although genetic, environmental, and most recently, microbiome elements have been recognized as contributors to this rising epidemic. The role of the gut microbiome in weight-loss or weight-gain warrants investigation, and bariatric surgery provides a good model to study influences of the microbiome on host metabolism. The underlying goals of my research were to analyze (i) the factors that change the microbiome after bariatric surgery, (ii) the effects of different types of bariatric surgeries on the gut microbiome and metabolism, (iii) the role of the microbiome on the success of bariatric surgery, and (iv) temporal and spatial changes of the microbiome after bariatric surgery. Roux-en-Y gastric bypass (RYGB) rearranges the gastrointestinal tract and reduces gastric acid secretions. Therefore, pH could be one of the factors that change microbiome after RYGB. Using mixed-cultures and co-cultures of species enriched after RYGB, I showed that as small as 0.5 units higher gut pH can aid in the survival of acid-sensitive microorganisms after RYGB and alter gut microbiome function towards the production of weight loss-associated metabolites. By comparing microbiome after two different bariatric surgeries, RYGB and laparoscopic adjustable gastric banding (LAGB), I revealed that gut microbiome structure and metabolism after RYGB are remarkably different than LAGB, and LAGB change microbiome minimally. Given the distinct RYGB alterations to the microbiome, I examined the contribution of the microbiome to weight loss. Analyses revealed that Fusobacterium might lessen the success of RYGB by producing putrescine, which may enhance weight-gain and could serve as biomarker for unsuccessful RYGB. Finally, I showed that RYGB alters the luminal and the mucosal microbiome. Changes in gut microbial metabolic products occur in the short-term and persist over the long-term. Overall, the work in this dissertation provides insight into how the gut microbiome structure and function is altered after bariatric surgery, and how these changes potentially affect the host metabolism. These findings will be helpful in subsequent development of microbiome-based therapeutics to treat obesity. / Dissertation/Thesis / Doctoral Dissertation Microbiology 2016

Salmonellosis or “food poisoning” is a foodborne infection brought on by the pathogen Salmonella from the ingestion of the bacterium on contaminated foods such as vegetables. Infection from Salmonella leads to the highest incidence of hospitalizations and deaths each year, compared to any other bacterial foodborne illness. South Florida is the second largest agricultural winter vegetable producer in the United States, and contamination of vegetables is often observed in preharvest practices. A hardy bacterium, Salmonella, has been shown to live up to 6 weeks in soil and water up to 42°C without a host. The Florida Everglades is a tropical wetland that plays a large role in South Florida’s watershed. It can be divided into agricultural, conservation, and urban areas that connect Lake Okeechobee to Florida Bay by canals, swamps, and rivers. Inland canals tightly regulate water levels in South Florida as a means of flood control for residential and agricultural land. With the influences of anthropomorphic run off from agricultural and urban use, we hypothesized that microbial communities would significantly differ between three select sites in western (Collier county) versus three sites in more urban eastern Florida (Broward county): natural standing water, manmade drainage canal in agricultural areas, and manmade drainage canals in urban areas. We also hypothesized that pathogenic like Salmonella would be present in these habitats. Deep sequencing and ecological genetics analyses of the 16s rRNA V4 region yielded a total of 163,320 unique bacterial OTUs from a total of 139 samples collected monthly for one year in 2015 and part of 2016. Salmonella is not considered an abundant taxon within the microbial population. With the knowledge that Salmonella resides within the microbial population isolates were cultured from soil and water samples that were taken monthly from each site using a modified version of the Food and Drug Administration Bacterial Analytical Methods manual (FDA-BAM). The culturing resulted in 234 isolates obtained and 31 different serovars of Salmonella. Culturing showed that Salmonella favored months with high standing water and high-water temperatures that would lead to the ideal environment for survival. The most commonly occurring isolates within the sample set are those associated with agricultural animals. Though Salmonella may be a rare taxon within the microbial population given the correct environmental conditions such as warm temperatures it is possible to observe Salmonella year round within the South Florida environment.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A castanheira-do-Brasil (Bertholletia excelsa H.B.K.), Lecythidaceae, é uma espécie nativa de potencial interesse para sistemas agroflorestais (SAFs) na Amazônia. Uma das dificuldades da propagação da castanheira é seu processo germinativo desuniforme, que constitui um problema no estabelecimento de mudas para cultivos. Pouco se sabe sobre os micro-organismos benéficos associados às raízes de castanheira-do-Brasil, sendo que o uso de micro-organismos promotores do crescimento vegetal pode ser uma biotecnologia positiva para a produção de mudas de qualidade. O objetivo desse trabalho foi caracterizar fenotípica e genotipicamente bactérias isoladas de raízes de castanheira-do-Brasil cultivadas e nativas e avaliar a presença de características para promoção do crescimento vegetal. Foram isoladas 303 bactérias de raízes de castanheiras em Roraima, sendo 81 oriundas da área de monocultivo de castanheira, 154 de SAF e 68 de área de floresta nativa. As bactérias foram isoladas utilizando meios semissólidos NFB, LGI, JMV e DYGS. A caracterização fenotípica dos isolados foi avaliada através das análises de proteína totais por SDS-PAGE, solubilização de fosfato de cálcio em meio sólido, solubilização de fosfato de alumínio em meio liquido e produção de ácido indol acético. Baseado nos perfis de proteínas (SDS-PAGE) foi estimado o índice de diversidade de Shannon e análise de rarefação. A caracterização genotípica foi realizada através da avaliação da presença ou ausencia do gene nifH e pelo sequenciamento parcial do 16S rRNA. Das 303 bactérias foram obtidas o perfil proteico de 290. Através da análise dos perfis proteicos foram gerados quatro dendrogramas referentes a cada meio de isolamento, JMV, LGI, DYGS e NFB. A partir desses dendrogramas, foi realizada uma seleção de bactérias a partir de grupos formados a 80% de similaridade em cada meio de isolamento, sendo selecionadas 100 bactérias. Na avaliação da diversidade em cada ambiente, os índices de diversidade foram similares para as três localidades de onde foram isoladas as bactérias. Das 100 bactérias avaliadas quanto a capacidade de solubilização de fosfato de cálcio em meio sólido, observou-se que 69% apresentaram esta característica e 90% apresentaram capacidade de solubilizar fosfato de alumínio em meio liquido. Setenta bactérias sintetizaram acido indol acético, onde 44 sintetizaram em meio sem triptofano, 61 em meio com triptofano e 35 apresentaram nos dois meios. Das 100 bactérias submetidas à amplificação da região do gene nifH apenas 18 apresentaram bandas específica de amplificação correspondente do gene nifH, sendo oito isoladas em meio semisseletivo para Azospirilum sp. (NFB e LGI), e 10 isoladas de meio não seletivo (DYGS). Entre as bactérias avaliadas, 24 se destacaram para futuros testes de inoculação na produção de mudas, por apresentarem no mínimo duas ou mais caracaterísticas de promoção do crescimento vegetal. Através da análise do gene 16S rRNA, as sequências de nucleotídeos obtidas resultaram na distribuição das bactérias em 18 gêneros distintos. Foram detectadas sequências de gêneros pertencentes às classes α-Proteobacteria, β-Proteobacteria, γ-Proteobacteria, Bacilli e Actinobacteria. Estes resultados indicam uma elevada diversidade de gêneros de bactérias endofíticas promotoras do crescimento presentes em raízes de castanheira-do-Brasil. Trata-se do primeiro trabalho de caracterização de bactérias endofíticas promotoras de crescimento em raízes de castanheira-do-Brasil.

A superfície das folhas de árvores (filosfera) oferece uma grande área de habitat para os micro-organismos, mas constitui um ambiente extremo. O desenvolvimento de comunidades microbianas é dependente de fonte de carbono e de certos nutrientes essenciais inorgânicos comumente liberados pela planta para a sua superfície. No entanto, um grupo especial de bactérias, Cyanobacteria, é menos dependente da planta para sua nutrição, pois vários destes organismos são autotróficos para carbono e nitrogênio. Portanto, Cyanobacteria é particularmente interessante para se avaliar neste ambiente. Neste estudo, linhagens de cianobactérias presentes na superfície das folhas secretoras de sal da planta de manguezal Avicennia schaueriana foram isoladas e caracterizadas morfológica, molecular e ultraestruturalmente. O potencial destes isolados para sintetizar moléculas bioativas também foi avaliado. Para isso, folhas de A. schaueriana foram coletadas em um manguezal com histórico de contaminação por petróleo, localizado próximo ao Rio Iriri, em Bertioga-SP. O isolamento das cianobactérias foi realizado usando quatro meios de cultura (BG-11, SWBG-11, BG-11o e SWBG-11o) e dois métodos: a) esfregaço das folhas nos meios sólidos em placas de Petri; e b) submersão das folhas em frascos Erlenmeyer contendo meios líquidos. Após a obtenção de culturas puras, os isolados foram crescidos em meios líquidos, as células foram concentradas e usadas para extração de DNA genômico. O gene de RNAr 16S de cada isolado foi amplificado por PCR usando iniciadores específicos (27F/1494Rc), clonado e sequenciado. As sequências de RNAr 16S foram usadas na construção de árvore filogenética. O potencial dos isolados para sintetizar moléculas bioativas foi acessado pela amplificação de PCR usando iniciadores específicos para sequências gênicas codificadoras de peptídeo sintetase (NRPS), policetídeo sintase (PKS), cianopeptolina, aeruginosina, saxitoxina, anatoxina-a/homoanatoxina-a e microcistina. Como resultado, trinta morfotipos foram isolados em meio líquido e quatro em meio sólido. Estes morfotipos foram identificados como pertencentes a quatro ordens diferentes (12 Nostocales, 9 Pseudanabaenales, 8 Chroococcales e 5 Oscillatoriales). Entre os isolados, alta abundância de linhagens potencialmente fixadoras de N2 foi encontrada, indicando que elas possivelmente são uma importante fonte de nitrogênio neste habitat. As sequências do gene de RNAr 16S de vinte e quatro isolados ficaram distribuídas em onze clados distintos na árvore filogenética e mostraram baixas similaridades com gêneros já descritos. Na análise da ultraestrutura destas linhagens, destacou-se a presença de grânulos de elevado volume em uma cianobactéria unicelular e de um arranjo de tilacoides incomum em uma cianobactéria filamentosa homocitada com morfologia aparentemente simples. Sequências gênicas codificadoras de PKS foram detectadas em dezessete linhagens, de aeruginosina em sete linhagens e de cianopeptolina em dez linhagens. Entretanto, sequências gênicas codificadoras de NRPS e das cianotoxinas microcistina, saxitoxina e anatoxina-a/homoanatoxina-a não foram detectadas. A superfície das folhas de A. schaueriana apresenta elevado número de cianobactérias não descritas, provavelmente um resultado das condições peculiares tanto da filosfera quanto do manguezal estudado. Este é o primeiro relato de isolamento de cianobactérias da superfície de folhas de A. schaueriana / The tree leaf surface (phyllosphere) offer a large habitat area for microorganisms but constitute an extreme environment. The development of microbial communities is dependent of carbon source and certain essential inorganic nutrients commonly released from the plant to its surface. However, a special group of bacteria, Cyanobacteria, is less dependent of the plant for their nutrition since several of these organisms are autotrophic for carbon and nitrogen. Therefore, cyanobacteria are particularly interesting to be evaluated in this environment. In this study, cyanobacterial strains present in the salt-excreting leaf surface of the mangrove Avicennia schaueriana were isolated and morphologically, molecularly and ultrastructurally characterized. The potential of these isolates to synthesize bioactive molecules was also evaluated. To this purpose, A. schaueriana leaves were collected in a mangrove with history of oil contamination located near to the Iriri river in Bertioga-SP. The isolation of cyanobacteria was achieved using four culture media (BG-11, SWBG-11, BG-11o and SWBG-11o) and two methods: a) smearing of leaves into solid media in Petri dishes; and b) submersion of leaves in Erlenmeyer flasks containing liquid media. After obtaining pure cultures, the isolates were grown into liquid media, and the cells were concentrated and used for genomic DNA extraction. The gene of rRNA 16S of each isolate was amplified by PCR using specific primers (27F/1494Rc), cloned and sequenced. The 16S rRNA sequences were used for the construction of a phylogenetic tree. The potential of the isolates to synthesize bioactive molecules was assessed by PCR amplification using primers specific for gene sequences encoding non-ribosomal peptide synthetase (NRPS), polyketide synthase (PKS), cyanopeptolin, aeruginosin, saxitoxin, anatoxin-a/homoanatoxin-a and microcystin. As results, thirty morphotypes were isolated in liquid media and four in solid media. These morphotypes were identified as belonging to four different orders (12 Nostocales, 9 Pseudanabaenales, 8 Chroococcales and 5 Oscillatoriales). Among the isolates, it was found a high abundance of potentially N2-fixing strains, what indicates that they possibly are an important source of nitrogen in this habitat. The 16S rRNA gene sequences of twenty-four isolates were distributed into eleven distinct clades in the phylogenetic tree and showed low similarities with described genera. In the ultrastructural analyses of these strains, the highlight was the presence of granules of high volume in a unicellular cyanobacterium and an unusual thylakoid arrangement in a homocytous filamentous cyanobacterium with apparently simple morphology. Gene sequences encoding for PKS were detected in seventeen strains, for aeruginosine in seven strains and cyanopeptolin in ten strains. Gene sequences encoding for NRPS and for the cyanotoxins microcystin, saxitoxin, and anatoxin-a/homoanatoxin-a were not found. The leaf surface of A. schaueriana presents a high number of undescribed cyanobacteria, probably as a result of the peculiar conditions of the phyllosphere and the studied mangrove. This is the first report of isolation of cyanobacteria from the leaf surface of A. schaueriana

As cianobactérias são microrganismos fotossintetizantes oxigênicos com ampla plasticidade metabólica e estrutural, que apresentam potencial biotecnológico para exploração na biossorção de metais pesados e biodegradação de poluentes orgânicos. Devido as suas fortes interações com cátions e ao contínuo suprimento de biomassa barata,as cianobactérias podem ser candidatas promissoras à biossorventes para remoção de metais e radionuclídeos. Dessa maneira, numa tentativa de encontrar uma cianobactéria com esse perfil para remover 226Ra de uma mina de urânio desativada da Unidade de Tratamento de Minérios (UTM) pertencente às Indústrias Nucleares do Brasil (INB), Caldas, MG, doze linhagens de cianobactérias foram isoladas desse ambiente. Essas linhagens foram morfologicamente caracterizadas como Aphanothece sp. CENA75, Rhabdoderma sp. CENA114, Synechococcus cf. lividus CENA79, Aphanocapsa cf. holsatica CENA80, Geitlerinema acutissimum CENA85, Pseudanabaena galeata CENA84, Pseudanabaena sp. CENA81, Leptolyngbya cf. tenerrima CENA76, Leptolyngbya sp. CENA83, Phormidium formosum CENA86, Phormidium violaceum CENA82 e Nostoc sp. CENA87. A análise molecular dos isolados, baseada em seqüências quase completas do gene RNAr 16S (1325 pb), estava de acordo com a análise morfológica, com exceção das linhagens Rhabdoderma sp. CENA114 e Phormidium violaceum CENA82. As seqüências de RNAr 16S dessas duas linhagens mostraram valores baixos de identidades (<92%) com seqüências do GenBank, o que pode representar novas espécies de cianobactérias. Altas percentagens de identidades (>96%) das seqüências do gene de RNAr 16S foram encontradas entre as linhagens restantes e as do GenBank. A árvore filogenética construída usando o método ?Neighbour Joining? mostrou que as linhagens unicelulares das ordens Chroococcales e as filamentosas da Oscillatoriales eram polifiléticas, conforme já relatado. A distribuição e abundância da população de cianobactérias nos efluentes da UTMINB foram investigadas pelo método da contagem de células viáveis (número mais provável, NMP). O NMP mostrou uma população de cianobactérias variando de 4.0 x 100 to ?2.4 x 108 cells?mL-1. Os locais Cava da Mina, com pH médio de 3,88, e o sistema de tratamento da usina, com pH 8,0, mostraram os mais baixos e mais altos valores de NMP, respectivamente. Para identificar os isolados de cianobactérias prejudiciais, um teste imunológico (ELISA) foi realizado para detectar microcistinas, uma hepatotoxina que causa envenenamento em humanos. A produção de microcistinas foi detectada em três isolados, Pseudanabaena galeata CENA84, Pseudanabaena sp. CENA81 e Leptolyngbya cf tenerrima CENA76. Esse resultado é inédito, pois não há relatos dos gêneros Pseudanabaena e Leptolyngbya como produtores de microcistinas. / Cyanobacteria are oxygenic photosynthetic microorganisms with wide metabolic and structural plasticity, which have biotechnological potential for exploration in metals biosorption and organic pollutants biodegradation. Due to its strong interactions with cations and a reliable supply of cheap biomass, cyanobacteria may be a promising biosorbent candidate for removing metals and radionuclides. In this way, in an attempt to find a cyanobacteria with this profile to remove 226Ra from a deactivated uranium mine effluents of the Ores Treatment Unit (UTM) belonging to the Nuclear Industries of Brazil (INB), Caldas, MG, twelve cyanobacterial strains were isolated from this environment. These strains were characterized morphologically as Aphanothece sp. CENA75, Rhabdoderma sp. CENA114, Synechococcus cf. lividus CENA79, Aphanocapsa cf. holsatica CENA80, Geitlerinema acutissimum CENA85, Pseudanabaena galeata CENA84, Pseudanabaena sp. CENA81, Leptolyngbya cf. tenerrima CENA76, Leptolyngbya sp. CENA83, Phormidium formosum CENA86, Phormidium violaceum CENA82 and Nostoc sp. CENA87. The molecular analysis of the isolates, based on the sequences of nearly complete 16S rRNA gene (1325 bp), was in agreement with the morphological analysis, with exception of Rhabdoderma sp. CENA114 and Phormidium violaceum CENA82 strains. The 16S rRNA sequences of these two strains showed low identities scores (<92%) with sequences from GenBank, which may represent novel cyanobacterial species. High percentages of identities (>96%) of 16S rRNA gene sequences were found between the remaining strains and of the GenBank. The phylogenetic tree of 16S rRNA sequences constructed using Neighbour-Joining method showed that unicellular strains of the orders Chroococcales and filamentous Oscillatoriales were polyphyletic, as reported earlier. The distribution and abundance of cyanobacterial population in the effluents of UTMINB were investigated by viable cells counting (most probable number, MPN) method. The MPN showed a cyanobacterial population range from 4.0 x 100 to ?2.4 x 108 cells?mL-1. The locations of the Pit Mine with pH 3.88 and the Plant System Treatment with pH 8.0 showed the lowest and highest MPN values, respectively. To identify harmful cyanobacterial isolates, an immunological test (ELISA) was carried out to detect microcystins, a hepatotoxin which cause human poisoning. Microcystins production was found in three isolates, Pseudanabaena galeata CENA84, Pseudanabaena sp. CENA81 and Leptolyngbya cf. tenerrima CENA76. This is a novel result since there is no report for both genera, Pseudanabaena and Leptolyngbya, as microcystin producers. Based on the obtained results, the Aphanothece CENA75 strain found in all UTM-INB effluents sampled, including in the Pit Mine location, which has an acidic pH (average of 3.88) and high level of uranium (5.68 mg?L-1) and radium, was selected for the 226Ra biosorption assays. The experiments performed in pH 3.5 and 5.0 showed that dried biomass of Aphanothece CENA75 behaves as a weakly acid resin. The ratio (final concentration/initial concentration) of 226Ra adsorption after 135 min in pH 3.5 and 5.0 was 0.86 and 0.82, respectively. These results showed that the dried biomass of Aphanothece CENA75 adsorbed low amount of 226Ra in both studied pH values. However, the increase of the radionuclide retention in pH 5.0 suggests that more adsorption may occur in pH above of this value.

A microbiota intestinal humana desempenha papel essencial no organismo saudável, pois sintetiza vitaminas, influencia no desenvolvimento e maturação do sistema imune da mucosa intestinal, além de exercer importante função protetora, competindo por nutrientes e receptores com bactérias patogênicas. A colonização desta microbiota se inicia na criança recém-nascida e alcança estabilidade em torno do segundo ano de vida, com consequência para a saúde da criança e do adulto. As diferenças na composição da microbiota estão relacionadas a diferentes níveis de contaminação ambiental e de diferentes fatores endógenos. O objetivo do nosso estudo foi analisar a microbiota fecal de crianças com idade entre 2 dias a 1 ano de idade, que vivem em baixas condições socioeconômicas em São Paulo, Brasil. Foram coletadas amostras de fezes de crianças saudáveis, nos seguintes pontos pós-nascimento: 2º e 7º dias, 1 mês, 3 meses, 6 meses e 1 ano de vida. O DNA bacteriano foi extraído diretamente a partir das amostras de fezes e as bibliotecas 16S rRNA foram construídas utilizando 2 iniciadores bactéria-específicos. Os clones foram selecionados aleatoriamente, parcialmente sequenciados e analisados com base em bibliotecas de gene 16S rRNA. Os principais grupos filogenéticos identificados foram Escherichia, Clostridium, Streptococcus e Bacteroides, do 1º ao 30º dia de vida. A partir do 3º mês, Streptococcus e bactérias não cultiváveis, além do gênero Escherichia, ganharam relevância na microbiota. Estes dados, em conjunto com as informações nutricionais, intercorrências clínicas e ambientais, sugerem a influência da contaminação ambiental e interpessoal no aumento da complexidade na composição da microbiota fecal. Essa abordagem molecular permitiu a análise da microbiota fecal do grupo selecionado, encontrando perfil bacteriano diferente do que é descrito nos países desenvolvidos. / The human intestinal microbiota plays essential role in healthy body since it synthesizes vitamins, influences on the development and maturation of the immune system of the intestinal mucosa. Furthermore, it also plays an important protective function competing for nutrients and receptors with pathogenic bacteria. The colonization of this microbiota starts in the newborn child and achieves stability around the second year of life, with consequence for the health of children and adults. The differences in the microbiota composition are related to different levels of environmental contamination and different endogenous factors. The aim of our study was to analyze the fecal microbiota of children ranging from 2º days to 1º year old living in low socioeconomic status in São Paulo, Brazil. We collected fecal samples of healthy children at the following points after birth: 2º e 7º days, 1 month, 3 months, 6 months and one year of life. Bacterial DNA was extracted directly from stool samples, and the 16S rRNA libraries were made using 2 bacterium-specific primers. The clones were randomly selected, and partially sequenced and analyzed based on 16S rRNA libraries. The main phylogenetic groups identified were Escherichia, Clostridium, Streptococcus, Bacteroides ranging from the 1º to 30º days of life. From the third month Streptococcus and uncultured bacteria, and, besides, Escherichia gender gained relevance in the microbiota. These data together with nutritional information, environmental and clinical intercurrents suggest the influence of interpersonal and environmental contamination in the increase of complexity in fecal microbiota composition. This molecular approach allowed the fecal microbiota analysis. This bacterial profile is different from described in developed countries.

L’ARNr 5S est une petite molécule d'ARN codée par le noyau, qui est partiellement adressée dans les mitochondries dans les cellules humaines. Bien que le mécanisme d'importation a été étudié, la fonction de ce phénomène est mal comprise. Les données publiées suggèrent que l’ARNr 5S importé pourrait participer à la synthèse des protéines mitochondriales, éventuellement être associé aux mitoribosomes. Cependant, les études structurelles ne supportent pas ces observations. Pour comprendre le rôle de l’ARNr 5S dans les mitochondries humaines, nous avons exploité plusieurs approches. Nous avons montré que seule une petite fraction de l’ARNr 5S pourrait être associé à mitoribosomes humaines, insuffisante pour former un complexe stoechiométrique 1:1. Nous avons appliqué l’approche de chromatographie d'affinité à l’ARN MS2 pour identifier les partenaires protéiques de l’ARNr 5S importé. Les résultats suggèrent que l’ARNr 5S pourrait être associé à des protéines ribosomales et des facteurs mitochondriaux d'assemblage du ribosome mitochondrial. Cela nous a permis de formuler une hypothèse sur la participation de l'ARNr 5S dans la biogenèse mitoribosomale. Ces données ont été partiellement validées par des expériences de co-immunoprécipitation, bien que plusieurs expériences sont encore nécessaires pour valider la participation ARNr 5S dans cette voie. / 5S rRNA is a small nuclear-encoded RNA molecule, which is partially imported into mitochondria from cytoplasm in human cells. Although the import mechanism was studied in details, the functional significance of this phenomenon is poorly understood. Published data suggest that imported 5S rRNA might participate in mitochondrial protein synthesis, possibly associating with mitoribosomes. However, structural studies do not support these observations. We have exploited several approaches to figure out the function of 5S rRNA in human mitochondria. Our studies showed that only a minor part of 5S rRNA pool could be associated with human mitoribosomes, insufficient to form stoichiometric 1:1 complex. We applied MS2 affinity chromatography approach to identify protein partners of 5S rRNA in human mitochondria. The results suggested that 5S rRNA could associate with mitochondrial ribosomal proteins and mitochondrial ribosome assembly factors. This allowed us to formulate a hypothesis about possible participation of 5S rRNA in mitoribosome biogenesis. The data were partially validated by co-immunoprecipitation experiments, although subsequent studies are required to validate 5S rRNA involvement in this pathway.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Coal is a fossil fuel extracted from underground by mining processes with the greatest availability in the world. In the state of Rio Grande do Sul, Brazil, the main source is the Candiota Mine with reserves of a billion tonnes likely to be mined in the open. This activity results in built environments with a high degree of disorganization, acid pH, heavy metal toxicity and susceptible to erosion. Exposing this weathering and sulphide minerals facilitates the formation of acid mine drainage, changing and affecting the quality of water, soil and air. Aiming to restore and preserve the desirable environmental conditions it is necessary to recover those mining areas. An alternative is the introduction of nitrogen-fixing bacteria that benefit the growth of leguminous plants. In this perspective, this study aimed to investigate the biodiversity of nitrogen fixing bacteria in soils built after coal mining. Contruidos soil samples were collected and after, in solution, added on substrate with plants-baits siratro and vetch to perform the isolation of nitrogen-fixing bacteria (BNF) through nodes. After the isolates were subjected to the pH (3.0, 4.0, 5.0, 7.0 and 9.0) testing and heavy metals (Cd, Cu, Pb, Ni and Zn in 3 different dosages ). Later isolates efficient at pH test were subjected to molecular characterization and verification of Noda and nifH genes. The data obtained showed a large number of isolates tolerant to different pH values and different metals with different concentrations. Molecular characterization showed 4 phyla α-β-γ-Proteobacteria and Firmicutes, whichever phylum α-proteobacteria, mainly Bradyrhizobium sp. Due greater adaptation in acidic environments, the predominant characteristic of degraded areas after coal mining. / O carvão é o combustível fóssil extraído do subsolo por processos de mineração com a maior disponibilidade no mundo. No estado do Rio Grande do Sul, Brasil, a principal fonte é a Mina de Candiota, com reservas de um bilhão de toneladas passíveis de serem mineradas a céu aberto. Essa atividade resulta em ambientes construídos com elevado grau de desestruturação, pH ácido, toxicidade de metais pesados e susceptível a processos erosivos. A exposição deste às condições atmosféricas e a minerais sulfetados possibilita a formação de drenagem ácida de mina, alterando e comprometendo a qualidade da água, do solo e do ar. Visando reestabelecer e preservar as condições ambientais desejáveis torna-se necessário a recuperação dessas áreas de mineração. Uma alternativa é a introdução de bactérias fixadoras de nitrogênio que beneficiam o crescimento de plantas leguminosas. Nesta perspectiva, este trabalho teve por objetivo investigar a biodiversidade de bactérias fixadoras de nitrogênio em solos construídos após a mineração de carvão. As amostras de solos contruídos foram coletadas e após, em solução, adicionadas em substrato com plantas-iscas de siratro e de ervilhaca para realizar o isolamento das bactérias fixadoras de nitrogênio (FBN) através dos nódulos. Após, os isolados foram submetidos ao teste de pH (3,0; 4,0; 5,0; 7,0 e 9,0) e de metais pesados (Cd, Cu, Pb, Ni e Zn, em 3 dosagens diferentes). Posteriormente os isolados mais eficientes no teste de pH foram submetidos a caracterização molecular e verificação dos genes nodA e nifH. Os dados obtidos apresentaram um amplo número de isolados tolerantes a diversos valores de pH e diferentes metais com variadas concentrações. A caracterização molecular demonstrou 4 filos α-β-γ-Proteobacteria e Firmicutes, prevalecendo o filo α-proteobacteria, principalmente por Bradyrhizobium sp., devido maior adaptação em ambientes ácidos, característica predominante das áreas degradadas após a mineração de carvão.