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Recent research in microbial ecology has focused on how aquatic bacterial communities are assembled. Only a few of these studies follow a “Gleasonian” approach where the roles of single bacterial populations are in focus. In this thesis, novel molecular tools were used to describe the distribution and evolutionary relationships of microbes in productive aquatic environments. Many new phylogenetic groups of bacteria were identified, likely representing bacterial populations restricted to productive freshwaters. I also addressed the dynamics and functional role of individual bacterial populations in eutrophic lakes and brackish environments with a focus on either biogeochemically significant or potentially pathogenic representatives. Flavobacteria blooms were observed, on occasions characterized by high heterotrophic production. In addition to high temporal dynamics microbial community composition and function differed on the spatial scale, as exemplified by free-living and Cyanobacteria-associated habitats. At the community scale, microbial processes, such as biomass production and substrate uptake could be predicted from the presence and absence of individual bacterial populations. I also studied the niches of potentially pathogenic Vibrio populations in various coastal waters. Using a novel culture-independent method, a V. cholerae population was detected along the entire Swedish coastline. Results from an environmental survey and a laboratory mesocosm experiment reveal that phytoplankton-derived dissolved organic matter enhance the growth of V. cholerae and other Vibrio spp. and hence create a largely overlooked niche for these heterotrophic bacteria. This thesis and future work on the role of individual bacterial populations will facilitate predictions of biogeochemical cycles and the distribution of bacteria in the context of global climate change and local eutrophication.

Résumé : Le nucléole est considéré comme étant une « usine » à produire des ribosomes. Cette production est la fonction la plus énergivore de la cellule. Elle met en jeu les trois ARN polymérases et représente 80% de l’activité de transcription au sein d’une cellule. Les trois quarts de cette activité de transcription correspondent à la synthèse des ARNr par l’ARN polymérase I (ARNPI). Ainsi mieux comprendre les mécanismes cellulaires se déroulant à l’intérieur de ce compartiment permettra le développement de nouveaux traitements contre le cancer. La synthèse d’ARNr par l’ARNPI est régulée à trois niveaux : l’initiation de la transcription, l’élongation et le nombre de gènes de l’ARNr en transcription. La plupart des travaux qui se sont intéressés à ces niveaux de régulation ont été réalisés avec des cellules en phase exponentielle de croissance. Au cours de mes travaux, je me suis attardé sur la régulation de la transcription par l’ARNPI au cours de la phase G1 du cycle cellulaire et au début de la phase S. Ainsi mes résultats ont montré que si la chromatine des gènes de l’ARNr est essentiellement dépourvue de nucléosomes, la régulation de l’ARNPI diffère dans des cellules en G1 et au début de la phase S. J’ai pu de ce fait observer qu’en G1, la transcription de l’ARNPI se concentre sur un nombre réduit de gènes en transcription. Dans des cellules arrêtées au début de la phase S avec de l’hydroxyurée, la transcription de l’ARNPI est perturbée par un défaut de maturation de l’ARNR. Fort de ces résultats sur la nature des gènes ribosomaux en phase G1, je me suis attardé à la réparation de ces gènes lors de cette phase. Alors que dans des cellules en phase exponentielle de croissance irradiées avec des UVC, la chromatine des gènes de l’ARNr se ferme ; je n’ai pas observé la formation de nucléosomes suite à l’irradiation de cellules synchronisée en G1. Mes résultats montrent également que la réparation est plus efficace. Parallèlement, j’ai exploré l’assemblage du complexe de réparation par excision de nucléotides. Toutefois, les résultats obtenus sont peu concluants. / Abstract : The nucleolus is thought to be a “factory” involve in the production of ribosomes. This production is the most energetically consuming process in the cell. The three RNA polymerases are involved and this represents 80% of the total transcription activity of the cell. Three quarters of this transcriptional activity correspond to the synthesis of rRNA by the RNA polymerase I (RNAPI). So a better understanding of the cellular mechanisms taking place in this compartment may help for the development of new drugs against cancer. The synthesis of rRNA by RNAPI is regulated at three levels: initiation of transcription, elongation and the number of rRNA genes in transcription. Most of the works that characterized those levels of regulation were done in exponentially growing cells. During my work, I studied the regulation of RNAPI during the G1 phase of the cell cycle and during the early S phase. So my results have shown that if the chromatin of the rRNA genes mostly depleted of nucleosomes, the regulation of the RNAPI differs in cells in G1 and early S phase. I could observe that in G1, RNAPI transcription concentrates on a reduced number of transcribed rRNA genes. In cells arrested in early S phase with hydroxyurea, RNAPI transcription is disrupted by a defect in rRNA processing. With this results on the nature of the ribosomal genes in G1, I started the analysis of the DNA repair of those genes during this phase of the cell cycle. In UVC irradiated exponentially growing cells, the rRNA genes are closing. But in cells synchronized in G1, I could not observe the deposition of nucleosomes after UVC irradiation. Moreover, my results show an increase repair of the locus. In parallel, I have explored the assembly of the complex of nucleotide excision repair. However, the results were not conclusive.

Bacterial resistance to 4,6-type aminoglycoside antibiotics, which target the 30S ribosomal subunit, has been traced to the arm/rmt family of rRNA methyltransferases. These plasmid-encoded enzymes transfer a methyl group from S-adenosylmethionine to N7 of the buried G1405 in the aminoglycoside binding site of 16S rRNA in the 30S ribosomal subunit. Neither 16S rRNA alone nor intact 70S ribosome is an efficient substrate for armA methyltransferase. To more fully characterize this family of enzymes, xiii we have investigated the substrate requirements of ArmA. We determined the Mg2+ dependence of ArmA activity and found that the enzyme could recognize both translationally active and translationally inactive forms of 30S subunits. To identify the site of interaction between ArmA and the 30S subunit, we used hydroxyl radical cleavage of 16S rRNA mediated by ferrous iron chelated to several sites on the ArmA molecule that were mutated to cysteine. This data suggests that significant conformational changes in 30S structure are involved in binding of ArmA. We hypothesized that a precursor intermediate in the biogenesis of the 30S subunit might be the optimal substrate for ArmA enzymes in vivo. To test this, we prepared 30S particles partially depleted of proteins by treatment with increasing concentrations of LiCl and assayed them for ArmA methylation. Even low concentrations of LiCl alter the 30S particles and greatly diminish their susceptibility to methylation. Additionally, a previously identified pre-30S particle isolated from an E. coli culture was assayed for its ability to support methylation by ArmA and found to be inferior to intact 30S particles as a methylation substrate. Thus, testing of immature particles prepared from in vitro and in vivo sources suggest that ArmA works very late in the 30S biogenesis pathway. Initiation factor 3 (IF3), a factor that only binds fully mature 30S particles, does not inhibit the ArmA methylation, while kasugamycin methyltransferase (KsgA) abolishes ArmA activity by sharing the same binding site with ArmA. From aforementioned experiments, we conclude that ArmA is most active toward 30S ribosomal subunits that are at or very near full maturation.

PURPOSE: The purpose of this study is to describe the subgingival bacterial biodiversity in untreated chronic periodontitis patients through the use of next generation 16S rRNA molecular analysis, and to determine similarities or differences between deep and shallow pockets within the same patients. METHODS: The analysis involved paired subgingival plaque samples from 24 subjects diagnosed with Generalized Moderate to Severe Chronic Periodontitis. One sample was selected from a single site having a probing depth >5 mm (i.e. Deep Site), and the other from a site with a probing depth <3mm (i.e. Shallow Site) within each subject. Bacterial DNA amplification of the V4-V6 region of the 16S rRNA was performed. The amplicons were sequenced via 454 Roche Genome Sequencer FLX System. The identified sequences were evaluated, and then compared to calculated false discovery rates. RESULTS: A total of 119 independent microbial genera were identified within the samples analyzed. Seven genera were identified to be statistically significant (p<0.05) in their association to deep or shallow sites following t-test and boot strap randomization: Actinomyces (p=0.004), Methylobacterium (p=0.028), Veillonella (p=0.028), and Rothia (p=0.038), and Streptococcus (p=0.033) in Shallow sites; while Mycoplasma (p=0.007) and Fusobacterium (p=0.016) were associated with deep sites. However, taking into account the calculated false discovery rates, it is suggested that none of the 119 microbial genera identified in this study were significantly associated with either deep nor shallow sites. CONCLUSION: The microbial genera identified within this study to be associated with deep and shallow sites follows the traditional pattern anticipated from the literature. However, the calculated false discovery rates suggest that these results may have occurred by chance and not due to a true difference.

The aim of presented work is to provide characteristics of the karyotypes of scorpions of the genus Euscorpius. Genus Euscorpius is a typical representative of scorpions in Europe. Its occurrence is wide throughout Europe. Until now, 18 species of this genus have been described. In this work six species were karyologically analyzed and one species was shown to possess only basic diploid number of chromosomes: E. carpathicus - 2n=90, E. concinnus - 2n=88, E. hadzii - 2n=68, E. sicanus - 2n=66, E. tergestinus - 2n=60, E. naupliensis - 2n=60, E. italicus - 2n=36. Description of the karyotypes revealed that all species studied exhibit achiasmatic meiosis; no presence of sex chromosomes was detected. The basic hypothesis of phylogenetic relationships and karyotype evolution of the genus Euscorpius was outlined. High interspecies variability in chromosome total count was found and by analysis of the 16S rRNA gene the taxonomic status of the species was confirmed. Hence, it seems that cytogenetic methods can contribute to the understanding of species diversity and differentiation of possible cryptic species within the genus Euscorpius.

Orientador: Marcos Rogério André / Resumo: Micoplasmas hemotrópicos (hemoplasmas) são bactérias Gram-negativas que parasitam a superfície de eritrócitos de uma ampla variedade de mamíferos. Mycoplasma wenyonii e ‘Candidatus Mycoplasma haemobos’ são espécies de hemoplasmas relatadas em bovinos, podendo causar anemia hemolítica e redução na produção de carne e leite e, consequentemente, prejuízos econômicos. O presente estudo teve como objetivo investigar a ocorrência de hemoplasmas em bovinos de corte no Pantanal brasileiro, área endêmica para tripanossomíase bovina na América do Sul. Adicionalmente, objetivou-se caracterizar molecularmente os genótipos de hemoplasmas encontrados. Para tal, amostras de sangue e soro de 400 bovinos de corte foram colhidas em cinco propriedades do município de Corumbá, sub-região da Nhecolândia, estado de Mato Grosso do Sul, centro-oeste brasileiro. As amostras de sangue foram submetidas à extração de DNA e a ensaios de PCR convencional para hemoplasmas baseados no gene 16S rRNA. As sequências obtidas foram submetidas a inferências filogenéticas, análises de distância e de diversidade genotípica. O Ensaio Imunoenzimático Indireto (iELISA) mostrou a presença de anticorpos IgG anti-Trypanosoma vivax em 89,75% dos animais amostrados, confirmando a endemicidade para o referido agente na região sob estudo. Dentre as 400 amostras de sangue de bovinos testadas, 2,25% (9/400) mostraram-se positivas na cPCR para hemoplasmas. A análise filogenética das sequências obtidas confirmou a presença de DN... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Hemotrophic mycoplasmas (haemoplasmas) are Gram-negative bacteria that parasitize the surface of erythrocytes of a wide variety of mammals. Mycoplasma wenyonii and 'Candidatus Mycoplasma haemobos' are hemoplasma species reported in cattle, which can cause hemolytic anemia and reduction in meat and milk production and, consequently, economic losses. The present study aimed to investigate the occurrence of haemoplasmas in beef cattle in the Brazilian Pantanal, an area endemic for bovine trypanosomiasis in South America. In addition, the work aimed to characterize molecularly the genotypes of haemoplasmas found. For this purpose, blood and serum samples of 400 beef cattle were collected from five properties in the municipality of Corumbá, sub-region of Nhecolândia, in the state of Mato Grosso do Sul, central-western Brazil. Blood samples were subjected to DNA extraction and cPCR assays for haemoplasmas based on 16S rRNA gene. The sequences obtained were submitted to phylogenetic inferences, distance analysis and genotype diversity. The Indirect Immunoenzymatic Assay (iELISA) showed the presence of anti-Trypanosoma vivax IgG antibodies in 89.75% of the animals sampled, confirming the endemicity for this agent in the studied region. Among the 400 bovine blood samples tested, 2.25% (9/400) were positive in cPCR for haemoplasmas. Phylogenetic analysis of the sequences obtained confirmed the presence of DNA 'C. M. haemobos' and M. wenyonii in 0.5% (2/400) and 1.75% (7/400) animals, r... (Complete abstract click electronic access below) / Mestre

Submitted by WALLYNSON EDUARDO SILVA ALMEIDA null (wallynson@yahoo.com.br) on 2017-02-24T16:09:51Z No. of bitstreams: 1 Dissertação_Wallynson_Eduardo_Silva_Almeida.pdf: 1951842 bytes, checksum: 7628e3c85a4699b4530e564dcd6cc11a (MD5) / Approved for entry into archive by LUIZA DE MENEZES ROMANETTO (luizamenezes@reitoria.unesp.br) on 2017-03-07T12:39:08Z (GMT) No. of bitstreams: 1 almeida_wes_me_jabo.pdf: 1951842 bytes, checksum: 7628e3c85a4699b4530e564dcd6cc11a (MD5) / Made available in DSpace on 2017-03-07T12:39:08Z (GMT). No. of bitstreams: 1 almeida_wes_me_jabo.pdf: 1951842 bytes, checksum: 7628e3c85a4699b4530e564dcd6cc11a (MD5) Previous issue date: 2017-01-25 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Os microrganismos têm participação ativa nas transformações do fósforo (P) no solo, influenciando sua disponibilidade para as plantas e seu fluxo na natureza. As transformações resultam de decomposição e mineralização de compostos orgânicos, imobilização na microbiomassa e solubilização das formas inorgânicas dos minerais. A base desse estudo consistiu em avaliar 10 isolados bacterianos, visando caracterizá-los quanto: padrão de crescimento em meio Tryptone Yeast (TY) (padrão de crescimento em meio NBRIP (National Botanical Research Institute’s Phosphate); solubilização de fosfato de hidróxido de cálcio (Ca5OH(PO4)3); variação do pH nos diferentes meios; sequenciamento parcialmente do gene 16S rRNA, e influência do crescimento, em casa de vegetação, para as culturas da soja (Glycine max) e milho (Zea mays). Os melhores solubilizadores em 120 h de cultivo foram os isolados LGA- 11-V0522, 33,81 mg/ml, Chromotacterium Violaceum, LGA13-V20F, 32,65 mg/ml, Arthrobacter echigonensis, LGA1-V4-V20J, 32,19 mg/ml, Arthrobacter echigonensis, e LGA05-V0513, 30,96 mg/ml, Bacilus Cereus. Em casa de vegetação, os isolados que apresentaram melhores resultados foram, com maior desenvolvimento de parte aérea, LGA13-V20F, 22,67 cm, Arthrobacter echigonensis, LGA09-V20L, 22,0 cm, Bacillus thuringiensis, LGA07-V0508, 21,25 cm, Bacillus thuringiensis, e LGA12-V05D 19,25, Bacillus thuringiensis. / The microorganisms have an active participation in the transformations of the phosphorus (P) in the soil, influencing its availability to the plants and their flow in nature. The transformations result from decomposition and mineralization of organic compounds, immobilization in the microbiomass and solubilization of inorganic forms of minerals. The base of this study was to evaluate 10 bacterial isolates, aiming to characterize them as: growth pattern in Tryptone Yeast (TY) medium (growth pattern in NBRIP medium; solubilization of calcium hydroxide phosphate Ca5OH (PO4) 3), pH variation in the different media, partial sequencing of the 16S rRNA gene, and growth influence in the greenhouse for soybean (Glycine max) and maize (Zea mays) crops.The best solubilizers In 120 h of culture were the isolates LGA-11-V0522, 33.81 mg / ml, Chromotacterium Violaceum, LGA13-V20F, 32.65 mg / ml, Arthrobacter echigonensis, LGA1-V4-V20J, 32.19 mg / ml , Arthrobacter echigonensis, and LGA05- V0513, 30.96 mg / ml, Bacilus Cereus, and LGA13-V20F, 22.67 cm. In the greenhouse, LGA09-V20L, 22.0 cm, Bacillus thuringiensis, LGA07-V0508, 21.25 cm, Bacillus thuringiensis, and LGA12-V05D 19.25, Bacillus thuringiensis.

A character data set suitable for cladistic analysis of tardigrades at the family level was developed. The data matrix consisted of 50 morphological characters from 15 families of tardigrades and was analyzed by maximum parsimony. Kinorhynchs, loriciferans and gastrotrichs were used as outgroups. The results agree with the currently accepted hypothesis that Eutardigrada and Heterotardigrada are distinct monophyletic groups. Among the eutardigrades, Eoyhypsibiidae was found to be a sister group to Macrobiotidae + Hypsibiidae, while Milnesiidae was the basal eutardigrade family. The basal heterotardigrade family was found to be Oreellidae. Echiniscoideans grouped with some traditional Arthrotardigrada (Renaudarctidae, Coronarctidae + Batillipedidae) suggesting that the arthrotardigrades are not monophyletic. An 18S rRNA phylogenetic hypothesis was developed and supports the monophyly of Heterotardigrada and of Parachela versus Apochela within the Eutardigrada. Mapping of habitat preference suggest that terrestrial tardigrades are the ancestral state. Molecular analysis of a sediment sample with an unusually large population of tardigrades had a higher diversity when compared to manual sorting and counting.

<p>Recent research in microbial ecology has focused on how aquatic bacterial communities are assembled. Only a few of these studies follow a “Gleasonian” approach where the roles of single bacterial populations are in focus. In this thesis, novel molecular tools were used to describe the distribution and evolutionary relationships of microbes in productive aquatic environments. Many new phylogenetic groups of bacteria were identified, likely representing bacterial populations restricted to productive freshwaters. I also addressed the dynamics and functional role of individual bacterial populations in eutrophic lakes and brackish environments with a focus on either biogeochemically significant or potentially pathogenic representatives. <i>Flavobacteria</i> blooms were observed, on occasions characterized by high heterotrophic production. In addition to high temporal dynamics microbial community composition and function differed on the spatial scale, as exemplified by free-living and <i>Cyanobacteria</i>-associated habitats. At the community scale, microbial processes, such as biomass production and substrate uptake could be predicted from the presence and absence of individual bacterial populations. I also studied the niches of potentially pathogenic <i>Vibrio </i>populations in various coastal waters. Using a novel culture-independent method, a <i>V. cholerae</i> population was detected along the entire Swedish coastline. Results from an environmental survey and a laboratory mesocosm experiment reveal that phytoplankton-derived dissolved organic matter enhance the growth of <i>V. cholerae</i> and other <i>Vibrio</i> spp. and hence create a largely overlooked niche for these heterotrophic bacteria. This thesis and future work on the role of individual bacterial populations will facilitate predictions of biogeochemical cycles and the distribution of bacteria in the context of global climate change and local eutrophication.</p>