Bacterial type IV pili (T4P) are filamentous surface appendages with a variety of functions including motility, surface attachment, and biofilm formation. In many species of bacteria a clear understanding of how the functions of T4P in lifestyle switching are regulated remains to be elucidated. Here, we focus on understanding the regulation of the T4P assembly ATPase PilB. We examined its interactions with the secondary messenger cyclic-di-GMP (cdG). Specifically we investigated how cdG binding regulates PilB functions not only as the assembly ATPase, but also as an EPS signaling molecule in Myxococcus xanthus biofilm regulation. Chapter 2 focuses on the development of a microplate-based biofilm assay for M. xanthus. This new assay allows for the analysis of the M. xanthus submerged biofilms under vegetative conditions in a high throughput format which has been absent in the published literature. M. xanthus biofilm formation tightly correlates with EPS production, suggesting that the assay can be used as a convenient method of examining EPS production. Chapter 3 examines the regulation of M. xanthus PilB (MxPilB) by cdG binding in vivo. We carried out a mutational analysis of the MshEN cdG binding domain in MxPilB. Mutations were created that either diverge with or converge from the MshEN consensus sequence. These two classes of MxPilB variants are expected to either decrease or increase cdG binding affinity, respectively. We examined the motility, EPS production, and piliation phenotypes of these mutants. Our results were consistent with a model where the function of MxPilB is altered in response to cdG binding, and suggesting that PilB responds to different thresholds of cdG concentration. In Chapter 4, we examine the ligand binding to the N-terminal cdG binding domain and C-terminal ATPase domain of Chloracidobacterium thermophilum PilB (CtPilB) in vitro. Our results confirm that these two domains bind to their respective ligands specifically, and demonstrate these domains communicate with each other in response to ligand binding. The results from all of the studies help us to establish a model where cdG binding fine tunes the functions of PilB to regulate the switch of bacteria between the motile and planktonic states. In addition to their roles in motility and biofilm formation, T4P are key virulence factors in many significant human pathogens. Antivirulence chemotherapeutics are considered to be a promising alternative to antibiotics, as they target disease processes rather than bacterial viability. Because PilB is essential for T4P biogenesis, we sought to identify PilB inhibitors for the development of antivirulence therapies. In Chapter 5, we describe the development of the first high throughput screen (HTS), for PilB inhibitors. This assay is uses the reduction of the binding of a fluorescent ATP analog to CtPilB in vitro, leading to the discovery of the plant flavonoid quercetin as a PilB inhibitor. Using M. xanthus as a model a bacterium, quercetin was found to inhibit T4P-dependent motility and T4P assembly in vivo. Builds on this initial success with CtPilB, Chapter 6 describes the development and implementation of a second HTS based on the inhibition of CtPilB as an ATPase. Screening a large chemical library led to the identification of benserazide and levodopa as CtPilB inhibitors. We show that both compounds inhibit T4P assembly in M. xanthus without any detrimental effects on bacterial growth. Furthermore we demonstrate that both levodopa and benserazide inhibit T4P-mediated motility in Acinetobacter nosocomialis, a human pathogen, providing the first evidence that the compounds identified with CtPilB can be effective against a pathogenic bacterium. Both of these studies validate the effectiveness not only of our HTSs, with of CtPilB as a model protein for the identification of PilB inhibitors. / Doctor of Philosophy / Bacteria can be motile or sessile. Motile bacteria can use hair like structures on their surface, called pili, to move in their natural environment, whereas sessile bacteria produce intricate structures attached to solid surfaces known as biofilms. Bacteria are able to switch between being motile and sessile states depending on their environment conditions. However, it isn't clear how this switch is controlled in bacteria that use pili to move. To answer this question, we studied how PilB the protein that assembles pili, might control this switching process. We specifically investigated PilB because it has two known roles. The first is that it can assemble pili, to enable pili-mediated motility. The second is that it can stimulate or promote biofilm formation. This places PilB at the intersection of these two lifestyles, suggesting that this protein may play a key role in deciding whether a bacterium is to be motile or sessile. Another important reason to understand how PilB functions is because pili are used by some antibiotic resistant pathogenic bacteria. Since PilB is essential for the formation of pili, if the actions of PilB could be blocked, bacteria would be unable to make pili. This could stop bacteria from causing disease. By searching for new chemicals which stop PilB from creating pili, we can potentially identify new drugs to treat bacterial infections.

Myxococcus xanthus displays social (S) motility, a form of surface motility that is key to the multicellular behaviors of this organism. S motility requires two cellular structures: type IV pili (TFP) and exopolysaccharides (EPS). Previous studies have shown that M. xanthus does not use glucose or any other sugar as a primary carbon source. However, eight monosaccharides, namely glucose, mannose, arabinose, galactose, xylose, rhamnose, N-acetyl-glucosamine, and N-acetyl-mannosamine, are found in M. xanthus EPS. In this study, pathways that M. xanthus could use to produce the activated sugar monomers to form EPS are proposed based on genomic data. Of the eight sugars, pathways for seven were disrupted by mutation and their effects on the EPS-dependent behaviors were analyzed. The results indicate that disruption of the two pathways leading to the production of activated rhamnose (GDP- and TDP-rhamnose) affected fruiting body formation (GDP form only) and dye binding ability (both forms) but not S motility. Disruptions of the xylose, mannose, and glucose pathways caused M. xanthus to lose S motility, fruiting body formation, and dye binding abilities. An interruption in the pathway for galactose production created a mutant with properties similar to a lipopolysaccharide (LPS) deficient strain. This discovery led us to study the phenotypes of all mutant strains for LPS production. The results suggest that all mutants may synthesize defective LPS configurations. Disruption of the UDP-N-acetyl-mannosamine pathway resulted in a wild type phenotype. In addition, it was discovered that interruption of the pathway for N-acetyl-glucosamine production was possible only by supplementing this amino-sugar in the growth medium. In an attempt to determine if other mutants could be recovered by sugar supplementation, it was discovered that the Δpgi mutant can be rescued by glucose supplementation. The Dif chemotaxis-like pathway is known to regulate EPS production in M. xanthus. DifA is the upstream sensor of the pathway. Previous studies had created a NarX-DifA chimeric protein, NafA, that enables the activation of the Dif pathway by nitrate, the signal for NarX. In this study, we constructed a Δpgi difA double mutant containing NafA. This strain was then subjected to various incubations with glucose and/or nitrate to determine whether the point of EPS regulation by the Dif pathway is down- or up-stream of the step catalyzed by Pgi (phosphoglucose isomerase). Preliminary results from this study are inconclusive. / Master of Science

O cromo é utilizado em diferentes setores industriais, inclusive aplicado em curtumes como curtente de couros, cujo processamento gera resíduos sólidos e líquidos contendo concentrações deste metal, que se lançados no meio aquático sem o devido tratamento, pode atingir os sedimentos e afetar a biota aquática. É nesse contexto que em 2001, cerca de 150 toneladas de aparas de couro foram lançadas no solo e a partir do runoff, lixiviadas para o córrego Monte Alegre, localizado no município de Araraquara (SP), contaminando o sedimento e ocasionando alterações na comunidade de macroinvertebrados aquáticos do córrego. O presente trabalho visou avaliar os efeitos da contaminação de cromo em macroinvertebrados aquáticos no córrego Monte Alegre por meio do estudo da estrutura da comunidade e por testes de toxicidade aguda e crônica em larvas de Chironomus xanthus utilizando sedimento contaminado. As amostras foram coletadas em dois períodos do ano e em três pontos distintos do córrego, sendo um ponto à nascente (Ponto 1), um intermediário (Ponto 2) e outro próximo à área contaminada (Ponto 3). A comunidade de macroinvertebrados aquáticos foi coletada em campo utilizando draga Ekman-Birge (225 cm²) e levada ao laboratório, onde passaram por triagem em bandejas transiluminadas. Os organismos foram identificados utilizando chaves de identificação específicas. O sedimento para a análise ecotoxicológica também foi coletado utilizando draga Ekman-Birge e levado ao laboratório para realização dos testes. Os organismos foram expostos ao sedimento contaminado para avaliação da mortalidade, em testes agudos com duração de 96 h, preparados em frascos de 500 mL onde foram adicionados, 240 mL de água deionizada, 60 g de sedimento, 5 mL de alimento (solução de Tetramin 5g L-1) e 6 larvas de IV instar. O teste crônico foi desenvolvido sob as mesmas condições do agudo, exceto pela adição de 6 larvas de Chironomus xanthus de I instar, aeração (4 mL s-¹) e duração de 8 dias. Os resultados da concentração de cromo no sedimento e da composição da comunidade de macroinvertebrados aquáticos sugerem que o cromo proveniente das aparas de couro depositado no solo em 2001, no assentamento Monte Alegre, não interferiu na estrutura da comunidade de macroinvertebrados aquáticos, uma vez que, no ponto 1 (nascente) também foi encontrado concentrações de cromo. Neste ponto, a riqueza e a diversidade de espécies foram mais elevadas quando comparadas aos demais pontos, apontando para uma boa qualidade ambiental. O sedimento do córrego apresentou indícios de toxicidade para as larvas de Chironomus xanthus no ponto 1 (nascente), tóxico no ponto 2 (intermediário) e ponto 3 (área impactada). Outros fatores como cultivos de cana-de-açúcar e banana, a utilização de fertilizantes e a ausência de mata ciliar podem ter contribuído para a baixa riqueza e diversidade de macroinvertebrados aquáticos e para o aumento da toxicidade do sedimento nos pontos 2 e 3. Destaca-se a importância das matas ciliares na preservação da qualidade da água e como mecanismo de barreira e proteção contra a entrada de metais para os corpos de água. Ressalta-se ainda a necessidade do adequado manejo de produtos agrícolas, de resíduos de curtume e dos impactos que podem causar, não só para o meio ambiente modificando a paisagem e a qualidade ambiental, mas também para a comunidade do assentamento que utiliza o solo para a produção e comercialização de alimentos e a água para o consumo. / Chromium is used in different industrial sectors, including applied to tanneries such as leather tanning, whose processing generates solid and liquid residues containing concentrations of this metal, which if launched into the aquatic environment without due treatment, can reach the sediments and affect the aquatic biota. It is in this context that in 2001 about 150 tons of leather chips were released into the soil and from the runoff, leached to the Monte Alegre stream, located in the city of Araraquara (SP), contaminating the sediment and causing changes in the community of aquatic macroinvertebrates of the stream. The present work evaluate the effects of chromium contamination on aquatic macroinvertebrates in the Monte Alegre stream through of the community structure study and by acute and chronic toxicity tests on Chironomus xanthus larvae using contaminated sediment. The samples were collected in two periods of the year and at three distinct points of the stream, one point at the nascent (Point 1), one intermediate (Point 2) and one near the contaminated area (Point 3). The community of aquatic macroinvertebrates was collected in the field using Ekman-Birge drag (225 cm²) and taken to the laboratory, where they were screened in transiluminated trays. The organisms were identified using specific identification keys. The sediment for the ecotoxicological analysis was also collected using Ekman-Birge drag and taken to the laboratory for testing. The organisms were exposed to the contaminated sediment for mortality evaluation in acute tests with a duration of 96 h, prepared in 500 mL pots adding 240 mL of deionized water, 60 g of sediment, 5 mL of food (Tetramin\'s solution 5g L-1) and 6 instar IV larvae. The chronic test was developed under the same acute conditions, except for the addition of larvae of Chironomus xanthus of I instar, aeration (4 mL s-1) and duration of 8 days. The results of the concentration of chromium in the sediment and of the community composition of aquatic macroinvertebrates suggest that the chromium from the leather chips deposited in the soil in 2001 in the Monte Alegre stream did not interfere in the structure of the aquatic macroinvertebrate community, since the concentrations of chromium were also found in point 1 (nascent). At this point, the richness and diversity of species were higher when compared to the other points, pointing to a good environmental quality. The stream sediment showed evidence of toxicity for Chironomus xanthus larvae at point 1 (nascent) toxic at point 2 (intermediate) and point 3 (impacted area). Other factors such as sugarcane and banana plantation, the use of fertilizers and the absence of riparian vegetation may have contributed to the low richness and diversity of aquatic macroinvertebrates and to the increase toxicity in the sediment in points 2 and 3. Highlights the importance of riparian vegetation in the preservation of water quality and as a barrier mechanism and protection against the entry of metals into watercourse. The need for adequate management of agricultural products, tannery residues and the impacts they can cause, not only for the environment by modifying the landscape and environmental quality, but also for the local community that uses the soil to the production and commercialization of food and water for consumption.

Frente ao cenário ambiental observado no Brasil e a uma crescente preocupação com os problemas de poluição, amostras de sedimento do rio Paraíba do Sul foram caracterizadas para levantamento de dados que permitam inferir a qualidade do rio e da região como um todo, visto que esta matriz desempenha papel fundamental na qualidade da água e acumula contaminantes ao longo do tempo, gerando maior número de informações. O rio Paraíba do Sul é considerado um dos mananciais mais importantes da região sudeste, uma vez que está localizado entre as duas maiores capitais brasileiras: São Paulo e Rio de Janeiro. A qualidade dos sedimentos deste manancial encontra-se bastante comprometida devido ao desmatamento, mau uso e ocupação do solo em áreas urbanas e rurais, uso de insumos agrícolas e pelo lançamento diário de alta carga de efluentes domésticos. Esta situação é agravada pelo baixo índice de tratamento dos efluentes lançados na bacia do Rio Paraíba do Sul. O descarte de efluentes, ricos em nutrientes como fósforo e nitrogênio, pode contribuir diretamente no processo de eutrofização. O objetivo deste trabalho foi avaliar as características do sedimento no trecho paulista do rio Paraíba do Sul, entre os municípios de Lorena, Cachoeira Paulista e Cruzeiro através da determinação e análise dos parâmetros físico-químicos e ecotoxicológicos das amostras, além de determinar a ocorrência de metais potencialmente biodisponíveis. As coletas de sedimento foram realizadas a montante e a jusante do rio em cada município nos meses de agosto/16 (estação seca) e novembro/16 (estação chuvosa). Os parâmetros avaliados para sedimento foram granulometria, teor de silte e argila, matéria orgânica, carbono orgânico total, fósforo total, nitrogênio total por métodos convencionais e metais (Mn, Fe, Ni, Cu, Zn e Pb) por ICP-OES, além de bioensaios de toxicidade crônica utilizando a espécie Chironomus xanthus. Para água intersticial foram determinados pH, condutividade e oxigênio dissolvido. As coletas foram efetuadas em triplicata e foram calculados a média e o desvio padrão das amostras para cada parâmetro. Os resultados foram estatisticamente analisados através dos Componentes Principais (ACP), Correlação de Spearman e Grupamento de Cluster. Os resultados mostraram correlação positiva na estação seca entre a condutividade e a concentração de Mn e Fe. pH, a condutividade e a concentração de Mn tiveram forte correlação positiva na estação chuvosa. Fósforo total e nitrogênio kjeldhal total mostraram-se abaixo do valor alerta de acordo com o estabelecido pela resolução CONAMA 344/04. Os resultados demonstraram que a precipitação influenciou nas características das amostras de sedimento, como na granulometria e teores de matéria orgânica (silte + argila). Uma maior disparidade do ponto a jusante de Cruzeiro em relação aos outros pontos pode estar associada à presença de indústrias na região da cidade, cujos resíduos contêm alta concentração de metais. O planejamento fatorial 23 feito para otimizar as condições de cultivo do C. xanthus mostrou que dentre os dois níveis para as variáveis temperatura, dias de alimentação e água (com e sem tratamento) mostrou que a água foi o efeito significante, com índice de mortalidade menor quando se utilizou água da mina sem tratamento preliminar. Os resultados dos bioensaios de toxicidade crônica não apresentaram efeito tóxico para o C. xanthus, a nível de 95% de confiança. Os resultados sugerem que os sedimentos no trecho estudado estão dentro das especificações preconizadas pela legislação ambiental. / Considering the environmental scenario observed in Brazil and increasing concern to pollution problems, sediment samples from the Paraíba do Sul river were characterized for data collection that allow to infer a quality of the river and the region as a whole, since this matrix plays a role water quality and accumulates contaminants over time, generating more information. The Paraíba do Sul river is considered one of the most important springs of the Southeast region, since it is located between the two largest brazilian capitals: São Paulo and Rio de Janeiro. The quality of the sediments of this source is very compromised due to the deforestation, misuse and occupation of the soil in urban and rural areas, use of agricultural inputs and the daily launch of high domestic effluent load. This situation is aggravated by the low treatment rate of effluent released in the Paraíba do Sul River Basin. Discharge of effluents, rich in nutrients such as phosphorus and nitrogen, can directly contribute to the eutrophication process. The objective of this work was to evaluate the characteristics of the sediment in the São Paulo stretch of the Paraíba do Sul river, between the municipalities of Lorena, Cachoeira Paulista and Cruzeiro, through the determination and analysis of the physicochemical and ecotoxicological parameters of the samples, besides determining the occurrence of Potentially bioavailable metals. Sediment collections were carried out upstream and downstream of the river in each municipality in the months of August / 16 (dry season) and November / 16 (rainy season). The standard values of nitrogen by conventional methods and metals (Mn, Fe, Ni, Cu, Zn and Pb) by ICPOES, in addition to a total of total nitrogen, by conventional methods and metals. From chronic toxicity bioassays using a Chironomus xanthus species. For interstitial water, pH, conductivity and dissolved oxygen were determined. The samples were collected in triplicate and the mean and standard samples were calculated for each parameter. The results were statistically analyzed through the Principal Components (PCA), Spearman Correlation and Cluster Grouping. The results show a positive correlation in the dry season between a conductivity and a concentration of Mn and Fe. pH, a conductivity and a concentration of Mn had a strong positive correlation in the rainy season. Total phosphorus and total Kjeldhal nitrogen were below the alert value according to CONAMA Resolution 344/04. The results showed that the precipitation influenced the characteristics of sediment samples, such as grain size and organic matter content (silt + clay). A greater disparity of the downstream point of Cruzeiro in relation to other points may be associated with the presence of the industries in the region of the city, whose residues contain high concentration of metals. The factorial design 23 did to optimize as C. xanthus culture conditions showed that among the two levels for the variables temperature, daily feed and water (with and without treatment) showed that water was the significant effect, with mortality index Minor when using mine water without preliminary treatment. The results of the chronic toxicity bioassays showed no toxic effect for C. xanthus at 95% confidence. The results suggest that the sediments in the studied section are within the specifications recommended by the environmental legislation.

O cromo é utilizado em diferentes setores industriais, inclusive aplicado em curtumes como curtente de couros, cujo processamento gera resíduos sólidos e líquidos contendo concentrações deste metal, que se lançados no meio aquático sem o devido tratamento, pode atingir os sedimentos e afetar a biota aquática. É nesse contexto que em 2001, cerca de 150 toneladas de aparas de couro foram lançadas no solo e a partir do runoff, lixiviadas para o córrego Monte Alegre, localizado no município de Araraquara (SP), contaminando o sedimento e ocasionando alterações na comunidade de macroinvertebrados aquáticos do córrego. O presente trabalho visou avaliar os efeitos da contaminação de cromo em macroinvertebrados aquáticos no córrego Monte Alegre por meio do estudo da estrutura da comunidade e por testes de toxicidade aguda e crônica em larvas de Chironomus xanthus utilizando sedimento contaminado. As amostras foram coletadas em dois períodos do ano e em três pontos distintos do córrego, sendo um ponto à nascente (Ponto 1), um intermediário (Ponto 2) e outro próximo à área contaminada (Ponto 3). A comunidade de macroinvertebrados aquáticos foi coletada em campo utilizando draga Ekman-Birge (225 cm²) e levada ao laboratório, onde passaram por triagem em bandejas transiluminadas. Os organismos foram identificados utilizando chaves de identificação específicas. O sedimento para a análise ecotoxicológica também foi coletado utilizando draga Ekman-Birge e levado ao laboratório para realização dos testes. Os organismos foram expostos ao sedimento contaminado para avaliação da mortalidade, em testes agudos com duração de 96 h, preparados em frascos de 500 mL onde foram adicionados, 240 mL de água deionizada, 60 g de sedimento, 5 mL de alimento (solução de Tetramin 5g L-1) e 6 larvas de IV instar. O teste crônico foi desenvolvido sob as mesmas condições do agudo, exceto pela adição de 6 larvas de Chironomus xanthus de I instar, aeração (4 mL s-¹) e duração de 8 dias. Os resultados da concentração de cromo no sedimento e da composição da comunidade de macroinvertebrados aquáticos sugerem que o cromo proveniente das aparas de couro depositado no solo em 2001, no assentamento Monte Alegre, não interferiu na estrutura da comunidade de macroinvertebrados aquáticos, uma vez que, no ponto 1 (nascente) também foi encontrado concentrações de cromo. Neste ponto, a riqueza e a diversidade de espécies foram mais elevadas quando comparadas aos demais pontos, apontando para uma boa qualidade ambiental. O sedimento do córrego apresentou indícios de toxicidade para as larvas de Chironomus xanthus no ponto 1 (nascente), tóxico no ponto 2 (intermediário) e ponto 3 (área impactada). Outros fatores como cultivos de cana-de-açúcar e banana, a utilização de fertilizantes e a ausência de mata ciliar podem ter contribuído para a baixa riqueza e diversidade de macroinvertebrados aquáticos e para o aumento da toxicidade do sedimento nos pontos 2 e 3. Destaca-se a importância das matas ciliares na preservação da qualidade da água e como mecanismo de barreira e proteção contra a entrada de metais para os corpos de água. Ressalta-se ainda a necessidade do adequado manejo de produtos agrícolas, de resíduos de curtume e dos impactos que podem causar, não só para o meio ambiente modificando a paisagem e a qualidade ambiental, mas também para a comunidade do assentamento que utiliza o solo para a produção e comercialização de alimentos e a água para o consumo. / Chromium is used in different industrial sectors, including applied to tanneries such as leather tanning, whose processing generates solid and liquid residues containing concentrations of this metal, which if launched into the aquatic environment without due treatment, can reach the sediments and affect the aquatic biota. It is in this context that in 2001 about 150 tons of leather chips were released into the soil and from the runoff, leached to the Monte Alegre stream, located in the city of Araraquara (SP), contaminating the sediment and causing changes in the community of aquatic macroinvertebrates of the stream. The present work evaluate the effects of chromium contamination on aquatic macroinvertebrates in the Monte Alegre stream through of the community structure study and by acute and chronic toxicity tests on Chironomus xanthus larvae using contaminated sediment. The samples were collected in two periods of the year and at three distinct points of the stream, one point at the nascent (Point 1), one intermediate (Point 2) and one near the contaminated area (Point 3). The community of aquatic macroinvertebrates was collected in the field using Ekman-Birge drag (225 cm²) and taken to the laboratory, where they were screened in transiluminated trays. The organisms were identified using specific identification keys. The sediment for the ecotoxicological analysis was also collected using Ekman-Birge drag and taken to the laboratory for testing. The organisms were exposed to the contaminated sediment for mortality evaluation in acute tests with a duration of 96 h, prepared in 500 mL pots adding 240 mL of deionized water, 60 g of sediment, 5 mL of food (Tetramin\'s solution 5g L-1) and 6 instar IV larvae. The chronic test was developed under the same acute conditions, except for the addition of larvae of Chironomus xanthus of I instar, aeration (4 mL s-1) and duration of 8 days. The results of the concentration of chromium in the sediment and of the community composition of aquatic macroinvertebrates suggest that the chromium from the leather chips deposited in the soil in 2001 in the Monte Alegre stream did not interfere in the structure of the aquatic macroinvertebrate community, since the concentrations of chromium were also found in point 1 (nascent). At this point, the richness and diversity of species were higher when compared to the other points, pointing to a good environmental quality. The stream sediment showed evidence of toxicity for Chironomus xanthus larvae at point 1 (nascent) toxic at point 2 (intermediate) and point 3 (impacted area). Other factors such as sugarcane and banana plantation, the use of fertilizers and the absence of riparian vegetation may have contributed to the low richness and diversity of aquatic macroinvertebrates and to the increase toxicity in the sediment in points 2 and 3. Highlights the importance of riparian vegetation in the preservation of water quality and as a barrier mechanism and protection against the entry of metals into watercourse. The need for adequate management of agricultural products, tannery residues and the impacts they can cause, not only for the environment by modifying the landscape and environmental quality, but also for the local community that uses the soil to the production and commercialization of food and water for consumption.

Frente ao cenário ambiental observado no Brasil e a uma crescente preocupação com os problemas de poluição, amostras de sedimento do rio Paraíba do Sul foram caracterizadas para levantamento de dados que permitam inferir a qualidade do rio e da região como um todo, visto que esta matriz desempenha papel fundamental na qualidade da água e acumula contaminantes ao longo do tempo, gerando maior número de informações. O rio Paraíba do Sul é considerado um dos mananciais mais importantes da região sudeste, uma vez que está localizado entre as duas maiores capitais brasileiras: São Paulo e Rio de Janeiro. A qualidade dos sedimentos deste manancial encontra-se bastante comprometida devido ao desmatamento, mau uso e ocupação do solo em áreas urbanas e rurais, uso de insumos agrícolas e pelo lançamento diário de alta carga de efluentes domésticos. Esta situação é agravada pelo baixo índice de tratamento dos efluentes lançados na bacia do Rio Paraíba do Sul. O descarte de efluentes, ricos em nutrientes como fósforo e nitrogênio, pode contribuir diretamente no processo de eutrofização. O objetivo deste trabalho foi avaliar as características do sedimento no trecho paulista do rio Paraíba do Sul, entre os municípios de Lorena, Cachoeira Paulista e Cruzeiro através da determinação e análise dos parâmetros físico-químicos e ecotoxicológicos das amostras, além de determinar a ocorrência de metais potencialmente biodisponíveis. As coletas de sedimento foram realizadas a montante e a jusante do rio em cada município nos meses de agosto/16 (estação seca) e novembro/16 (estação chuvosa). Os parâmetros avaliados para sedimento foram granulometria, teor de silte e argila, matéria orgânica, carbono orgânico total, fósforo total, nitrogênio total por métodos convencionais e metais (Mn, Fe, Ni, Cu, Zn e Pb) por ICP-OES, além de bioensaios de toxicidade crônica utilizando a espécie Chironomus xanthus. Para água intersticial foram determinados pH, condutividade e oxigênio dissolvido. As coletas foram efetuadas em triplicata e foram calculados a média e o desvio padrão das amostras para cada parâmetro. Os resultados foram estatisticamente analisados através dos Componentes Principais (ACP), Correlação de Spearman e Grupamento de Cluster. Os resultados mostraram correlação positiva na estação seca entre a condutividade e a concentração de Mn e Fe. pH, a condutividade e a concentração de Mn tiveram forte correlação positiva na estação chuvosa. Fósforo total e nitrogênio kjeldhal total mostraram-se abaixo do valor alerta de acordo com o estabelecido pela resolução CONAMA 344/04. Os resultados demonstraram que a precipitação influenciou nas características das amostras de sedimento, como na granulometria e teores de matéria orgânica (silte + argila). Uma maior disparidade do ponto a jusante de Cruzeiro em relação aos outros pontos pode estar associada à presença de indústrias na região da cidade, cujos resíduos contêm alta concentração de metais. O planejamento fatorial 23 feito para otimizar as condições de cultivo do C. xanthus mostrou que dentre os dois níveis para as variáveis temperatura, dias de alimentação e água (com e sem tratamento) mostrou que a água foi o efeito significante, com índice de mortalidade menor quando se utilizou água da mina sem tratamento preliminar. Os resultados dos bioensaios de toxicidade crônica não apresentaram efeito tóxico para o C. xanthus, a nível de 95% de confiança. Os resultados sugerem que os sedimentos no trecho estudado estão dentro das especificações preconizadas pela legislação ambiental. / Considering the environmental scenario observed in Brazil and increasing concern to pollution problems, sediment samples from the Paraíba do Sul river were characterized for data collection that allow to infer a quality of the river and the region as a whole, since this matrix plays a role water quality and accumulates contaminants over time, generating more information. The Paraíba do Sul river is considered one of the most important springs of the Southeast region, since it is located between the two largest brazilian capitals: São Paulo and Rio de Janeiro. The quality of the sediments of this source is very compromised due to the deforestation, misuse and occupation of the soil in urban and rural areas, use of agricultural inputs and the daily launch of high domestic effluent load. This situation is aggravated by the low treatment rate of effluent released in the Paraíba do Sul River Basin. Discharge of effluents, rich in nutrients such as phosphorus and nitrogen, can directly contribute to the eutrophication process. The objective of this work was to evaluate the characteristics of the sediment in the São Paulo stretch of the Paraíba do Sul river, between the municipalities of Lorena, Cachoeira Paulista and Cruzeiro, through the determination and analysis of the physicochemical and ecotoxicological parameters of the samples, besides determining the occurrence of Potentially bioavailable metals. Sediment collections were carried out upstream and downstream of the river in each municipality in the months of August / 16 (dry season) and November / 16 (rainy season). The standard values of nitrogen by conventional methods and metals (Mn, Fe, Ni, Cu, Zn and Pb) by ICPOES, in addition to a total of total nitrogen, by conventional methods and metals. From chronic toxicity bioassays using a Chironomus xanthus species. For interstitial water, pH, conductivity and dissolved oxygen were determined. The samples were collected in triplicate and the mean and standard samples were calculated for each parameter. The results were statistically analyzed through the Principal Components (PCA), Spearman Correlation and Cluster Grouping. The results show a positive correlation in the dry season between a conductivity and a concentration of Mn and Fe. pH, a conductivity and a concentration of Mn had a strong positive correlation in the rainy season. Total phosphorus and total Kjeldhal nitrogen were below the alert value according to CONAMA Resolution 344/04. The results showed that the precipitation influenced the characteristics of sediment samples, such as grain size and organic matter content (silt + clay). A greater disparity of the downstream point of Cruzeiro in relation to other points may be associated with the presence of the industries in the region of the city, whose residues contain high concentration of metals. The factorial design 23 did to optimize as C. xanthus culture conditions showed that among the two levels for the variables temperature, daily feed and water (with and without treatment) showed that water was the significant effect, with mortality index Minor when using mine water without preliminary treatment. The results of the chronic toxicity bioassays showed no toxic effect for C. xanthus at 95% confidence. The results suggest that the sediments in the studied section are within the specifications recommended by the environmental legislation.

Tous les organismes, les animaux, les plantes et les microbes, sont composés de cellules polarisées, en affichant un positionnement asymétrique des organites sub-cellulaires ou des structures. Le contrôle de polarité a été étudié chez les eucaryotes pendant une longue période, et a été montré pour être impliqués dans de nombreux processus physiologiques, tels que l'embryogenèse, le cancer métastatique et les maladies dégénératives des neurones. Chez les procaryotes, des études de polarité ne sont apparues récemment avec le développement de la microscopie à fluorescence sensibles. Ces études ont révélé que les cellules procaryotes sont en fait très organisé et une masse croissante de la littérature a montré que les cellules bactériennes également utiliser des radeaux lipidiques, courbure membranaire, la paroi cellulaire et un cytosquelette complexe pour diriger le positionnement spécifique de structures subcellulaires.Petites GTPases de la superfamille Ras sont des éléments réglementaires polarisation répandue chez les eucaryotes. Malgré l'existence depuis longtemps de ces petites GTPases dans les génomes procaryotes, leur fonction a jamais été étudiée. Pendant ce travail de thèse, nous avons trouvé, pour la première fois, qu'une petite GTPase, MglA et de sa protéine apparentée Activation GTPase (GAP) MglB, directe une dynamique axe antéro-postérieur à la motilité directe en forme de tige deltaproteobacterium Myxococcus xanthus. Dans ce processus, MglA s'accumule dans son état lié au GTP au niveau du pôle leader de cellules, en activant les machineries motilité. Ce schéma de localisation est maintenue par MglB, qui localise le pôle opposé, le blocage de l'accumulation MglA à ce pôle à travers son activité GAP. Remarquablement, les deux protéines passer leur localisation synchrone, ce qui correspond à un changement dramatique dans la direction du mouvement cellulaire (inversion). Ce commutateur est réglementé par un système chimiosensoriels-like, Frz. Dans une deuxième partie de ce travail, nous avons identifié un régulateur de protéine de réponse, RomR qui est essentiel pour le regroupement polaire de MglA. Interdépendances complexes entre la localisation RomR, MglA et MglB indiquent que ces protéines pourraient constituer un complexe de polarité dynamique de trois protéines qui reçoit Frz de signalisation pour passer l'axe de polarité. En conclusion, les résultats de ce travail de thèse suggère que M. xanthus intégré un module de polarité eucaryotes-like (MglAB) dans un procaryote spécifique (Frz) réseau de signalisation pour réguler sa motilité. Une telle réglementation est distincte sous forme de petites protéines G des règlements, qui sont généralement couplés à la protéine G récepteurs couplés (GPCR) chez les eucaryotes. Enfin, ce travail ouvre la voie pour comprendre comment la réglementation seule la motilité cellulaire sont intégrés pour générer des comportements commandés multicellulaires donnant naissance à des structures primitives de développement, par exemple, la morphogenèse du corps fructifères. D'autre part, ce travail fournit également un exemple d'analyser les étapes évolutives donnant lieu à des réseaux de signalisation. / All organisms, animals, plants and microbes, are composed of polarized cells, displaying asymmetric positioning of sub-cellular organelles or structures. Polarity control has been studied in eukaryotes for a long time, and has been shown to be involved in many physiological processes, such as embryogenesis, cancer metastasis and neuron degenerative diseases. In prokaryotes, polarity studies only emerged recently with the development of sensitive fluorescent microscopy. These studies revealed that prokaryotic cells are in fact highly organized and a growing body of literature has shown that bacterial cells also use lipid rafts, membrane curvature, the cell wall and a complex cytoskeleton to direct the specific positioning of subcellular structures.Small GTPases of the Ras superfamily are widespread polarization regulatory elements in eukaryotes. Despite the long known existence of such small GTPases in prokaryotic genomes, their function has never been studied. During this thesis work, we found, for the first time, that a small GTPase, MglA and its cognate GTPase Activating Protein (GAP) MglB, direct a dynamic anterior- posterior axis to direct motility of the rod-shaped deltaproteobacterium Myxococcus xanthus. In this process, MglA accumulates in its GTP-bound state at the leading cell pole, activating the motility machineries. This localization pattern is maintained by MglB, which localizes at the opposite pole, blocking MglA accumulation at this pole through its GAP activity. Remarkably, both proteins switch their localization synchronously, which correlates with a dramatic change in the direction of cell movement (reversal). This switch is regulated by a chemosensory-like system, Frz. In a second part of this work, we identified a response regulator protein, RomR which is essential for the polar clustering of MglA. Intricate localization interdependencies between Romr, MglA and MglB indicate that these proteins might constitute a dynamic three-protein polarity complex that receives Frz-signaling to switch the polarity axis. In conclusion, the results from this thesis work suggest that M. xanthus integrated a eukaryotic-like polarity module (MglAB) into a prokaryotic- specific (Frz) signaling network to regulate its motility. Such regulation is distinct form small G- protein regulations, which are generally coupled to G-protein coupled receptors (GPCRs) in eukaryotes. Finally, this work paves the way to understand how single cell motility regulations are integrated to generate ordered multicellular behaviors giving rise to primitive developmental structures, for example fruiting body morphogenesis. On the other hand, this work also provides an example to analyze the evolutionary steps giving rise to signaling networks.

Le mécanisme de la motilité de type gliding chez Myxococcus xanthus est longtemps resté incompris, du fait que ce type de déplacement ne requière aucune organelle extracellulaire. Nous avons démontré que le gliding est énergisée par un canal à protons, composé par les protéines AglRQS. Ce moteur coopère avec le cytosquelette d’actine bactérien pour transporter de manière directionnelle le complexe de l’enveloppe Glt à la surface de la cellule. Ce transport est traduit en motilité car les complexes Glt transportés interagissent avec un polysaccharide de surface qui agit comme une colle et immobilise les complexes Glt transportés contre le substrat.Nous avons également fait l’étonnante découverte que le moteur AglRQS est également essentiel à la sporulation, processus cellulaire durant lequel les cellules s’arrondissent et sont recouvertes d’un épais polysaccharide (le spore coat), qui leur confère une résistance face à des conditions défavorables. Nous avons démontré une interaction directe entre le moteur AglRQS et le complexe de l’enveloppe Nfs, un proche homologue du complexe Glt. Nous avons démontré que le moteur AglRQS transporte le complexe Nfs de manière directionnelle autour de la spore. Le spore coat étant sécrété en différents foci autour de la surface de la spore, son transport par la machinerie Agl-Nfs assure la formation d’une couche de « spore coat » compacte autour de la future spore.Ces résultats démontrent l’existence d’un moteur bactérien impliqué dans le transport directionnel de complexes protéiques associés à des sucres. Ces moteurs modulaires pourraient être adaptés à des fonctions spécifiques, en fonction du complexe avec lequel ils interagissent. / How gliding motility on solid surfaces is achieved in Myxococcus xanthus has long remained enigmatic, mostly because movement does not involve obvious extracellular organelles. Recently, we demonstrated that motility in M. xanthus is driven by a proton channel composed by the AglRQS proteins. This motor cooperates with the bacterial actin cytoskeleton to transport an envelope-spanning Glt motility complexes at the cell surface directionally. Motility is produced as a motility machinery surface tip-bound polysaccharide acts like a glue to immobilize the transported Glt complexes against the substratum.In the course of this study, we also made the surprising discovery that the AglRQS motor is essential not only for motility but also for sporulation, a cellular process during which the cells become surrounded by a thick polysaccharide (the spore coat) that confers resistance during unfavourable conditions. We demonstrated a direct interaction between the AglRQS motor and the Nfs envelope complex, a close homolog of the Glt complex. Transmission electron microscopy, time-lapse microscopy and localization studies, showed that the AglRQS motor rotates the Nfs complex directionally around the spore surface. Since the main spore coat polymer is secreted at discrete sites around the spore surface, its transport by the Agl-Nfs machinery ensures the formation of a compact spore coat layer around the future spore.These results highlight the existence of new class of bacterial motors involved in intracellular and directional transport of sugar-associated complex. These modular motors can be adapted to specific functions based which output complex they interact with.

Os estudos ecológicos, com vistas para a conservação do meio ambiente, vêm sendo intensificados desde meados do século XX. Devido à integração com várias outras ciências durante o processo de formação do conhecimento ecológico, esses estudos têm se tornado essenciais para o desenvolvimento de tecnologias e de políticas adequadas ao gerenciamento dos recursos naturais, em especial dos recursos hídricos. Estudos que contemplem a comparação entre sistemas naturais e artificiais são importantes na medida em que permitem investigar como esses sistemas reagem a perturbações antropogênicas de curto ou longo prazo. Há um consenso geral de que a camada de sedimento dos corpos d\'água desempenha um papel fundamental na dinâmica de transporte, acumulação e disponibilização de contaminantes nesses sistemas. A tríade de qualidade de sedimento é considerada uma das melhores abordagens para o estudo de sistemas aquáticos impactados por efluentes oriundos de atividades antrópicas. Habitats dulcícolas são particularmente suscetíveis à introdução de espécies exóticas de invertebrados. Uma possível rota de introdução dessas espécies é o cultivo em laboratório para testes de toxicidade. Substituí-las por espécies nativas com sensibilidade similar pode prevenir a introdução acidental de espécies exóticas, além de facilitar a obtenção de inóculos para as renovações da cultura, e de serem mais significativos na extrapolação dos resultados laboratoriais para situações em campo. Duas espécies nativas de organismos bentônicos foram estudadas com relação à aspectos de suas biologia e a adequação como organismos-teste em ensaios ecotoxicológicos: Chironomus xanthus (Díptera) e Branchiura sowerbyi (Oligochaeta). Os resultados dos testes de toxicidade ao pentaclorofenol para C. xanthus permitiram calcular uma CL50 (96h) de 0,111 mg/L com uma faixa de sensibilidade entre 0,082 e 0,141 mg/L. Os testes de sensibilidade de B. sowerbyi ao cloreto de potássio resultaram em uma CL50 (96h) de 0,364 g/L com uma faixa de sensibilidade entre 0,177 e 0,551 g/L. Realizou-se também um balanço energético geracional cumulativo para C. xanthus, obtendo-se os seguintes valores para as eficiências metabólicas calculadas: coeficiente instantâneo de assimilação de 98,14%, coeficiente de produção bruta de 14,76% e coeficiente de produção líquida de 15,03%. Além dos ensaios toxicológicos com os dois organismos, realizou-se um estudo comparativo entre o sistema de reservatórios em cascata do rio Tietê com o sistema de lagos naturais do Vale do Rio Doce, aplicando-se os princípios da tríade de qualidade de sedimentos. Os resultados dessa análise permitiram classificar os reservatórios estudados de acordo com o nível de qualidade ambiental sendo que o reservatório de Barra Bonita (SP) é considerado como o de pior qualidade, enquanto que os lagos do Vale do Rio Doce (MG) encontram-se ainda relativamente preservados. / The ecological studies, with views for the conservation of the environment, have been intensified from middles of the century XX. Due to the integration with several other sciences during the process of ecological knowledge formation, those studies have become essential to the development of technologies and appropriate politics for the administration of the natural resource. Studies that contemplate the comparison among natural and artificial systems are important in the way it is allowed to investigate as those systems react to antropogenic disturbances of short or long periods. There is a general consensus that the sediment of the water bodies plays a fundamental part in the transport dynamics, accumulation and disposal of pollutants in these systems. The triad of sediment quality is considered one of the best approaches for the study the influence of pollutants originated from of human activities in aquatic ecossystems. Freshwater habitats are particularly susceptible to the introduction of exotic species of invertebrates. A possible route of introduction of those species is the cultivation in laboratory for toxicity tests. To substitute them for native species with similar sensibility can prevent the accidental introduction of exotic species, besides facilitating the inocula obtaining for the renewals of the culture, and may be more significant in the extrapolation of the laboratories results for field situations. Two native species of benthic organisms were studied regarding to aspects of their biology and the adaptation as organism-test in ecotoxicological assays: Chironomus xanthus (Díptera) and Branchiura sowerbyi (Oligochaeta). The results of the toxicity tests to the pcp for C. xanthus allowed to calculate a LC50 (96h) of 0,111 mg/L with a sensitivity range of 0,082 - 0,141 mg/L. The sensitivity testes for B. sowerbyi to the KCl resulted in a LC50 (96h) of 0,364 g/L with a sensitivity range of 0,177 - 0,551 g/L. An energy budget of C. xanthus was performed, being obtained the following values for the calculated metabolic efficiencies: instantaneous coefficient of assimilation of 98,14%, coefficient of production of 14,76% and coefficient of liquid production of 15,03%. A comparative study among the system of reservoirs in cascade of the Tietê river with the system of natural lakes of Rio Doce valley was applied following the principles of the triad of quality of sediments. The results of that analyze allowed to classify the reservoirs studied in agreement with the level of environmental quality and the reservoir of Barra Bonita (SP) is considered as the one of worse quality, while the lakes of Rio Doce Valley (MG) still meet relatively preserved.

O crescimento populacional, o desenvolvimento industrial e a expansão agrícola resultaram no aumento da geração de resíduos sólidos e na necessidade de se efetuar seu descarte de forma adequada e não prejudicial ao meio ambiente, principalmente aos recursos hídricos. O uso de aterros controlados é um dos métodos de disposição final de resíduos no solo, e possui falhas nas estruturas de apoio ambiental como sistemas de drenagem, impermeabilização, controle de entrada de resíduos, produção de líquidos percolados. Os líquidos percolados apresentam elevado potencial poluidor, e compreende, geralmente, o chorume, a água de infiltração e o material lixiviado, portanto, é importante avaliar sua toxicidade em relação aos ecossistemas aquáticos. Os testes de toxicidade foram realizados com Ceriodaphnia silvestrii, Chironomus xanthus e Daphnia similis e permitiram confirmar a toxicidade aguda destas amostras para os três organismos-teste realizados. Os testes de toxicidade crônica apresentaram diferença significativa, tanto na reprodução quanto na sobrevivência em relação ao controle, com Ceriodaphnia silvestrii. Nos testes com Daphnia similis, foi registrada uma diferença na reprodução apenas na presença de determinados líquidos percolados. Em alguns casos a diferença de precipitação não interferiu na toxicidade, porém nos testes efetuados com Chironomus xanthus foi verificada que a falta de precipitação pode ter aumentado a toxicidade dos líquidos percolados. / Industrial, agrarian and population growth is responsible for the increased amount of solid waste what demands suitable procedures for waste disposal so as to preserve the environment, particularly and water resources. Landfill is one of the methods for final disposal of solid waste, but it is not adequate because it lacks drainage systems, waste input and out put controls, percolation control, etc. Liquid waste from those landfills has a very high polution potential, and includes the leachate, the infiltration water, and the overflow waste. That is why it is important to assess the degree of toxicity of those solid wastes in relation to the aquatic ecosystems. Toxicological tests were carried out with Ceriodaphnia silvestrii, Chironomus xanthus and Daphnia similis allowed to confirm high toxic level in those samples for the three tested organisms. Chronic toxicological tests presented statistic significant difference regarding reproduction and survival in relation to the control samples for Ceriodaphnia silvestrii. Daphnia similis, however presented significant difference in the reproduction in relation to some substances only. In some cases, precipitation did not interfere in the toxicological assessment, but the tests with Chironomus xanthus showed that the lack of precipitation might have affected the toxicology.