Submitted by Bruna Buch (bruna.buch@gmail.com) on 2019-01-31T14:18:48Z No. of bitstreams: 1 BrunaBuch_Tese.pdf: 6059154 bytes, checksum: ab596bb37969e3a6ac3aea0557b5363e (MD5) / Rejected by Elza Mitiko Sato null (elzasato@ibilce.unesp.br), reason: Solicitamos que realize correções na submissão seguindo as orientações abaixo: Problema 01) Solicitamos que corrija o ano descrita na capa para 2019, o ano de entrega da dissertação na Seção Técnica de Pós-Graduação Problema 02) Segundo a Portaria nº 206, de 4 de setembro de 2018, todos os trabalhos que tiveram financiamento CAPES deve constar nos agradecimentos a expressão: "O presente trabalho foi realizado com apoio da Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Código de Financiamento 001 Agradecemos a compreensão. on 2019-01-31T16:28:27Z (GMT) / Submitted by Bruna Buch (bruna.buch@gmail.com) on 2019-01-31T16:59:59Z No. of bitstreams: 1 BrunaBuchTese.pdf: 6058711 bytes, checksum: 91a5b502646692883f387f5ae5a634f6 (MD5) / Approved for entry into archive by Elza Mitiko Sato null (elzasato@ibilce.unesp.br) on 2019-01-31T17:24:13Z (GMT) No. of bitstreams: 1 buch_b_dr_sjrp.pdf: 6058711 bytes, checksum: 91a5b502646692883f387f5ae5a634f6 (MD5) / Made available in DSpace on 2019-01-31T17:24:13Z (GMT). No. of bitstreams: 1 buch_b_dr_sjrp.pdf: 6058711 bytes, checksum: 91a5b502646692883f387f5ae5a634f6 (MD5) Previous issue date: 2018-12-05 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / As cianobactérias são importantes componentes das comunidades aquáticas em diversos ecossistemas, graças a longa história evolutiva do grupo que desenvolveu diversas adaptações fisiológicas e citológicas que permitiram a sua dominância ao redor do globo. As cianobactérias são também os bactérias fotossintetizantes com a morfologia mais diversificada e, embora essa qualidade tenha sido extensivamente utilizada pelos cientistas para a delimitação dos táxons e reconstrução da história evolutiva, vem perdendo cada vez mais espaço para o uso de marcadores moleculares, os quais são capazes de inferir relações filogenéticas mais robustas e que mais proximamente refletem o percurso evolutivo traçado por esses microrganismos. Desse modo, o objetivo da realização deste estudo foi caracterizar taxonomicamente populações de cianobactérias bentônicas de ambientes lênticos da região noroeste do estado de São Paulo, utilizando uma abordagem polifásica, por meio do uso do gene rRNA 16S e da estrutura secundária do ITS 16S-23S, além de considerar aspectos morfológicos e ecológicos. Como resultado deste trabalho, 41 populações de cianobactérias bentônicas foram avaliadas, sendo alocadas em 15 gêneros distribuídos em 11 famílias e cinco ordens. A ordem Oscillatoriales foi a mais representativa entre as populações estudadas (68,3%, 28 populações), seguida pela ordem Synechococcales (19,5%, 8 populações). A análise filogenética do gene RNAr 16S foi capaz de revelar a presença de táxons crípticos, que embora apresentem morfologia correspondente com táxons já descritos, formaram clados separados, indicando se tratarem de táxons ainda não conhecidos, e esse foi o caso de 13 populações aqui estudadas. Parte dos táxons crípticos foi trabalhada em maior profundidade, resultando em três manuscritos apresentados na forma de capítulos que correspondem à descrição dos novos gêneros e espécies Koinonema pervagatum (Capítulo III) e Blennothricopsis periphytica (Capítulo IV), além da descrição de três novas espécies para o gênero Phormidium (Capítulo V), com o registro da primeira espécie bentônica produtora de microcistina para o estado de São Paulo. Embora a prospecção dos genótipos tóxicos, utilizando marcadores específicos para os genes mcyE e sxtA responsáveis pela síntese de microcistinas e saxitoxinas, respectivamente, tenha revelado apenas uma linhagem tóxica, esse resultado é positivo do ponto de vista de impactos relacionados à presença de cianobactérias em corpos d’água para uso público. Entretanto, mostra a importância dos estudos de prospecção de toxinas em cianobactérias bentônicas no Brasil, ainda pouco explorados. / Cyanobacteria are important components of aquatic communities in different ecosystems, thanks to its long evolutionary history that provided several physiological and ecological adaptations, allowing them to spread around the globe. Cyanobacteria are also the most morpological diversified photossintetic bacterial group and, despite many taxonomists have extensively used this character to delimit taxa and to reconstruct their evolutionary history, it has been losing its prominence to molecular markers, which are most suited to infer robust phylogenetic relationships that properly reflect the evolutionary path followed by these organisms. Therefore, the aim of the study was to taxonomically characterize benthic cyanobacterial populations in lentic habitats from the Northwest region of São Paulo state, using a polyphasic approach, through the phylogenetic analysis of the 16S rRNA gene and the 16S 23S ITS secondary structure, aside from morphological and ecological aspects. As result, 41 benthic cyanobacterial populations were evaluated and assigned to 15 genera distributed in 11 families and five orders. The Oscillatoriales order was the most representative among all (69.3%, 28 populations), followed by the Synechococcales (19.5%, eight populations). The 16S rRNA phylogenetic analysis revealed the presence of cryptotaxa, which, despite morphologically ressembling previously described taxa, formed separated clades, suggesting not yet acknowledged taxa. That was the case of 13 populations studied. Some of these cryptotaxa were deeper evaluated in three manuscripts presented here as chapters and described as the new genera and species Koinonema pervagatum (Chapter III) and Blennothricopsis periphytica (Chapter IV), the description of three new species from the Phormidium genus (Chapter V) and the first record of a benthic microcystin producing cyanobacteria in the São Paulo state. Although the toxic genotypes prospection using specific molecular markers for the mcyE and sxtA genes, responsible for microcystin and saxitoxin production, respectively, revealed only one toxic strain, it demonstrates a positive result regarding the impact caused by toxic cyanobacterial strains in waters for public use worldwide. However, it highlights the importance of studies on potentially toxic benthic cyanobacterial communities, still little explored in Brazil.

Submitted by Évelyn Lira (costa_eve@yahoo.com.br) on 2018-11-03T15:34:17Z No. of bitstreams: 3 TESE_Évelyn Costa Lira.pdf: 5199119 bytes, checksum: 3542649866fd36a1d250872b70b7f22e (MD5) Carta de Encaminhamento.pdf: 120410 bytes, checksum: d948e19f5bae8bee2cb485c166efda6d (MD5) Diploma de Doutorado ou Ata de Defesa.pdf: 129558 bytes, checksum: 3cdafbe7ae427bf6a30e4dd767a402e5 (MD5) / Approved for entry into archive by PPGBIOTEC Biotecnologia (ppg_biotec.ufam@yahoo.com.br) on 2018-11-06T20:43:46Z (GMT) No. of bitstreams: 3 TESE_Évelyn Costa Lira.pdf: 5199119 bytes, checksum: 3542649866fd36a1d250872b70b7f22e (MD5) Carta de Encaminhamento.pdf: 120410 bytes, checksum: d948e19f5bae8bee2cb485c166efda6d (MD5) Diploma de Doutorado ou Ata de Defesa.pdf: 129558 bytes, checksum: 3cdafbe7ae427bf6a30e4dd767a402e5 (MD5) / Rejected by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br), reason: A carta anexada deve ser a "Carta de Encaminhamento para o Autodepósito" disponível em http://biblioteca.ufam.edu.br/servicos/teses-e-dissertacoes Dúvidas? ddbc@ufam.edu.br on 2018-11-07T12:20:35Z (GMT) / Submitted by Évelyn Lira (costa_eve@yahoo.com.br) on 2018-11-07T13:48:25Z No. of bitstreams: 3 TESE_Évelyn Costa Lira.pdf: 5199119 bytes, checksum: 3542649866fd36a1d250872b70b7f22e (MD5) Diploma de Doutorado ou Ata de Defesa.pdf: 129558 bytes, checksum: 3cdafbe7ae427bf6a30e4dd767a402e5 (MD5) Carta de Encaminhamento para Autodepósito.pdf: 146139 bytes, checksum: 0b1a55433f2ef8e007ce7d6045af2443 (MD5) / Approved for entry into archive by PPGBIOTEC Biotecnologia (ppg_biotec.ufam@yahoo.com.br) on 2018-11-07T14:13:03Z (GMT) No. of bitstreams: 3 TESE_Évelyn Costa Lira.pdf: 5199119 bytes, checksum: 3542649866fd36a1d250872b70b7f22e (MD5) Diploma de Doutorado ou Ata de Defesa.pdf: 129558 bytes, checksum: 3cdafbe7ae427bf6a30e4dd767a402e5 (MD5) Carta de Encaminhamento para Autodepósito.pdf: 146139 bytes, checksum: 0b1a55433f2ef8e007ce7d6045af2443 (MD5) / Rejected by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br), reason: O Documento anexado como "Carta de Autodepósito" não é o documento exigido. O documento que deve ser anexado é a "Carta de Encaminhamento para o Autodepósito" disponível em http://biblioteca.ufam.edu.br/servicos/teses-e-dissertacoes Dúvidas? ddbc@ufam.edu.br on 2018-11-07T14:56:48Z (GMT) / Submitted by Évelyn Lira (costa_eve@yahoo.com.br) on 2018-11-07T15:22:23Z No. of bitstreams: 3 TESE_Évelyn Costa Lira.pdf: 5199119 bytes, checksum: 3542649866fd36a1d250872b70b7f22e (MD5) Diploma de Doutorado ou Ata de Defesa.pdf: 129558 bytes, checksum: 3cdafbe7ae427bf6a30e4dd767a402e5 (MD5) Carta de Encaminhamento para Autodepósito.pdf: 33896 bytes, checksum: cbb2fa1f7645982868cec33dd021fd5d (MD5) / Approved for entry into archive by PPGBIOTEC Biotecnologia (ppg_biotec.ufam@yahoo.com.br) on 2018-11-12T19:17:48Z (GMT) No. of bitstreams: 3 TESE_Évelyn Costa Lira.pdf: 5199119 bytes, checksum: 3542649866fd36a1d250872b70b7f22e (MD5) Diploma de Doutorado ou Ata de Defesa.pdf: 129558 bytes, checksum: 3cdafbe7ae427bf6a30e4dd767a402e5 (MD5) Carta de Encaminhamento para Autodepósito.pdf: 33896 bytes, checksum: cbb2fa1f7645982868cec33dd021fd5d (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2018-11-12T20:47:54Z (GMT) No. of bitstreams: 3 TESE_Évelyn Costa Lira.pdf: 5199119 bytes, checksum: 3542649866fd36a1d250872b70b7f22e (MD5) Diploma de Doutorado ou Ata de Defesa.pdf: 129558 bytes, checksum: 3cdafbe7ae427bf6a30e4dd767a402e5 (MD5) Carta de Encaminhamento para Autodepósito.pdf: 33896 bytes, checksum: cbb2fa1f7645982868cec33dd021fd5d (MD5) / Made available in DSpace on 2018-11-12T20:47:54Z (GMT). No. of bitstreams: 3 TESE_Évelyn Costa Lira.pdf: 5199119 bytes, checksum: 3542649866fd36a1d250872b70b7f22e (MD5) Diploma de Doutorado ou Ata de Defesa.pdf: 129558 bytes, checksum: 3cdafbe7ae427bf6a30e4dd767a402e5 (MD5) Carta de Encaminhamento para Autodepósito.pdf: 33896 bytes, checksum: cbb2fa1f7645982868cec33dd021fd5d (MD5) Previous issue date: 2017-11-30 / FAPEAM - Fundação de Amparo à Pesquisa do Estado do Amazonas / The role of the human microbiota in health and disease processes has received increased attention due to the ease of characterization of microbial communities using independent culture methods, which are based on the analysis of hypervariable regions of the gene 16S rRNA. The vaginal microbiota is inhabited by microbial communities that play a very important role in the maintenance of vaginal homeostasis and in the presence of colonization by pathogenic microorganisms, but the mechanisms by which they exert this influence are not yet so well defined. in this context, an understanding of their relative abundance and variations is necessary for the recognition of potential pathogenic microorganisms and the physiological processes of protection of this microbiota. This study evaluated the vaginal microbial diversity in four different conditions: I Microbiota Normal II Vaginal Candidosis Microbiota III Bacterial vaginosis microbiota IV microbiota presenting pre-malignant and malignant lesions of the cervix. Cervical samples from 187 women were collected, characterized and diagnosed at the molecular level for the presence of HPV, C. trachomatis, T. vaginalis e N. gonorrhoeae. HPV positive samples were genotyped at ABI 3500 and HPV co-infection were related to the presence of other pathogens and socio-economic and clinical factors obtained through the questionnaire filled out by the volunteers. Significance and association analyzes were performed using the Chi-square Test of Pearson, exact of Fischer’s Test and Kruskal-Wallis test using R 2.9.0. After the groups were composed, four libraries of amplicons of the regions V1-V2 of the gene 16S rRNA and, later sequenced on Ion PGM platform. The sequences generated were analyzed using the QIIME and classified by comparison in the Greengenes database. the prevalence found for HPV was 51.33%, and the most prevalent types were HPV 16, 58 and 33. The prevalence found for CT and TV were 6.42% and 12.83%, respectively, with co- HPV / CT infection in 5.20% and HPV / VT in 6.25% of the women. The DNA of N. gonorrhoeae were not found in samples. as to the estimation of microbial diversity, the number of samples sequenced was enough to guarantee coverage of the total diversity found. Overall abundancy revealed a predominance of the genus lactobacillus on the four study groups, followed by the genres Ureaplasma, Prevotella and Shuttleworthia, followed by the study groups. the greatest microbial diversity was found in the vaginosis group, with the most abundant genera Prevotella, Shuttleworthia and Megasphaera, followed by the lesion group, abundance of genders Ureaplasma, Prevotella e Shuttleworthia. The differences at the species level are extremely necessary for the understanding of the physiological role of the vaginal microbial in the maintenance of autochthonous balance and of pathogenic mechanisms in the development of diseases related to vaginal microbiota. In addition, an understanding of the functionality of the types of CST is necessary to complement what we already know about its structure. Studies are needed to investigate the changes and stability of this microbiota. / O papel da microbiota humana nos processos de saúde e doença tem recebido maior atenção devido à facilidade para a caracterização das comunidades microbianas utilizando os métodos independentes de cultivo, que se baseiam na análise das regiões hipervariáveis do gene 16S rRNA. A microbiota vaginal é habitada por comunidades microbianas que exercem papel importantíssimo para a manutenção da homeostase vaginal e prevenção da colonização por microrganismos patogênicos, mas os mecanismos pelos quais exercem essa influência ainda não são tão bem definidos. Neste contexto, uma compreensão quanto a sua abundância relativa e variações é necessária para o reconhecimento de microrganismos patogênicos potenciais e de processos fisiológicos de proteção dessa microbiota. Este estudo avaliou a diversidade microbiana vaginal em quatro condições distintas: I. microbiota autóctone; II. Microbiota apresentando candidose vaginal; III. microbiota apresentando vaginose bacteriana e; IV. microbiota apresentando lesões pré-malignas e malignas do colo do útero. Amostras cervicais de 187 mulheres foram coletadas, caracterizadas e diagnosticadas a nível molecular quanto à presença de HPV, C. trachomatis, T. vaginalis e N. gonorrhoeae. Amostras HPV+ foram genotipadas em ABI 3500 e a co-infecção do HPV foi relacionada à presença dos outros patógenos e fatores sócio-econômicos e clínicos obtidos através do questionário preenchido pelas voluntárias. Análises de significância e associação foram realizadas através dos Testes Qui-Quadrado de Pearson, Exato de Fischer e Kruskal-Wallis utilizando o R 2.9.0. Após a composição dos grupos, foram montadas quatro bibliotecas de amplicons das regiões V1-V2 do gene 16S rRNA e, posteriormente sequenciadas em plataforma Ion PGM. As sequências geradas foram analisadas utilizando o QIIME e classificadas por comparação no banco Greengenes. A prevalência encontrada para HPV foi de 51,33% sendo que os tipos mais prevalentes foram HPV 16, 58 e 33. As prevalências encontradas para CT e TV foram de 6,42% e 12,83%, respectivamente, sendo observadas co-infecção HPV/CT em 5,20% e HPV/TV em 6,25% das mulheres. O DNA de N. gonorrhoeae não foi encontrado nas amostras. Quanto à estimativa da diversidade microbiana, o número de amostras sequenciado foi suficiente para garantir a cobertura da diversidade total encontrada. A abundância geral revelou predominância do gênero Lactobacillus nos quatro grupos de estudo, seguido pelos gêneros Ureaplasma, Prevotella e Shuttleworthia entre os grupos estudados. A maior diversidade microbiana foi encontrada no grupo Vaginose, tendo como gêneros mais abundantes Prevotella, Shuttleworthia e Megasphaera, seguido pelo grupo Lesão, com abundância dos gêneros Ureaplasma, Prevotella e Shuttleworthia. As diferenças a nível de espécie são extremamente necessárias para o entendimento do papel fisiológico da microbiota vaginal na manutenção do equilíbrio autóctone e de mecanismos patogênicos no desenvolvimento de doenças relacionadas à microbiota vaginal. Além disso, uma compreensão da funcionalidade dos tipos de CST é necessária para complementar o que já sabemos sobre sua estrutura. Estudos são necessários para investigar as mudanças e a estabilidade desta microbiota.

Os fungos micorrízicos arbusculares (FMAs, filo Glomeromycota) formam associações simbióticas com a maioria das plantas vasculares. Normalmente, as hifas dos FMAs crescem no solo e colonizam o interior das raízes. No entanto, não se sabe se as espécies mais abundantes detectadas no solo, por meio da identificação com base na morfologia dos esporos assexuais, são também as mais abundantes no interior das raízes, devido às dificuldades para a identificação dos FMAs com base nas estruturas intrarradiculares. Assim, o objetivo do presente trabalho foi avaliar a estrutura da comunidade de FMAs em cana-de-açúcar sob dois manejos de colheita por meio da identificação das espécies que estão no solo na forma de esporos assexuais e aquelas que estão nas raízes usando o sequenciamento de clones do gene rRNA 18S. Amostras de solo e raízes de cana-de-açúcar de três variedades e dois manejos de colheita: SEM QUEIMA prévia e COM QUEIMA prévia à colheita, foram coletadas em um experimento localizado no município de Novo Horizonte, SP. Foram utilizadas três abordagens para a identificação dos FMAs no interior das raízes: emprego de (1) iniciador específico para fungos em geral, (2) iniciador específico para FMAs e (3) iniciadores específicos para grupos de FMAs. O número de esporos por 50 g de solo, a riqueza de espécies observada e estimada e a diversidade de esporos não diferiram significativamente entre os manejos SEM QUEIMA e COM QUEIMA. Efeitos significativos de variedades de cana-de-açúcar ou na interação dos fatores manejo e variedade não foram observados. A análise de ordenação com base nos esporos identificados também não indicou separação das amostras em função dos tratamentos. Entretanto, plantas do tratamento sob manejo SEM QUEIMA apresentaram as maiores taxas de colonização micorrízica arbuscular, quando comparadas às plantas do tratamento sob manejo COM QUEIMA. Esses dados indicam que a taxa de colonização micorrízica arbuscular é um indicador mais sensível à mudança de manejo de colheita da cana-de-açúcar do que os outros indicadores avaliados. Após a extração de DNA das raízes, o uso dos iniciadores específicos para fungos em geral, para FMAs e iniciadores específicos para grupo de FMAs não resultou em sequências de Glomeromycota. Mesmo assim, a comunidade de fungos associados às raízes detectada por sequenciamento do gene rRNA 18S foi avaliada. Os resultados indicam que a estrutra da comunidade fúngica associada às raízes de cana-de-açúcar diferiu significativamente entre os manejos de colheita SEM QUEIMA e COM QUEIMA prévia, apesar de não haver diferenças na riqueza e índices de diversidade de unidades taxonômicas operacionais observadas. Em geral, estudos adicionais devem ser feitos para otimizar as condições para amplificação do gene rRNA 18S de FMAs para melhor entender a ecologia dos mesmos. / Arbuscular mycorrhizal fungi (AMF, Glomeromycota) form mutualistic symbioses with most land plants. AMF hypha generally grow through the soil and colonize the cortical tissue of the plant roots. However, it is not known whether the most abundant species in the soil, determined based on the morphology of asexual spores are the most abundant inside the roots, due the difficulties in identifying AMF based on intraradical structures. Therefore, the aim of this study was to evaluate the AMF community structure in sugarcane rhizosphere and roots under two harvesting managements, based on spores in the soil and sequencing of 18S rRNA gene clones, respectively. Sugarcane rhizosphere soil and roots were sampled from three varieties, under two harvesting managements: without pre-harvesting burning and with pre-harvesting burning, at an experimental field located in Novo Horizonte (São Paulo, Brazil). Three approaches were used to identify AMF inside the roots: (1) using fungi-specific primers, (2) using AMF-specific primers and (3) using AMF group-specific primers. The number of spores in the soil, the observed and estimated species richness and the diversity of AMF spores in the treatments without and with pre-harvesting burning were not statistically different. Statistically significant effects of sugarcane varieties or the interaction of the factors Harvesting Management and Varieties were not observed. Ordination analysis based on the identified spores did not show clustering by treatments. However, intraradical root colonization rates were higher in the treatment without pre-harvesting burning, as compared to the treatment with pre-harvesting burning. These data indicate that intraradical colonization rate may be used as a more sensitive indicator of environmental changes due to harvesting management, as compared to the other indicators evaluated. The use of fungi-specific, AMF-specific and AMF group-specific primers did not allow the detection of Glomeromycota in the sugarcane roots sampled from the field experiment. Nonetheless, the fungal communities associated with sugarcane roots detected by 18S rRNA gene clone sequencing were evaluated. The results indicate that the fungal communities associated with sugarcane roots from the treatments without and with pre-harvesting burning were statistically different, even though no differences in operational taxonomic unit richness and diversity indices were observed. In general, additional studies are necessary to optimize AMF 18S rRNA gene amplification for a better understanding of their ecology.

Dentre os contaminantes de água subterrânea de maior importância está o tetracloroeteno (PCE), o qual é altamente tóxico e potencialmente carcinógeno. As comunidades bacterianas de águas subterrâneas contaminadas com PCE e a diversidade de bactérias capazes de degradar esses organoclorados são pouco conhecidas. O objetivo deste trabalho é comparar a estrutura das comunidades de bactérias de amostras de água subterrânea em uma área contaminada com PCE e selecionar um consórcio microbiano capaz de degradar eficientemente o PCE em reator horizontal de leito fixo (RHLF). Amostras de água subterrânea de oito poços de monitoramento, instalados em uma área contaminada com PCE foram coletadas e analisadas para determinação de oxigênio dissolvido, potencial redox, condutividade elétrica, pH e concentração de tetracloroeteno, tricloroeteno, cis-dicloroeteno e cloreto de vinila (COVs). As amostras foram analisadas também para a determinação da estrutura das comunidades de bactéria por PCRDGGE e seqüenciamento de clones do gene rRNA 16S. Os parâmetros físico-químicos oscilaram consideravelmente ao longo do tempo em todos os poços de monitoramento (PM). Tetracloroeteno e tricloroeteno foram detectados apenas no PM6. As estruturas das comunidades bacterianas dos PMs analisados mostraram tanto variação temporal quanto espacial. As análises das comunidades bacterianas nos PM6 e PM8, contaminado e não-contaminado com PCE, revelaram resultados semelhantes aos obtidos por DGGE. Uma maior riqueza estimada de espécies bacterianas foi observada nas amostras do PM8, pelo menos em duas épocas de amostragem, sugerindo que a contaminação com PCE está associada com a redução da diversidade bacteriana em água subterrânea. Cultivos de enriquecimento e ensaios de degradação do PCE foram realizados utilizando-se um RHLF, o qual foi preenchido com sedimento do PM6 imobilizado em espuma de poliuretano e enriquecido com meio mineral básico suplementado com PCE. A análise das alterações nas comunidades de bactérias nos reatores foi feita por PCRDGGE e seqüenciamento parcial do gene rRNA 16S. No ensaio de degradação do PCE no RHLF foi utilizado meio com PCE suplementado ou não com lactato e acetato. Tanto pelo DGGE quanto pelo seqüenciamento, foi observada a seleção de bactérias específicas no reator. A partir das análises de seqüenciamento, essas bactérias foram identificadas como Alphaproteobacteria e Sphingobacteria. No ensaio de degradação do PCE, os parâmetros físico-químicos do meio não mostraram variações ao longo do comprimento dos reatores. As análises de COVs mostraram uma grande eficiência na degradação do PCE (98%), com um tempo de retenção de 12 horas, não havendo diferença significativa na percentagem de degradação em meio com lactato ou acetato, com relação ao controle sem fonte de carbono. No processo de degradação nenhum dos produtos da via de degradação do PCE foi detectado, o que sugere uma via alternativa de degradação do PCE, a qual ocorre em aerobiose. / Tetrachloroethene (PCE) is one of the most important contaminants of groundwater, since it is highly toxic and potentially carcinogenic. The bacterial communities of PCE contaminated groundwater and the diversity of bacteria capable of degrading this contaminant are barely known. The objective of this work is to compare the structure of bacterial communities from groundwater samples from a PCE contaminated site and select a microbial consortium capable to degrading efficiently PCE in a horizontal fixed bed reactor (HFBR). Groundwater samples from eight monitoring wells, installed in a PCE contaminated site were collected and analyzed for determination of dissolved oxygen, redox potential, electrical conductivity, pH, and concentrations of tetrachloroethene, trichloroethene (TCE), cis- and trans-dichloroethene, vinyl chloride (VOCs). The structure of the bacterial communities was determined by PCR-DGGE and 16S rRNA gene clone sequencing. The physical-chemical parameters oscillated considerately throughout time in all the monitoring wells (MW). PCE and TCE were detected only in MW6. The bacterial community structures in the groundwater from the MWs analyzed showed temporal and spatial variation. The analysis of the bacterial communities in MW6 and MW8, contaminated and non-contaminated with PCE, respectively, based on sequencing of 16S rRNA gene clones revealed results to the ones observed by DGGE. Estimated richness of bacterial species was higher in samples from MW8, at least in two sampling times, suggesting that the contamination with PCE is associated with reduction of bacterial diversity in groundwater. Enrichment cultures and PCE biodegradation assays were performed in a HFBR, which was filled with sediment from MW6 immobilized onto polyurethane foam and enriched with basic mineral medium supplemented with PCE. Shifts in bacterial community structure were analyzed using PCRDGGE and partial sequencing of 16S rRNA gene clones. In the PCE biodegradation assays in the HFBR, were performed in medium containing lactate or acetate. DGGE and 16S rRNA gene clone sequencing data suggest selection of specific bacteria in the reactor. Sequencing data showed that these bacteria belong to Alphaproteobacteria and Sphingobacteria. In the PCE biodegradation assays, media physical-chemical parameters did not show variation along the reactor length. VOC analyses showed a great efficiency in the degradation of PCE (98%) with a residence time of 12 hours in the reactor, and no significant differences were observed in the presence of lactate or acetate, as compared to the medium without a carbon source. During the biodegradation process, none of the products from the anaerobic pathway of PCE reductive dechlorination was detected, suggesting that an alternative PCE biodegradation pathway is occurring in aerobiosis.

Staphylococcus aureus has been extensively studied, yet it remains unclear why certain individuals continually carry the bacteria while others do not. Livestock workers are known to be at an increased risk of S. aureus colonization, but have not been as studied as other high risk groups, including hospitalized patients, have been. Culture based studies have shown other bacteria may decrease the likelihood of S. aureus colonization. Here, we utilize 16s rRNA sequencing to better characterize the ecologic relationships between S. aureus and the other microbes in the nares and oropharynx in a population of livestock workers. A cross-sectional, epidemiological study was conducted enrolling 59 participants (26 of which had livestock contact) in Iowa. Participants were enrolled in one of four ways: from an existing prospective cohort study (n=38), from the Iowa Department of Natural Resources Animal Feeding Operations database (n=17), through Iowa county fairs (n=3), and through snowball sampling (n=1). We collected two sets of swabs from the nares and oropharynx of each participant. The first set of swabs was used to assess the microbiome via 16s rRNA sequencing and the second was used to culture S. aureus. We observed livestock workers to have greater diversity in their microbiomes compared to those with no livestock contact. In the nares, there were 26 operational taxonomic units found to be different between livestock workers and non-livestock workers with the greatest difference seen with Streptococcus and Proteobacteria. In the oropharynx, livestock workers with swine exposure were more likely to carry several pathogenic organisms. We also observed colonized livestock workers to be more likely to carry P. gingivalis which may act as a bridge allowing S. aureus to adhere to Streptococcus in the oral cavity. While we observed no significant differences when comparing colonized persons to non-colonized persons in either the nares or oropharynx, Corynebacterium was more abundant in the colonized persons. Colonized individuals also had greater diversity in their nasal microbiome compared to non-colonized individuals. However, when comparing persistently colonized persons to intermittently colonized persons, we found Corynebacterium argentorantense to be more abundant in the persistently colonized individuals. We hypothesized the genera present in the nares and oropharynx of S. aureus carriers would be different from that of non-carriers and there would be differences in the nasal and oropharyngeal microbiomes based on livestock contact and carrier state (persistent, intermittent, and non-carrier). While there were no significant differences between carriers and non-carriers, we were able to identify several operational taxonomic units that were different between livestock worker carrier and non-carriers as well as differences by carrier state. The results of this study are the first to characterize the livestock worker nasal and oropharyngeal microbiomes. Additionally, the results shed light onto several organisms that may be influential in S. aureus carriage. However, further studies are needed to better understand these relationships and determine causality.

The bacterium Vibrio vulnificus is an autochthonous inhabitant of estuarine waters and also found in shellfish such as oysters. It is a human pathogen of importance in the seafood industry, and can also infect recreational water users. Currently, recognized methods of detection rely upon isolation of pure cultures which requires at least 24 hours. To reduce the time needed for identification of the pathogen and simultaneously ascertain the virulence potential of the strains present, real-time PCR assays and sample processing procedures were developed (Chapter 1). These assays discriminate between type A (environmental, generally lower virulence) and type B (clinical, higher virulence) isolates. The genetic relationships between environmental V. vulnificus strains isolated from permitted and prohibited shellfish harvesting areas was determined using BOX-PCR genomic fingerprinting coupled with sequence analysis of three proposed virulence markers: (1) the virulence correlated gene (vcg), (2) 16S rRNA type and (3) presence/absence of the vulnibactin gene (viuB) (Chapter 2). The real-time PCR assays were able to detect the presence of seeded V. vulnificus in environmental water at a concentration of 160 cells 100·ml-1. In seeded oyster homogenates, the assays were able to detect a minimum of 10³ cells and 10² cells per reaction of type A and type B respectively. The phylogenetic analysis separated the majority of type A/ vcgE strains isolated from permitted shellfish harvesting areas from those isolated from prohibited harvesting areas. The genomic (BOX-PCR) fingerprints of type A and type AB isolates were more similar to one another than to type B isolates. Only one type A/ vcgE isolate contained the viuB gene; however, eight type B/ vcgC isolates had that gene. No obvious grouping was discerned between type B/ vcgC isolates from permitted versus prohibited shellfish harvesting areas or between those possessing the viuB gene versus those lacking viuB. These data provide insight into the ecology and correlation between population biology and general water quality, as gauged by the classification of the shellfish growing area waters. The 16S typing assays can be used for routine rapid typing to aid in risk assessment and reduce infection frequency through consumption of contaminated seafood.

A Molecular Approach to Assessing Meiofauna Diversity in Marine Sediments Heather C. Hamilton Abstract The purpose of this study was to determine if a molecular approach could be applied to calculating the diversity of meiofauna in marine sediments from two sites in Tampa Bay, FL, similar to the approach of McCaig et al, 1999 in calculating the diversity of microbes in pastureland soils. The approach includes extracting total DNA directly from the sediment and amplifying the 18S rRNA gene by PCR. Clone libraries from the 18S gene would be created for each site and 300 sequences from each clone library would be obtained. These sequences would then be phylogenetically analyzed and assigned to an OTU, from which diversity indices can be calculated. The phylogenetic analysis of the sequences from the two sites revealed that of the 102 OTUs assigned from the sequences, only 7 OTUs included sequences from both sites, while 93 OTUs contained sequences from one site or from the other. Thus the sites were phylogenetically different from each other. Shannon diversity indices calculated for each site showed a difference between the two sites and paralleled diversity indices for macrofauna data for each site collected by the Hillsborough County Environmental Protection Commission. Sequences from 30 OTUs were completely sequenced and identified by phylogenetic comparison with a metazoan reference alignment. A discrepancy between the sequence data and data collected from preserved samples taken at each site was evident upon analysis: roughly 60% of each preserved sample consisted of nematodes and 10% consisted of copepods, while roughly 30% of the identified OTUs consisted of copepods and 10% consisted of nematodes. This discrepancy could be explained if the OTUs that were not identified consisted of nematode sequences or if a primer bias were present in the PCR amplification such that the regions flanking the primer site in the nematode sequences inhibited primer annealing.

16S rRNA gene analysis has shown that bacterial diversity in the GAB bores studied was limited to the genera Hydrogenobacter in the phylum Aquificae, Thermus in the phylum Deinococcus-Thermus, Desulfotomaculum in the phylum Firmicutes, the alpha-, beta- and gamma-classes of the phylum Proteobacteria and the phylum Nitrospirae. There was no clone closely related to members of the delta-proteobacteria and epsilon-proteobacteria classes detected. The number of bacterial strains directly isolated from the Fairlea and the Cooinda bores were far less than the numbers of distinctive phylotypes detected by the 16S rRNA gene characterisation. In addition none of the bacterial strains directly isolated from the water samples were represented in the 16S rRNA gene clone libraries. Similar discrepancies between the bacterial populations obtained from the 16S rRNA gene analysis and those obtained from direct isolation have been reported in the literature (Dunbar et al., 1999; Kampfer et al., 1996; Suzuki et al., 1997; Ward et al., 1998; Ward et al., 1997). However, in general, the phyla with which the isolates were affiliated were the same as those phyla to which the clones belonged. The environmental changes introduced (by bringing the artesian water up to the surface and exposing it to four types of metal coupons made of carbon steels identified by codes ASTM-A53B, ASTM-A53, AS-1074 and AS-1396 and commonly used in bore casings) led to changes in the bacterial community structures. In general, the species which proliferated in the communities before and after the changes were different. The diversity of the bacterial species in the community decreased following the environmental changes. Clones dominating the clone libraries constructed from newly established bacterial communities also differed from the clones dominating the libraries constructed from the bacterial communities which had existed naturally in the bores. These trends toward change in the bacterial communities were observed at both the Fairlea and the Cooinda bore sites. All four metal types incubated in the Fairlea bore water lost between 3.4 and 4.7% of their original weight. In contrast none of the metals incubated in Cooinda bore water lost weight. Clone library A1 showed that the natural population of the Fairlea bore was dominated by clone A1-3, which represented a novel species related to the isolate boom-7m-04. But after metal incubation (and recording of the metal weight loss), the bacterial community was dominated by clone PKA34B, which has a 95% similarity in its 16S rRNA gene sequence with Desulfotomaculum putei. Desulfotomaculum species are known to cause metal corrosion due to their byproduct H2S. But the low level of phylogenetic relatedness found does not provide enough information to speculate on whether the species represented by clone PKA34B is a member of the genus Desulfotomaculum or not. However, the fact that clone PKA34B dominated the PKA clone library by 50% makes the species it represents a suspected candidate likely to be involved with the metal weight loss at the Fairlea bore. In contrast, clone library 4381 showed that the natural population of the Cooinda bore was dominated by clone 4381-15 representing a species distantly related to a hydrogen oxidiser Hydrogenophaga flava (95% similarity). The dominating clone of the new community formed after metal incubation was clone COO25, which has 99% similarity with Thermus species that have not been reported to be involved with metal corrosion to my knowledge. In this project detection, identification and comparative quantification by 16S rRNA gene-targeted PCR probing with probes 23B and 34B were successfully developed for a Leptothrix-like species and for a Desulfotomaculum-like species represented by clones PKA23B and PKA34B respectively. This method of probing permits a fast, sensitive and reproducible detection, identification and at least a comparative quantification of the bacteria in the environment without the need for culturing. Therefore it is extremely suitable for use in bacterial population monitoring. PCR probing with the 34B probe has a potential commercial use as a means of screening for bores with a potential high risk of corrosion due to this Desulfotomaculum-like species. Direct isolation of bacteria from the GAB water has resulted in the isolation of seven strains from the Fairlea bore and eight from the Cooinda bore. Among these isolates, three novel strains were studied in detail. Reports on the characterisation of strain FaiI4T (T=Type strain) from the Fairlea bore (Kanso & Patel, 2003) and strain CooI3BT from the Cooinda bore have been published (Kanso et al., 2002). The data generated during this project add to our current information and extend our knowledge about the bacterial communities of the GAB's sub-surface environment. This information will provide a basis for further ecological studies of the GAB. Studies on involvement of certain groups of bacteria with the corrosion of metals used in bore casings could provide a foundation for further studies to develop maintenance and managing strategies for the GAB bores.

Comme le nucléole joue un rôle fondamental dans l’expression des protéines, via la synthèse des ARN ribosomiques, il n’est donc pas surprenant que des études aient révélé un lien étroit, entre des dysfonctionnements nucléolaires et l’origine de certaines maladies humaines. La découverte, il y a plusieurs années, d’un taux anormalement élevé de protéines nucléolaires dites argyrophiles ou AgNORs, dans les cellules tumorales, a permis d’envisager leur utilisation comme outil diagnostique ou pronostique du cancer. Détectées, de manière in vitro grâce à leur affinité pour l’argent, l’identification de quelques protéines AgNORs n’a pourtant pas permis d’établir une caractéristique commune à toutes les protéines argyrophiles détectées dans les extraits nucléolaires. Ainsi, bien que le test colorimétrique AgNOR soit utilisé dans de nombreux laboratoires académiques, l’absence d’identification de protéines AgNORs spécifiques du processus de cancérisation, a limité son utilisation en laboratoire clinique. Comme certaines limites technologiques et expérimentales ont limité leur caractérisation chez l’humain, nous avons donc décidé de reprendre les recherches sur ce sujet et de le réactualiser grâce aux avancées technologiques et scientifiques. Les protéines AgNORs étant étroitement liées à la biogenèse des ribosomes, nous avons donc décidé d’amorcer nos recherches chez la levure Saccharomyces cerevisiae, dans laquelle, la voie de biosynthèse des ribosomes a été particulièrement bien décrite. Devant l’intérêt biologique et médical de ces protéines, l’objectif de ce projet a donc été triple : 1-identifier des protéines AgNORs chez la levure 2-caractériser les propriétés physico-fonctionnelles et physico-chimiques de ces protéines AgNORs. 3-utiliser ces caractéristiques physico-chimiques pour rechercher de nouvelles AgNORs humaines, spécifiques de processus de cancérisation et potentiellement utilisables comme marqueurs tumoraux./The nucleolus is a subnuclear compartment that organized around ribosomal gene (rDNA) repeats NORs, which encode for ribosomal RNA. A peculiar group of acidic proteins which are highly argyrophilic are also localized at the same sites as NORs, thus allowing NORs to be very clearly and rapidly visualized by silver nitrate staining procedures. However, if three human argyrophilic proteins, UBF, C23 (nucleolin) and B23 (nucleophosmin), have been associated for staining of NOR, the exact number of AgNOR proteins and their intrinsic biochemical feature are unclear. Here, we have performed an heterologous screen in a genetically tractable eukaryotic organism (budding yeast) for the identification of novel AgNOR proteins and in vitro characterized an intrinsic feature that underlies silver binding and offers a strong predictive value for the identification of novel human AgNOR proteins.

The assembly of ribosomal RNA (rRNA) and ribosomal proteins (r-proteins) into ribosomal subunits (30S and 50S) is a complex process. Transcription of rRNA requires antitermination proteins and the primary transcripts are processed by ribonucleases. R-proteins and rRNAs are chemically modified, the r-proteins bind to the rRNAs and the formed RNA-protein complexes are folded into mature ribosomal subunits. All these processes are well-coordinated and overlapping. Non-ribosomal factors are required for proper assembly and maturation of the ribosomal subunits. Two of these factors are the RimM and RbfA proteins, which bind to 30S subunits and are important for efficient processing of 16S rRNA. Lack of either RimM or RbfA results in a reduced amount of polysomes and a lower growth rate. An increased amount of RbfA can partially compensate for deficiencies shown by a RimM lacking mutant. Here, mutations that alter phylogenetically conserved amino acids in RimM have been constructed. One of these (rimM120), which resulted in the replacement of two adjacent tyrosines by alanines, reduced the growth rate three-fold and also decreased the processing efficiency of 16S rRNA. The RimM120 mutant protein showed a much reduced binding to the 30S subunits. Suppression of the rimM120 mutant was achieved by increased amount of the RimM120 protein, by overexpression of rbfA, or by mutations that changed r-protein S19 or 16S rRNA. A variant of r-protein S13, which was previously isolated as a suppressor to a deletion of rimM (∆rimM), suppressed also the rimM120 mutation. The wild-type RimM protein, but not the RimM120 protein, was shown to bind r-protein S19 in the 30S subunits. The changes in S13, S19 and 16S rRNA that compensated for the deficiencies shown by the rimM mutants are all located within a small region of the head of the 30S subunit, suggesting that this region is the likely target for the RimM action. To isolate RbfA variants that show reduced association with the 30S subunits, phylogenetically conserved, surface exposed amino acid residues of RbfA were changed to alanines or, in some instances, to amino acids of the opposite charge to that in the wild-type protein. Alterations of F5, R31, D46 and D100 had the largest effect on growth. Mutations in the metY-nusA-infB operon, isolated as suppressors to the ∆rimM mutant, were shown to increase the amounts of RbfA. In a ∆rimM mutant, all RbfA protein was found associated with the 30S subunits and no free RbfA was detected. The RlmB protein was shown to be the methyltransferase responsible for the formation of Gm2251 in 23S rRNA in Escherichia coli. Unlike a Saccharomyces cerevisiae mutant that lacks the orthologue to RlmB, Pet56p, which methylates mitochondrial rRNA, a ∆rlmB mutant did not show any defects in ribosomal assembly.