Este trabalho foi desenvolvido nos reservatórios, em cascata, que fazem parte do Médio e Baixo Rio Tietê, incluindo os tributários, os Rios Tietê, Piracicaba e Bauru. Foram considerados os pontos de coleta, localizados à montante, jusante, bem como os pontos intermediários dos reservatórios estudados. Considerando-se os períodos referentes a outubro de 1999 fevereiro, maio e julho de 2000, as pesquisas nestes locais abordaram a qualidade ambiental dos reservatórios, com características limnológicas (água, sedimento), a fauna de macroinvertebrados bentônicos, além de testes ecotoxicológicos (Chironomus xanthus). As análises dos sedimentos permitiram evidenciar as frações de silte e argila, o elevado teor de matéria orgânica nos reservatórios situados no início da cascata, além do nitrogênio e fósforo e metais potencialmente biodisponíveis. Elevadas concentrações de nutrientes nitrogenados e fosfatados, material em suspensão (orgânico e inorgânico) e clorofila-a, foram observadas nas amostras de água, o que permitiu classificar os reservatórios como eutróficos, mesotróficos e oligotróficos, de acordo com sua posição no sistema bem como do período amostrado. As concentrações de alguns dos metais encontrados na água estiveram acima do permitido pela Resolução CONAMA (20/96), considerando-se os períodos em estudo. Quanto às relações bióticas, foram obtidas através do índice de Diversidade (H\'), da equitabilidade, a qual foi evidenciada pela distribuição agregadados organismos e a presença de indivíduos pertencentes à mesma espécie, evidenciou a baixa riqueza de taxa. Através do IET/Densidade de Oligochaeta constatou-se para os reservatórios em cascata e os principais tributários características de eutrofizados. A fauna bentônica dos rios Tietê, Piracicaba e Bauru e dos reservatórios dos locais de estudo caracterizou-se pela presença de Tubificidae (Oligochaeta) além de Chironomidae (Díptera), e do grupo Mollusca. Entre os Tubificidae, a espécie Limnodrilus hoffmeisteri, Branchiura sowerbyi e Limnodrilus udekemianus ocorreram em considerável dominância numérica no período de estudo. A Família Chironomidae, foi representada pelos gêneros Goeldichiromus e Coelotanypus e o grupo Mollusca pelo gênero, Haylacostoma e espécie Melanoides tuberculatus (Thiaridae), Corbicula fluminea (Corbiculidae), o gênero Biomphalaria (Planorbidae) e o gênero Lymnaea collumela, (Lymnaiidae). Quanto à Família Naididade foi representada pelas espécies (Prístina americana, Dero nívea, Dero digitata e Allonais chelata), pela família Alluroididae (Brinkhurstia americanus) e pela Família Opistocystidae (Opistocysta funiculus). Os testes de toxicidade com Chironomus xanthus confirmaram os resultados obtidos através das análises físicas e químicas, conferindo características de impactados aos reservatórios situados no início da cascata (Barra Bonita, Bariri e Ibitinga) e aos tributários (Tietê, Piracicaba e Bauru) moderadamente poluídos aos reservatórios intermediários (reservatório de Nova Avanhandava e Promissão) e, com ausência de poluição, o Reservatório de Três Irmãos, situado no final da cascata. Os resultados decorrem dos usos e ocupação do solo, atividades antropogênicas na Bacia Hidrográfica, do aporte de materiais para o sistema na qual se inserem os rios e os reservatórios estudados. / This work was developed in the reservoirs, at waterfalls, which are part of the Medium and Low Tiete River, including its tributaries, the Tiete, the Piracicaba river and the Bauru river. The collecting sites considered were upstream, downstream and those intermediary sites of the reservoirs. Taking into account the different periods of October 1999 and February, May and July of 2000, the researches in those sites were about the environmental quality of the reservoirs with limnologic characteristics (water, sediment), the bentonic macroinvertebrate fauna besides ecotoxilogic tests (Chironomus xanthus). The sediment analysis allowed to evidence the silt and clay fractions, the high organic matter content in the reservoirs located at the waterfall head in addition to nitrogen, phosphur and potentially bio-available metals. High nitrogened and phosphated nutrients, suspended matter (organic and inorganic) and chlorophyl-a were observed in the water samples, which allowed to classify the reservoirs as euthrophic, mesotrophic and oligotrophic according to their location in the system and the sampling period. The concentration of some metals found the water was above the level permitted by CONAMA Resolution (20/96) taking into account the periods of study. As to the biotic relations, they were obtained by means of the Diversity index (H\') of the equitability, which was evidenced by the aggregated distribution of the organisms and the presence of individuals belongingto the same species evidenced the low taxa richness. Through the IET/Oligochaeta abundance, the reservoirs and the tributaries were found to be eutrophitized. The Tiete, Piracicaba and Bauru rivers and the reservoirs studied sites bentonic fauna was characterized by the presence of Tubificidae (Oligochaeta) besides Chironomidae (Diptera) and the Mollusca group. Among the Tubificidae, the Limnodrilus hoffmeisteri, Branchiura sowerbyi e Limnodrilus udekemianus species occurred in considerable numerical dominance in the period of study. The Chironomidae family was represented by the genus Goeldichiromus and Coelotanypus and the Mollusca group by the genus Haylacostoma and Melamoides tuberculatus (Thiaridae) species, Corbicula fluminea (Corbiculidae), the genus Bionphalaria (Planorbidae) and the genus Lymnaea collumela (Lymnaiidae). The Naididae family was represented by the species (Pristina americana, Dero nivea, Dero digitata and Allonais chelata), by the family Alluroididae (Brinkhurstia americanus) and the family Opistocystidae (Opistocysta funiculus).The toxicity tests with Chironomus xanthus confirmed the results obtained through physical and chemical analyses granting characteristics of impacted to the reservoirs located at the waterfall head (Barra Bonita, Bariri and Ibitinga) and the tributaries (Tiete, Piracicaba and Bauru) moderately polluted to the intermediate reservoirs (Nova Avanhandava and Promissão reservoirs) and, absenceof pollution, the Tres Irmãos Reservoir located at the end of the waterfall. The results arise from the uses and occupation of the soli, anthropogenic activities in the hydrographic basis, disclosure of materials into the system in which are the rivers the reservoirs under study are located.

The surface gliding motility of Myxococcus xanthus is required for a multicellular developmental process initiated by unfavorable growth conditions. One form of the M. xanthus surface motility, social (S) gliding, is mediated by the extension and retraction of polarly localized type IV pili (Tfp). Besides Tfp, exopolysaccharides (EPS), another cell surface associated component, are also required for M. xanthus S motility. Previous studies demonstrated that the Dif chemotaxis-like signal transduction pathway is central to the regulation of EPS production in M. xanthus. Specifically, difA, difC and difE mutants were found to be defective in EPS production and S motility. DifA, DifC and DifE, homologous to methyl-accepting chemotaxis proteins (MCPs), CheW and CheA, respectively, are therefore positive regulators of EPS. This study, undertaken to better understand the regulation of EPS production, led to a few major findings. First, DifD and DifG, homologous to CheY and CheC, respectively, were found to be negative regulators of EPS production. Both DifD and DifG likely function upstream of the DifE kinase in EPS regulation. DifB, which has no homology to known chemotaxis proteins, was found not to be involved in EPS production. Secondly, this study led to the recognition that Tfp likely function upstream of the Dif pathway in the regulation of EPS production. Extracellular complementation experiments suggest that Tfp may act as sensors instead of signals for the Dif chemotaxis-like pathway. We propose a regulatory feedback loop that couples EPS production with Tfp function through the Dif signaling proteins. Lastly, we sought to identify additional genes involved in EPS production. Our efforts identified a mutation in a separate chemotaxis gene cluster as a suppressor of difA mutations, suggesting potential cross-talks among the multiple chemotaxis-like pathways in M. xanthus. In addition, we identified twenty-five previously uncharacterized genes that are predicted to be involved in M. xanthus EPS production. These genes appear to encode additional EPS regulators and proteins with biosynthetic function. / Ph. D.

The Myxococcus xanthus Dif and Frz chemosensory pathways play important roles in the regulation of gliding motility. The Dif system regulates the production of exopolysaccheride (EPS), which is essential for social motility and fruiting body formation. The Frz pathway controls reversal frequency, which is fundamental for directed movement by this surface-gliding bacterium. In addition, both pathways are involved in the chemotactic response towards several phosphatidylethanolamine (PE) species such that the Dif pathway is required for excitation while the Frz pathway is essential for adaptation. In this study we addressed three crucial questions regarding the signal processing of these two chemosensory pathways by focusing on DifA and FrzCD, the MCP homologs from their respective pathways. First, the receptor protein in the Dif pathway, DifA, lacks a perisplasmic domain, the typical signal-sensing structure. To examine whether DifA shares similar transmembrane signaling mechanism with typical transmembrane sensor proteins (MCPs and sensor kinases), we constructed a chimeric protein that is composed of the N-terminus of NarX (nitrate sensor kinase) and the C-terminus of DifA. This NarX-DifA chimera restores the DifA functionality (EPS production, agglutination, S-motility and development) to a "difA mutant in a nitrate-dependent manner, suggesting DifA shares a similar transmembrane signaling mechanism with typical MCPs and sensor kinases despite its unorthodox structure. Second, the M. xanthus chemotaxis is still controversial. It has been argued that the taxis-like response in this slowly gliding bacterium could result from physiological effects of certain chemicals. To study motility regulation by the Frz pathway, we constructed two chimeras between the N-terminus of NarX and C-terminus of FrzCD, which is the receptor protein of the Frz pathway. The two chimeras, NazDF and NazDR, are identical except that NazDR contains a G51R mutation in the otherwise wild-type NarX sensory module. This G51R mutation was shown to reverse the signaling output of a NarX-Tar chimera to nitrate. We discovered that nitrate specifically decreased the reversal frequency of NazDF-expressing cells and increased that of NazDR-expressing cells. These results show that directional motility in M. xanthus can be regulated independently of cellular metabolism and physiology. Surprisingly, the NazDR strain failed to adapt to nitrate in temporal assays, as did the wild type to known repellents. Therefore, the lack of temporal adaptation to negative stimuli is an intrinsic property in M. xanthus motility regulation. Third, the Dif and Frz pathways are both involved in the chemotactic response towards certain PE molecules such that the Dif pathway is required for excitation and while the Frz system is essential for adaptation. In addition, 12:0 PE, known to be sensed by DifA, results in increased FrzCD methylation. These findings suggested that in the regulation of PE response, two pathways communicate with each other to mediate adaptation. Here we provided evidence to indicate that DifA does not undergo methylation during EPS regulation and PE chemotaxis. On the other hand, using mutants expressing the NarX-DifA chimera, it was found that signal transduction through DifA, DifC (CheW-like) and DifE (CheA-like) modulates FrzCD methylation. Surprisingly, the attractant 12:0 PE can modulate FrzCD methylation in two ways distinguishable by the dependency on DifA, DifC and DifE. The DifACE-independent mechanism, which may result from specific sensing of 12:0 PE by FrzCD, increases FrzCD methylation as expected. Unexpectedly, 12:0 PE decreases FrzCD methylation with the DifACE-dependent mechanism. This "opposite" FrzCD methylation by DifACE-dependent signaling was supported by results from NafA-expressing mutants because nitrate, which acts as a repellent, increases FrzCD methylation. Based on these findings, we proposed a model for chemotaxis toward 12:0 PE (and 16:1 PE). In this model, DifA and FrzCD both sense the same signal and activate the pathways of excitation (Dif) and adaptation (Frz) independently. The two pathways communicate with each other via methylation crosstalk between DifACE and FrzCD in such a way that processes of excitation and adaptation can be coordinated. / Ph. D.

Made available in DSpace on 2016-06-02T19:29:38Z (GMT). No. of bitstreams: 1 3822.pdf: 3918575 bytes, checksum: d646b2b0a3b661d4dfa2d3c8a1ee8a69 (MD5) Previous issue date: 2011-03-28 / Universidade Federal de Sao Carlos / The Alto Jacaré-Guaçu Basin was evaluated regarding limnological and ecotoxicological aspects in four water and sediment sampling campaigns at 14 points distributed in the area. Toxicity tests with water, in the laboratory and in situ, showed chronic toxicity, with effects on the survival, growth and gill morphology of juveniles of both fish species examined (Danio rerio and Poecilia reticulata). The lowest survival percentages in the laboratory assays were observed for point L, and in the field tests for point J1. The analysis of the gills revealed first-stage alterations in the specimens of both species exposed at all the sample points, and there were second-stage alterations (aneurism and vascular congestion) at various points. The high concentrations of nutrients and metals in the basin s water and sediments reveal a system impacted by human activities in the surrounding region. The analyses of metals quantified Cd, Cr, Cu, Mn and Fe levels in the water above those permitted by the Brazilian National Environmental Council (CONAMA 357/05), and of Cd in the sediments above the threshold levels permitted both by national (CONAMA 344/04) and international regulations (CCME, 2003). Simple synthetic sediments were prepared composed of sand, clay and organic matter (obtained from the aquatic macrophyte Scirpus cubensis, at concentrations of 0, 0.25, 0.5 and 1%) to assess the toxicity of Cd. The synthetic sediments were viable for the growth and survival of Ceriodaphnia silvestrii, Chironomus xanthus and Danio rerio, and were suitable for use in spiked experiments. The evaluation of the acute toxicity of the sediments fortified with Cd indicated mean 48-h;EC50 for C. silvestrii of 3.09mg.Kg-1 in the sediments with 0% OM and 2.98mg.Kg-1 in the sediments with 1% OM. The mean 96-h;LC50 for C. xanthus was 18.67 mg.Kg-1 in the sediments with 0% OM and nearly four times lower in those with 1% OM (4.93 mg.Kg-1). A similar pattern was noted for D. rerio, a higher 48-h;LC50 in the sediments with 0% OM (114.88 mg.Kg-1) than in the sediments with 1% OM (75.31 mg.Kg-1). In subsequent experiments, the sediments were also spiked with 0.6mg.Kg-1 of Cd, a concentration considered safe for protection of aquatic life according to the applicable national and international regulations. In these tests, the survival of C. silvestrii was impaired in the Cd spiked sediments, so it was not possible to assess the effects on reproduction. There were also effects on the survival, biomass, mentum morphology and Cd concentration in the tissues of C. xanthus. For D. rerio, the survival and growth were compromised in the sediments with higher percentage of organic matter. Besides this, the organisms showed impaired gill morphology and higher Cd concentration in tissue in function of exposure to sediments spiked with the metal. Based on the results, it can be concluded that C. silvestrii was the most sensitive species to the sediments fortified with Cd, followed by C. xanthus and D. rerio, the least sensitive. These effects on the iv organisms at concentrations considered safe for the biota suggest more conservative thresholds of this metal should be established for tropical aquatic habitats. / A bacia do Alto Jacaré-Guaçu foi avaliada quanto aos aspectos limnológicos e ecotoxicológicos em quatro campanhas de amostragem de água e sedimento em 14 pontos distribuídos na área. Testes de toxicidade com água, tanto em laboratório como in situ, apresentaram toxicidade crônica com efeitos na sobrevivência, crescimento e morfologia de brânquias de juvenis de ambas as espécies de peixes avaliadas (Danio rerio e Poecilia reticulata). As menores porcentagens de sobrevivência nos testes em laboratório foram observadas no ponto L e, nos testes in situ, em J1. A análise das brânquias mostrou alterações de primeiro estágio nos organismos expostos em todos os pontos avaliados, e em diversos pontos foram verificadas alterações de segundo estágio (aneurisma e congestão vascular). As elevadas concentrações de nutrientes e metais na água e sedimentos da Bacia do Alto Jacaré-Guaçu revelam um sistema impactado pelas atividades humanas que se desenvolvem em seu entorno. As análises de metais quantificaram Cd, Cr, Cu, Mn e Fe acima da CONAMA 357/05 na água e Cd acima dos valores estabelecidos pela legislação nacional (CONAMA 344/04) e internacional (CCME, 2003) nos sedimentos. Foram desenvolvidos sedimentos sintéticos simples compostos de areia, argila e matéria orgânica (obtida a partir da macrófita aquática Scirpus cubensis, nas concentrações de 0; 0,25; 0,5 e 1%) para avaliar a toxicidade do Cd. Os sedimentos sintéticos se apresentaram viáveis para o crescimento e sobrevivência de Ceriodaphnia silvestrii, Chironomus xanthus e Danio rerio, sendo adequados para uso em experimentos de fortificação. A avaliação da toxicidade aguda dos sedimentos fortificados com Cd, indicaram CE50;48h média para C. silvestrii de 3,09mg.Kg-1 nos sedimentos sem MO e 2,98mg.Kg-1 nos sedimentos com 1% de MO. A CL50;96h média para C. xanthus foi de 18,67mg.Kg-1 nos sedimentos sem MO e, cerca de 4 vezes menor nos sedimentos com 1% de MO, 4,93mg.Kg-1. Para D. rerio também foi notada tendência semelhante, CL50;48h maior nos sedimentos sem MO, 114,88mg.Kg-1 e menor nos sedimentos com 1% de MO, 75,31mg.Kg-1. Os sedimentos também foram fortificados com 0,6mg.Kg-1 de Cd, concentração considerada segura para a proteção da vida aquática segundo legislações nacionais e internacionais, em experimentos posteriores. C. silvestrii teve a sobrevivência comprometida nos sedimentos fortificados com Cd, de modo que não foi possível avaliar os efeitos na reprodução. C. xanthus também apresentou efeitos na sobrevivência, biomassa, morfologia de mento e concentração de Cd nos tecidos. D. rerio teve a sobrevivência e crescimento comprometidos nos sedimentos com maior porcentagem de matéria orgânica. Além disso, os organismos apresentaram a morfologia de brânquias comprometida e concentração de Cd em ii seus tecidos elevada em função da exposição aos sedimentos fortificados com o metal. Com base nos resultados foi possível concluir que C. silvestrii foi a espécie mais sensível aos sedimentos fortificados com Cd, seguido de C. xanthus e, a menos sensível, D. rerio. Tais efeitos observados nos organismos em concentrações consideradas seguras para a biota sugerem a avaliação mais cuidadosa dos valores para espécies e climas tropicais.

Made available in DSpace on 2016-06-02T19:31:43Z (GMT). No. of bitstreams: 1 2493.pdf: 1820155 bytes, checksum: c4fbdc0a3fdb7a9316ba5c6c6a6905c8 (MD5) Previous issue date: 2009-05-15 / Universidade Federal de Sao Carlos / The objective of the present dissertation was to make a toxicity assessment of the application of calcium nitrate solution as a remediation procedure for sediments of a eutrophic aquatic ecosystem. The study was carried out using microcosms with superficial sediments and water from sediment-water interface of the Ibirité Reservoir located in the metropolitan area of Belo Horizonte (Minas Gerais, SE Brazil). The experiment lasted 135 days and the following treatment or incubation periods were applied: t=0, t=5, t=10, t=25, t=50, t=85 and t=135 days. In each period, one controlmicrocosm and three treatment-microcosms were disassembled and, chemically and ecotoxicologically analyzed. The organisms Ceriodaphnia silvestrii and Vibrio fischeri (Microtox® System) were used for the acute toxicity assessment of the water from sediment-water interface and the pore water of sediments, whereas the organism Chironomus xanthus was used for the toxicity assessment of bulk sediment. The toxicity tests were run in parallel with chemical analyzes of dissolved inorganic nitrogen species (nitrate, nitrate and ammonium), sulfate, and metals in the interface sediment-water and interstitial water samples. Acid volatile sulfide (AVS), simultaneously extracted metal (SEM) and potentially bioavailable metals were analyzed in bulk sediment. The overall results indicate that nitrate whose concentration reached 1,200 mg N-NO3 - L-1 in sediment pore water samples from treatment-microcosms is the most probable compound causing toxicity to the tests organisms. For Chironomus xanthus sediments were deleterious to the exposed organisms in all microcosm run, except in the period of t= 135 days. For the experimental conditions of this work, the application of calcium nitrate as a remediation procedure for sediments from Ibirité Reservoir indicated to be inadequate from the ecotoxicological pint of view. / O presente trabalho visou à avaliação da toxicidade da aplicação de solução de nitrato de cálcio, como procedimento de intervenção para remediação de sedimentos de um ambiente aquático eutrofizado. O estudo foi realizado através de microcosmos com sedimento e água de interface sedimento-água da Represa Ibirité, situada na região metropolitana de Belo Horizonte (Minas Gerais, Brasil). Os experimentos tiveram a duração total de 135 dias, divididos nos tempos de tratamento ou incubação de: t=0, t=5; t=10; t=25; t=50; t=85 e t=135 dias. Em cada tempo de tratamento foram analisados um microcosmo-controle e três microcosmostratamento. Os organismos Ceriodaphnia silvestrii e Vibrio fischeri (Sistema Microtox®) foram utilizados para avaliação da toxicidade aguda das águas de interface sedimento-água e intersticial dos sedimentos, enquanto o organismo Chironomus xanthus para avaliação do sedimento integral. Em paralelo aos testes de toxicidade foram realizadas análises químicas da série nitrogenada (nitrato, nitrito e amônia), sulfato, e metais nas amostras de água de interface sedimento-água e intersticial dos sedimentos, sulfetos volatilizáveis por acidificação (SVA), metais extraídos simultaneamente (MES) e metais potencialmente biodisponíveis nos sedimentos. Os resultados mostraram que o nitrato, chegando a concentração superior a 1.200 mg N-NO3 - L-1 nas amostras de água intersticial dos sedimentos dos microcosmos-tratamentos, foi considerado o causador mais provável da toxicidade das amostras dos microcosmos-tratamento para os organismos-teste empregados. Para o organismo Chironomus xanthus, os sedimentos em tratamento foram deletérios aos organismos expostos em todos os tempos de incubação, exceto no tempo t=135 dias. Estritamente do ponto de vista ecotoxicológico e para as condições experimentais deste trabalho, a aplicação do nitrato como forma de intervenção para remediação dos sedimentos da Represa Ibirité não se mostrou adequada.

Bacterial DNA replication can stall at DNA lesions, leading to cell death if the damage fails to be repaired. To circumvent this, bacteria possess a mechanism called translesion DNA synthesis (TLS) to allow DNA damage bypass. The ImuABC TLS mutasome comprises the RecA domain-containing protein ImuA, the inactive polymerase ImuB, and the error-prone polymerase ImuC. ImuA and ImuB are necessary for the mutational function of ImuC that can lead to antimicrobial resistance (AMR) as seen in high-priority pathogens Pseudomonas aeruginosa and Mycobacterium tuberculosis. Understanding how ImuA and ImuB contribute to this function can lead to new targets for antimicrobial development. This research aims to discover the molecular functions of ImuA and ImuB homologs from Myxococcus xanthus through structural modelling and biochemical analyses. ImuA was discovered to be an ATPase whose activity is enhanced by DNA. Based on predicted structural models of the ATPase active site, I identified the critical residues needed for ATP hydrolysis, and found that the ImuA C-terminus regulates ATPase activity. Further, ImuA and ImuBNΔ34 (a soluble truncation of ImuB) display a preference for longer single-stranded DNA and overhang DNA substrates, and their affinity for DNA was quantified in vitro. To better understand how ImuA and ImuB assemble in the TLS mutasome, bacterial two-hybrid assays determined that ImuA and ImuB can self-interact and bind one another. Mass photometry revealed that ImuA is a monomer and ImuBNΔ34 is a trimer in vitro. ImuA and ImuBNΔ34 binding affinity was quantified in vitro at 1.69 μM ± 0.21 by microscale thermophoresis, and removal of the ImuA C-terminus weakens this interaction. Lastly, ImuA and ImuBNΔ34 secondary structures were quantified using circular dichroism spectroscopy, and ImuA was modified to enable crystallization for future structural studies. Together, this research provides a better understanding of ImuABC-mediated TLS, potentially leading to novel antibiotics to reduce the clinical burden of AMR. / Thesis / Master of Science (MSc) / The antimicrobial resistance (AMR) crisis is fueled by the emergence of multi-drug resistant microbes, posing a major threat to global health and disease treatment. Bacteria can develop resistance to antibiotics through mutations in the genome. When the genome becomes damaged, bacteria can acquire these mutations by an error-prone replication mechanism called translesion DNA synthesis (TLS). In some bacteria, TLS involves a specialized enzyme complex, consisting of proteins ImuA, ImuB and ImuC, allowing replication past bulky DNA damage and lesions. The goal of this thesis is to investigate how the ImuA and ImuB proteins contribute to the functioning of this mistake-making machinery. I used biochemical and biophysical methods to identify ImuA and ImuB interactions with each other and themselves. I discovered that ImuA is an enzyme that uses energy to enhance its binding to DNA, and determined the specific amino acids involved in this function.