Submitted by Mario BC (mario@bc.ufrpe.br) on 2016-11-28T14:07:58Z No. of bitstreams: 1 Michelle Barros Pantoja.pdf: 1641953 bytes, checksum: 3ccb5972f0e3ceb3779eebd3c7ee502f (MD5) / Made available in DSpace on 2016-11-28T14:07:58Z (GMT). No. of bitstreams: 1 Michelle Barros Pantoja.pdf: 1641953 bytes, checksum: 3ccb5972f0e3ceb3779eebd3c7ee502f (MD5) Previous issue date: 2015-02-26 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / The peanut (Arachis hypogaea) is an oleaginous plant belonging to the Fabaceae family, native to South America. Its kernels are widely used as a food source and the production of oil. The main producing countries are China, India and the United States, which occupy the 1st, 2nd and 3rd places, respectively, in the ranking of world production. Brazil occupies the 17th position, in which the State of São Paulo stands out with 95% of total production of the country. The peanut is infected by several virus species, which limit the development of the crop in different parts of the world. The objective of this study was to detect the presence of virus in peanut plants in 10 counties of the State of São Paulo. Thirty-five samples (s) from 18 fields (f) were analyzed from Santa Adélia (3f/6s), Lusitânia (1f/1s), Jaboticabal (3f/6s), Itápolis e Pindorama (1f/2s, each), Tupã (4f/8s), Rancharia e Marília (1f/2s, each), Guaimbê (2f/4s) and Guarantã (1f/2s). The maintenance of the virus isolates was done in peanut plants graft inoculated. The isolates were submitted to biological, serological e molecular analyses. For host range study, Lactuca sativa, Capisicum annuum, C. annuum var. 679, C. annuum var. Ikeda, Chenopodium amaranticolor, C. quinoa, Datura stramonium, Gomphrena globosa, Nicotiniana tabacum, N. benthamiana, N. rustica, N. glutinosa, Physalis floridana and Solanum sculentum were mechanically inoculated and the symptoms analyzed. Based on Dot-ELISA results, the presence of the tospoviruses Groundnut ringspot virus (GRSV) (16s), Tomato chlorotic spot virus (TCSV) (6s) and do Tomato spotted wilt virus (TSWV) (3s) was detected. The comparison of the sequence obtained in the RT-PCR test with those deposited in the GenBank, revealed the occurrence of the GRSV. Furthermore, the metagenomic analysis showed the presence of the GRSV, as well as, the potyvirus Peanut motlle virus (PeMoV), both with complete genome. / O amendoim (Arachis hypogaeae L.) é uma oleaginosa pertencente à família Fabaceae, originária da América do Sul. Seus grãos são muito utilizados como fonte de alimento e na produção de óleo. Os principais países produtores são China, Índia e Estados Unidos, que ocupam o 1°, 2° e 3º lugar, respectivamente. O Brasil situa-se na 17ª posição, tendo o Estado de São Paulo contribuído com 95% da produção total do país. O amendoim é hospedeiro de um grande número de espécies de vírus, que limitam o desenvolvimento da cultura em diferentes partes do mundo. O objetivo deste trabalho foi detectar a presença de vírus em plantas de amendoim oriundas de 10 municípios paulistas, sendo analisadas amostras (am) de campos (cam) de Santa Adélia (3cam/6am), Lusitânia (1cam/1am), Jaboticabal (3cam/6am), Itápolis e Pindorama (1cam/2am, cada), Tupã (4cam/8am), Rancharia e Marília (1cam/2am, cada), Guaimbê (2cam/4am) e Guarantã (1cam/2am). Plantas de amendoim foram usadas para manutenção dos isolados virais, empregando-se transmissão sucessivas por meio de enxertias. Os isolados foram submetidos a análises biológicas em gama de hospedeiros, sorológicas pelo método Dot-ELISA e moleculares por RT-PCR e metagenômica. Os hospedeiros indicadores testado foram: Lactuca sativa, Capisicum annuum, C. annuum var. 679, C. annuum var. Ikeda, Chenopodium amaranticolor, C. quinoa, Datura stramonium, Gomphrena globosa, Nicotiniana tabacum, N. benthamiana, N. rustica, N. glutinosa, Physalis floridana e Solanum sculentum. Com base nos resultados do teste Dot-ELISA foi detectada a presença dos tospovírus Groundnut ringspot virus (GRSV) (16am), Tomato chlorotic spot virus (TCSV) (6am) e do Tomato spotted wilt virus (TSWV) (3am). Ao ser comparado os resultados obtidos no sequenciamento (Macrogen) do fragmento obtido no teste RT-PCR, com as sequências disponíveis no GenBank, ficou comprovada ocorrência do GRSV. Por outro lado, a análise metagenômica confirmou a presença do GRSV e detectou o potyvírus Peanut motlle virus PeMoV), ambos com genoma completo.

Submitted by isabel silva (isabel_manaus@yahoo.com.br) on 2017-06-22T15:28:22Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) CLONAGEM E CARACTERIZAÇÃO ENZIMÁTICA DE UMA LIPASE ISOLADA DE UMA BIBLIOTECA METAGENÔMICA DE TERRA PRETA DE ÍNDIO_Edson J1.pdf: 508907 bytes, checksum: 6b77566991052362063c90de82c2af97 (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2017-06-23T13:18:22Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) CLONAGEM E CARACTERIZAÇÃO ENZIMÁTICA DE UMA LIPASE ISOLADA DE UMA BIBLIOTECA METAGENÔMICA DE TERRA PRETA DE ÍNDIO_Edson J1.pdf: 508907 bytes, checksum: 6b77566991052362063c90de82c2af97 (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2017-06-23T13:18:48Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) CLONAGEM E CARACTERIZAÇÃO ENZIMÁTICA DE UMA LIPASE ISOLADA DE UMA BIBLIOTECA METAGENÔMICA DE TERRA PRETA DE ÍNDIO_Edson J1.pdf: 508907 bytes, checksum: 6b77566991052362063c90de82c2af97 (MD5) / Made available in DSpace on 2017-06-23T13:18:48Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) CLONAGEM E CARACTERIZAÇÃO ENZIMÁTICA DE UMA LIPASE ISOLADA DE UMA BIBLIOTECA METAGENÔMICA DE TERRA PRETA DE ÍNDIO_Edson J1.pdf: 508907 bytes, checksum: 6b77566991052362063c90de82c2af97 (MD5) Previous issue date: 2017-04-05 / CAPES / The advancement of molecular technologies in the scientific field has enabled the development of various forms of access to the genetic material of organisms in order to locate, map, isolate, characterize and decode the target genes of a particular individual or a set of individuals coexisting in a specific environment. Metagenomic studies are very promising in several areas of biotechnology, such as the discovery of new molecules of industrial biotechnological interest, the investigation of new antibiotics and drugs, the bioremediation of environments impacted with toxic metals and the prospection of various enzymes. The objective of this work was characterize enzymatically a previously screened lipase enzyme from Metagenomic Library of Terra Preta de Índio. Lipase gene sequence was isolated from the metagenomic library and expressed in Pichia pastoris under control of PGK promoter. Recombinant lipase was characterized by hydrolysis activity of lipid substrates and synthesis ability in organic solvent. In silico analyzes infer the identification of extracellular lipase belonging to the lipase family, superfamily -hydrolase and lipase activity molecular function. Enzyme was efficiently produced in P. pastoris and recombinant lipase showed activity of 374.59 U/mL hydrolyzing p-nitrophenyl palmitate, Vmax (ap.) 143,4 U/mL.min-1, Km (ap.) 1,4 mM and Kcat (ap.) 103,7 S-1. Enzyme had maximum activity at pH 8.0, temperature 90 ºC and remained stable at high temperatures. Synthesis ability was evaluated by the formation of ethyl laurate by esterification reaction of lauric acid with ethanol, yielding 70% conversion in 45 minutes. Recombinant protein is characterized as an alkaline, thermotolerant, activated by calcium, EDTA e detergents. / O avanço das tecnologias moleculares no campo científico, possibilitou o desenvolvimento de diversas formas de acesso ao material genético dos organismos, de forma a ser possível localizar, mapear, isolar, caracterizar e decodificar os genes alvo de um indivíduo particular ou de um conjunto de indivíduos coexistentes em um ambiente específico. Estudos metagenômicos são bastante promissores em diversas áreas da biotecnologia, como nas descobertas de novas moléculas de interesse biotecnológico industrial, na investigação de novos antibióticos e fármacos, na biorremediação de ambientes impactados com metais tóxicos e na prospecção de enzimas diversas. O objetivo deste trabalho foi caracterizar enzimaticamente uma enzima lipase previamente rastreada de uma Biblioteca Metagenômica de Terra Preta de Índio. A sequência correspondente ao gene de lipase foi isolada da biblioteca metagenômica, clonada e expressada em Pichia pastoris sob controle do promotor PGK. A lipase recombinante foi caracterizada pela atividade de hidrólise de substratos lipídicos e capacidade de síntese em solvente orgânico. Análises in silico da sequência proteica inferem a identificação de uma lipase extracelular pertencente à / - hidrolase e com função molecular para atividade lipásica. A enzima foi produzida eficientemente em P. pastoris e a lipase recombinante apresentou atividade de 374,59 U/mL hidrolisando p-nitrofenil palmitato, Vmax(ap.) 143,4 U/mL.min-1, Km(ap.) 1,4 mM e Kcat(ap.) 103,7 S-1. A enzima possuiu atividade máxima em pH 8,0, temperatura 90 ºC e se manteve estável em altas temperaturas. A capacidade de síntese foi avaliada pela formação de laurato de etila pela reação de esterificação do ácido láurico com etanol apresentando rendimento de 70% em 45 minutos de reação. A proteína recombinante se caracteriza como uma enzima alcalina, termotolerante, ativada por cálcio, EDTA e detergentes.

A construção de bibliotecas metagenômicas tornou possível o acesso ao potencial biotecnológico de micro-organismos não cultiváveis. O solo é um habitat pouco explorado, com elevada diversidade bacteriana que possuem genes codificadores de enzimas de interesse industrial, chamadas de biocatalisadores naturais. Dentre estas, destacam-se as enzimas lipolíticas, lipases e esterases, por promoverem a hidrólise de matérias-primas de alto valor agregado e com muitas aplicações biotecnológica. Devido a necessidade, existe uma constante busca para isolar novos genes com diferentes especificações. Neste trabalho, o gene lip4, isolado de biblioteca metagenômica de solo, foi superexpresso, purificado e identificado como membro da família V das lipases bacterianas. Tem atividade ótima hidrolisando triacilglicerol de cadeia média em pH alcalino sem presença de íons. A estrutura cristalográfica obtida identificou o dobramento conservado das α/β hidrolases e a tríade catalítica, Ser94-Asp217-His245, além da presença do domínio CAP, comum nas esterases. / Metagenomic libraries construction make possible the access to the biotechnological potential of not cultivated microorganisms. Soil environment has a big bacterial diversity with genes encoding industrial interest enzymes, named natural biocatalysts. Among these, lipolytic enzymes, that includes lipases and estarases, are able to catalyse different biochemistry reaction and promoting raw material hydrolysis with added values. In this overview, a search for new genes with different specifications, allowed isolation by functional screening in tributyrin agar, a gene lip4 from a soil metagenomic library. These gene was overexpressed, purified and identified as a member to family V of bacterial lipases. Presents better activity in alkaline pH with medium chain triacyglycerol without ions. Three dimensional structure of Lip4 identified a conserved α/β hydrolase backbone and the catalytic triad Ser94-Asp217-His245, besides the presence of CAP domain, common structure in esterase.

Considerando a dieta como um fator modulador do microbioma ruminal, neste trabalho objetivou-se investigar o impacto do bagaço da cana-de-açúcar sobre a composição e funcionalidade das espécies microbianas residentes no rúmen de carneiros (Ovis aries). Foram utilizados seis animais machos fistulados de O. aries, dos quais três foram alimentados com uma dieta composta por 70% de volumoso e 30% de concentrado (tratamento controle) e outros três animais alimentados com uma dieta similar a anterior, mas com 14% do volumoso substituído por bagaço de cana-de-açúcar (tratamento bagaço). O conteúdo ruminal (líquido e fibra) foram amostrados quinzenalmente durante 60 dias. A partir dessas amostras foram acessadas a estrutura e a composição da comunidade microbiana pela extração de DNA total e amplificação das regiões V3 e V6-V7 do gene 16S rRNA bacteriano e a região intergênica fúngica (ITS2). Além disso, foram feitas análises metagenômicas e metatranscriptômicas de comunidade microbianas enriquecidas em fibra ruminal para identificar enzimas lignocelulolíticas expressas. As frações líquida e fibrosa do conteúdo ruminal de O. aries revelaram uma comunidade bacteriana dominada principalmente por Bacteroidetes e Firmicutes ao longo de todo período experimental. Dois gêneros, Prevotella e Ruminococcus representaram 20% e 4% da comunidade bacteriana ruminal, respectivamente. Para a comunidade fúngica o filo Neocallimastigomycota representou 91% das sequências e os principais gêneros deste filo foram Piromyces, Neocallimastix, Orpinomyces, Anaeromyces, Caecomyces e Cyllamyces aderidos a fibra ruminal. O gênero Caecomyces, foi significativamente mais abundante na fibra ruminal de animais que se alimentaram de bagaço de cana-de açúcar. Além disso, foi observado um aumento significativo na frequência de enzimas como, por exemplo, 1,4-α-glucano, α-galactosidase, endo 1,4-β-xilanase, β- xilosidase, xilose isomerase, celobiose fosforilase e α-N-arabinofuranosidase no tratamento com bagaço de cana-de-açúcar. Considerando que a recuperação de enzimas a partir de comunidades microbianas naturalmente selecionadas para a degradação de biomassa é uma estratégia promissora para superar a atual ineficiência da ação enzimática na produção industrial de biocombustíveis, os resultados deste trabalho representam a possibilidade de aumentar a capacidade de recuperação ou descoberta de enzimas a partir de ruminantes, ou ainda, a possibilidade de manipular a estrutura do microbioma do rúmen para usá-lo como fonte de inóculo enriquecido em processos industriais de degradação de biomassa. / Considering the diet as a modulator of ruminal microbiome, this work aimed to investigate the impact of sugarcane bagasse on the composition and function of microbial species residents in the sheep (Ovis aries) rumen. Six cannulated male animals were used in the experiment, where three individuals were fed on a diet consisting of 70% forage and 30% concentrate (control treatment), and three were fed on a similar diet, but with sugarcane bagasse replacing 14% of the forage portion (bagasse treatment). The ruminal content (i.e., liquid and fiber) were sampled every two weeks during 60 days. From these samples, the structure and composition of the microbial community were assessed by total DNA extraction and amplification of V3 and V6-V7 regions of 16S rRNA gene from bacteria and the fungal intergenic region (ITS2). Furthermore, metagenomics and metatranscriptomics approaches were used to evaluate the enrichment of specific members of the microbial community in the ruminal fiber and genes related to lignocellulolytic enzymes. The liquid and fiber fractions of the O. aries rumen revealed a microbial community dominated mainly by Firmicutes and Bacteroidetes throughout the experimental period. The genera Prevotella and Ruminococcus accounted for 20% and 4% of the bacterial community of rumen, respectively. In the fungal community, the phylum Neocallimastigomycota accounted for 91% of sequences and its main genera adhered on the ruminal fiber were Piromyces, Neocallimastix, Orpinomyces, Anaeromyces, Caecomyces and Cyllamyces. The genus Caecomyces was significantly more abundant in the ruminal fiber in animals fed on sugarcane bagasse. Furthermore, there was a significant increase in the frequency of enzymes, such as α-1,4-glucan, α-galactosidase, endo- 1,4-β-xylanase, β-xylosidase, xylose isomerase, cellobiose phosphorylase and α- Narabinofuranosidase in the bagasse treatment. Considering that the recovery of enzymes from ecosystems naturally evolved for degradation of biomass is a promising strategy to overcome the current inefficient enzymatic action in industrial production of biofuels, the results of this study bring great possibilities to increase the discovery and or recovery of enzymes from ruminants, as well as the possibility of the ruminal microbiome structure manipulation to be used as source of an enriched inoculum for biomass degradation in industrial processes.

Orientador: Valéria Maia Merzel / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-25T14:04:53Z (GMT). No. of bitstreams: 1 Verde_LeandroCostaLima_D.pdf: 7821596 bytes, checksum: b0f165c3b35ff62438f4e8f59035eb82 (MD5) Previous issue date: 2014 / Resumo: O processo de biodegradação do petróleo em reservatórios pode resultar em mudanças na composição e propriedades físico-químicas de óleos brutos e gases naturais, as quais levam à diminuição do teor de hidrocarbonetos saturados, produzindo óleos mais pesados e com baixo valor econômico. O uso combinado de técnicas dependentes e independentes de cultivo pode nos permitir um melhor entendimento acerca da comunidade de micro-organismos que habita os reservatórios de petróleo, incluindo aqueles responsáveis por esta biodegradação. O conhecimento sobre a composição microbiana, suas funções e interações com outros micro-organismos e com o ambiente pode levar à definição de estratégias de monitoramento e/ou controle da biodegradação em reservatórios. Este estudo teve como finalidade avaliar a diversidade de micro-organismos e genes envolvidos na degradação de hidrocarbonetos presentes em amostras de petróleo provenientes de dois poços terrestres da Bacia Potiguar (RN), identificados como GMR75 (poço biodegradado) e PTS1 (poço não-biodegradado), através da construção de bibliotecas de genes catabólicos (alcano monooxigenases - alk, dioxigenases que hidroxilam anéis aromáticos ¿ ARHDs e 6-oxocyclohex-1-ene-1-carbonyl-CoA hidroxilase - bamA) e sequenciamento em larga escala de metagenoma e metatranscriptoma de enriquecimentos microbianos aeróbios. Os resultados obervados mostraram uma distribuição diferencial dos genes catabólicos entre os reservatórios, sendo o óleo biodegradado mais diverso para os genes alk e bamA. As sequências foram semelhantes aos genes alkB dos gêneros Geobacillus, Acinetobacter e Streptomyces, aos genes ARHD dos gêneros Pseudomonas e Burkholderia, e aos genes bamA do gênero Syntrophus. A análise quantitativa dos genes catabólicos de degradação de hidrocarbonetos presentes e expressos nos enriquecimentos microbianos em diferentes etapas da biodegradação do óleo, através de PCR Tempo Real, demonstrou maior atividade do gene que codifica a enzima dioxigenase nas comunidades microbianas enriquecidas, e os resultados obtidos pela técnica de microarray sugeriram a existência de novas sequências dos genes alk e ARHD provindas do reservatório de petróleo. As análises das sequências obtidas a partir do metagenoma e metatranscriptoma mostraram que a comunidade bacteriana recuperada no enriquecimento aeróbio é bastante diversa, com predominância do Filo Actinobacteria, seguido de Proteobacteria. As sequências com maior abundância e níveis de expressão foram relacionadas aos genes que codificam as proteínas ligase CoA de ácido graxo de cadeia longa, envolvida na degradação de compostos aromáticos; descarboxilase, envolvida com o ciclo do glioxilato, e o fator sigma da RNA polimerase, envolvida com a regulação da resposta ao estresse oxidativo, sugerindo uma adaptação da comunidade microbiana às condições do enriquecimento e um processo inicial de biodegradação dos hidrocarbonetos. Os resultados obtidos neste trabalho fornecem dados inéditos sobre a diversidade de genes catabólicos e de membros da comunidade microbiana potencialmente envolvidos com a degradação do óleo em reservatórios de petróleo / Abstract: The process of oil biodegradation in reservoirs may result in changes in the composition and physico-chemical properties of crude oils and natural gases, which lead to the decrease of the content of saturated hydrocarbons, producing heavy oils and with low economic value. The combined use of both dependent and independet cultivation techniques may allow us to better understand the microbial community inhabiting oil reservoirs, including those microorganisms responsible for oil degradation. The knowledge about the microorganisms, ther functions and interactions with other microorganisms and the environment may lead to the definition of monitoring and/or control strategies of biodegradation in oil reservoirs. This study aimed at evaluating the diversity of microorganisms and genes involved in the degradation of hydrocarbons present in oil samples from two onshore reservoirs at Potiguar Basin (RN), identified as GMR75 (biodegraded) and PTS1 (non- biodegraded), through the construction of catabolic gene libraries (alkane monooxygenases - alk, aromatic ring hydroxylating dioxygenases ¿ ARHD and 6-oxocyclohex-1-ene-1-carbonyl-CoA hydroxylase - bamA) and highthroughput sequencing of metagenome and metatranscriptome from aerobic microbial enrichments. Results observed showed a differential distribution of catabolic genes between the reservoirs, being the biodegraded oil more diverse for the alk and bamA genes. The sequences were similar to alkB genes from Geobacillus, Acinetobacter and Streptomyces genera, to the ARHD genes from Pseudomonas and Burkholderia genera, and to the bamA genes from Syntrophus genus. Quantitative analysis of the hydrocarbon degradation genes present and expressed in the microbial enrichments during the different phases of oil biodegradation by Real-Time PCR showed that there was a higher activity of dioxygenase enzymes in the enriched microbial communities and results from microarray assays suggested the existence of new alk and ARHD gene sequences originated from the oil reservoir. Metagenomic and metatranscriptomic analyses showed a highly diverse bacterial community, dominated by the Phylum Actinobacteria, followed by Proteobacteria. The most abundant and active sequences were affiliated to the Long-chain-fatty-acid-CoA ligase protein, involved in the degradation of aromatic compounds; decarboxylase, which is involved with the glyoxylate cycle, and RNA polymerase sigma factor, which is involved in regulating the oxidative stress response, suggesting an adaptation of the microbial community to the enrichment conditions and an initial process of biodegradation of hydrocarbon compounds. The results obtained in this work bring innovative data on the diversity of catabolic genes and microbial community members potentially involved with oil degradation in petroleum reservoirs / Doutorado / Genetica de Microorganismos / Doutor em Genetica e Biologia Molecular

Les picocyanobactéries marines des genres Prochlorococcus et Synechococcus sont les organismes photosynthétiques les plus abondants de la planète et ils contribuent de façon substantielle à la production primaire mondiale. Alors que le genre Prochlorococcus se caractérise par sa forte abondance dans les régions oligotrophes et son génome réduit, le genre Synechococcus se distingue par sa grande diversité génétique et pigmentaire ainsi qu'une aire de distribution plus étendue.Le principal objectif de ce travail a été de mettre en relation la diversité génétique de ces organismes avec leur niche écologique par des approches de génomique comparative et de métagénomique. Tout d'abord, le développement d'une méthode de scaffolding (WiseScaffolder) a permis de clore 32 nouveaux génomes de Synechococcus, lesquels ont été intégrés au système d'information Cyanorak, dédié à l'annotation de gènes orthologues. Ces nouveaux génomes, complétant les 65 génomes disponibles pour ces deux genres, ont fait l'objet d'analyses comparatives afin de mieux comprendre la diversité et l'évolution de ce phylum et de définir les gènes spécifiques de différents groupes phylogénétiques, potentiellement liés à leur adaptation à des niches écologiques distinctes.Ces génomes ont ensuite été utilisés comme référence, en conjonction avec le gène marqueur petB, pour analyser les données de métagénomique issues de l'expédition TARA-Océans. Ces analyses ont notamment mis en lumière la diversité génétique, la distribution et l'importance écologique de certains clades phylogénétiques. Ce travail soulève de nouvelles hypothèses quant au rôle des picocyanobactéries dans le fonctionnement global des océans. / Marine picocyanobacteria Prochlorococcus and Synechococcus genera are the most abundant photosynthetic organisms and contribute substantially to global primary production. While the genus Prochlorococcus is characterized by its high abundance in oligotrophic regions and its reduced genome, Synechococcus is characterized by a larger genetic and pigment diversity and a wider area of distribution.The main objective of this PhD thesis was to link the genetic diversity of these organisms to the environmental conditions of their ecological niche by comparative genomics and metagenomics approaches. Firstly, the development of a scaffolding method (WiseScaffolder) has allowed us to close 32 new genomes of Synechococcus which were integrated into the Cyanorak information system dedicated to the annotation of orthologous genes. These new genomes, supplementing the 65 genomes previously available for these two genera, allowed us to perform comparative analyses which led to a better understanding of the diversity and evolution of this phylum and to the definition of genes sets specific to different phylogenetic groups and thus potentially related to their adaptation to different ecological niches.These genomes were then used as reference, in conjunction with the marker gene petB gene, to analyze metagenomic data produced in the frame of the Tara-Oceans Expedition. In particular, these analyzes highlighted the genetic diversity, distribution and ecological importance of some phylogenetic clades. This work raises new hypotheses about the role of picocyanobacteria in the overall functioning of the oceans.

Chez les mammifères, les Eucaryotes représentent une composante importante des micro-organismes peuplant le tractus digestif. Au total, 16 champignons et 2 autres micro-eucaryotes ont été identifiés dans l’échantillon provenant de la personne saine. Par contre, peu d’espèces fongiques ont été identifiées via l’échantillon provenant du patient atteint de tuberculose.D'autre part, la diversité des eucaryotes qui peuplent les primates non humains tels que les grands singes demeure relativement inexploré .Pour ces raisons, nous avons entrepris une analyse moléculaire dans le but de détecter ces micro-organismes eucaryotes, dont certains demeurent pathogènes pour l’homme, en utilisant un seul échantillon fécal prélevé chez un gorille sauvage en provenance de l’ouest du Caméroun. Ces analyses ont été suivies d’une détection moléculaire spécifique du potentiel pathogène de ces eucaryotes du tractus gastro-intestinal des gorilles sauvages. En conséquence, ils ont permis d’identifier 87 espèces eucaryotes. Nous avons également signalé la présence de champignons pathogènes, et de parasites. Afin d’examiner d’une manière plus approfondie si ces gorilles abritaient d’autres parasites, nous avons analysé 91 échantillons fécaux à la recherche d’agents pathogènes comme la leishmaniose. Les résultats ont montré que 12 échantillons contenaient des parasites du genre Leishmania et 4 phlébotomes comme vecteurs. L’analyse moléculaire a été effectuée par enchaînement de 3 différentes réactions de polymérase en chaine (PCR) spécifiques aux agents de la leishmaniose. / Eukaryotes represent significant component of the mammalian intestinal tract. Their occurrence might have either beneficial or virulent parasitic effects on the host.A total of 16 fungal species and 2 other micro-eukaryotes were identified in healthy fecal sample. Contrary, a very few fungi were detected in the fecal sample from patient with resistant tuberculosis. On another hand , The diversity of eukaryotes inhabiting non-human primates such as great apes remains relatively unexplored .For these reasons we undertook an extensive molecular analysis for detecting eukaryotic microbiota including some human eukaryotic pathogens in a single fecal sample from a wild western lowland gorilla from Cameroon, and then followed by specific molecular detection of potential human eukaryotic pathogen in gastrointestinal tracts of wild population of gorillas. Our effort resulted in retrieving 87 eukaryotic species. We also reported the occurrence of pathogenic fungi, parasites. To further examine whether these gorillas harbor other sever parasites, we screened 91 of their fecal samples for the presence of blood borne pathogen such as Leishmania. The results showed that, 12 fecal samples contained Leishmania parasites, and 4 contained phlebotomine sand fly vectors. The molecular identity was determined by running 3 different polymerase chain reaction tests for detection of Leishmania major.

Magister Scientiae - MSc / The metagenomic approach to gene discovery circumvents conventional gene and gene product acquisition by exploiting the uncultured majority of microorganisms in the environment. It was demonstrated in this study that metagenomic methods are suitable for gene mining in extreme environments that harbor very high levels of unculturable microorganisms. DNA was extracted from Antarctic mineral soil samples taken from the Miers Valley, Antarctica. The metagenomic DNA was also used to construct a fosmid library comprising over 7900 clones with an average insert size of 29 kb. PCR amplification using bacterial and archaeal 16S rRNA gene specific primers and subsequent denaturing gradient gel electrophoresis (DGGE) of bacterial 16S rDNA amplicons showed that a small percentage of bacterial diversity was captured in the metagenomic fosmid library. Activity-based screening for lipase and esterase genes using a tributyrin plate assay yielded twelve positive clones. LD1, a putative, novel cold-active GDSL lipase/esterase was identified and sequenced. The C-terminal domain of the ORF was found to be an autotransporter similar to those associated with type V secretion systems in Gram negative bacteria. Sub-cloning of the gene resulted in lipolytic activity in E. coli. Preliminary enzyme assays have determined that LD1 hydrolyses p-nitrophenyl esters with chain lengths shorter than C10, an indication that the enzyme is an esterase. Complete purification and characterisation of this enzyme is subject to further study. / South Africa

>Magister Scientiae - MSc / A metagenomic gene library prepared from soil found beneath a mummified seal carcass in the Miers Valley, Antarctica, suggests an environment rich in uncharacterised biodiversity including enzymes with possible application to industrial processes. A sequence based gene mining investigation was performed on a clone, which archives a metagenomic sequence from this environment. The sequence was annotated using de novo bioinformatics and molecular biology techniques. A predicted NAD+-dependent DNA ligase, ligDB1 was selected for further characterisation. LigDB1 encodes a gene product that contains all the sequence features of a functional ligase. The protein was overexpressed in a heterologous E. coli host and purified to homogeneity. LigDB1 did not exhibit nick sealing activity, but was able to perform AMP-dependent DNA relaxation in the presence of high concentrations of enzyme. DNA modifying enzymes from cold environments perform optimally at low temperatures and may be of use as molecular tools in biotechnology. Complete characterisation of this enzyme is subject to further investigations

Metagenômica é a técnica que permite analisar os genomas de microorganismos que habitam determinados nichos do ambiente sem a necessidade de isolar e cultivar cada um separadamente. Ao conjunto de microorganismos que habita um determinado nicho se dá o nome de microbi- oma. Análises do perfil da diversidade taxonômica e funcional de comunidades microbianas em microbiomas são comuns em estudos de metagenômica. No entanto, atualmente as plata- formas de uso geral (como MG-RAST e IMG/M) tendem a separar as análises baseadas em reads (sequências não montadas) das baseadas em contigs (sequências montadas), isto dificulta as análises destes dados. Motivado por esta separação, desenvolvemos uma plataforma web, batizada de CARAVELA, que facilita a conexão entre os resultados de análises de diversidade taxonômica e funcional baseadas em reads e contigs respectivamente. Uma das principais fun- ções da plataforma CARAVELA é associar a identificação taxonômica de cada read com o contig que este read faz parte e, anotações funcionais do contig, quando existirem. Essa função deve permitir a rápida identificação de contigs potencialmente quiméricos bem como contigs taxonomicamente bem resolvidos. Também é possvel fazer buscas, tais como: listar todos os contigs que tenham um ou mais reads classificados como Pseudoxanthomonas suwonensis em sua composição e ainda, é possvel navegar nos contigs de maneira similar a navegadores de metagenomas tradicionais. Podem ser utilizados como arquivos de entrada a sada de outros programas, desde que o formato atenda certos padrões. A plataforma CARAVELA foi desenvol- vida com Java, HTML, CSS, Javascript e Mysql, e com o fim de testar a ferramenta, utilizamos o conjunto de dados metagnômicos obtidos a partir da operação de compostagem do Parque Zoológico de São Paulo. / The taxonomic diversity analysis (read-based) and functional analysis (contig / gene-based) from metagenomic studies usually generate information that is complementary. However, the tools that produce gene annotations (eg IMG / M) and taxonomic assignments (eg MyTaxa) do not allow easy integration of these results. Motivated by this split, we are develop a web platform called Caravela to facilitate the integration, search and visualization of information provided by read-based analyzes and contig / gene-based analyzes. The tool is able to display the list of contigs and for each contig, it displays annotated genes, reads participating in its composition and rate associated with each read (when such association exists). Such a capability enable manual / automated curation of assembly as well as taxonomic assignments (detection of possible mis-assignments). The platform able to accept output files from a variety of tools, as long as the file formats follow certain standards. The tests was performed on a dataset of metagenomic reads obtained from the composting operation of the São Paulo Zoological Park. The tool was implemented using Java technology, HTML, CSS and Javascript. Information was stored in a MySQL database.