L’évaluation de l’impact des polluants dans l’environnement est une des préoccupations majeures qui s’inscrit dans la Directive Européenne Cadre Eau 2000. Les réglementations préconisées ont notamment pour objectif de parvenir au bon état chimique et écologique des masses d’eau. Dans ce contexte, notre étude a consisté à développer une approche multibiomarqueur sur des juvéniles de poisson afin d’évaluer les effets biologiques de la pollution chimique en milieu littoral. Des paramètres moléculaires de détoxification (EROD, GST) et une enzyme antioxydante (CAT) ont été utilisés en tant que «système d’alarme » susceptibles de détecter une perturbation avant l’apparition de signes pathologiques irréversibles. En parallèle, différents biomarqueurs physiologiques (croissance somatique et récente, rapport ARN/ADN, indices morphométrique et lipidique) ont été analysés en considérant que ceux-ci pourraient révéler les dommages induit par les polluants sur l’état de santé des juvéniles. La sensibilité et la pertinence des biomarqueurs moléculaires et physiologiques ont été testés expérimentalement sur des juvéniles : i) de bar exposés à une pollution aigüe de pétrole, ii) de bar et de turbot soumis à des mélanges de contaminants en concentrations environnementales en conditions contrôlée et semi-contrôlée (« caging »). Nos résultats montrent la capacité de l’EROD, et à un degré moindre de la GST, à détecter une exposition courte (2 et 4 jours) des organismes au pétrole et à refléter ses effets délétères sur leur état de santé. Cette relation entre biomarqueurs moléculaires et physiologiques a par contre été plus difficilement établie dans un contexte de pollution multiple. Les indices de croissance et de condition utilisés se sont avérés plus sensibles aux différents niveaux de contamination analysés (métaux et HAPs). Leur utilisation a permis d’évaluer la condition affaiblie des organismes mis en cage en milieu portuaire pendant 38 jours. Cette expérience de « caging » s’est révélée concluante, notamment pour les juvéniles de bar, sur lesquels aucun stress physiologique de la mise en cage n’a été détecté dans la station de référence. Les effets délétères des contaminants chimiques sur l’état de santé des juvéniles de turbot ont également été observés en condition contrôlée après exposition de 21 jours aux mêmes sédiments portuaires et à un sédiment estuarien. En complément de ces expériences, une étude de terrain a été réalisée sur des juvéniles de flet prélevés dans des estuaires le long de la côte française et belge. Une diminution des indices morphométrique et lipidique des juvéniles de flet, issu des trois estuaires anthropisés, a été observée en relation avec des bioconcentrations en métaux plus élevées que l’estuaire de référence. Les résultats issus de ces différentes études montrent la potentialité des indices de croissance et de condition à révéler les effets biologiques des contaminants chimiques sur les juvéniles de poissons marins. Cependant, leur spécificité vis à vis des polluants étant plus faible que les paramètres de détoxification, leur utilisation peut être limitée. Ces travaux montrent ainsi le besoin d’utiliser des biomarqueurs à différents niveaux d’organisation biologique dans les programmes de biosurveillance. / Evaluation of pollutant effects in environment is one of the major issues of the European Water Framework Directive 2000. Regulations have particularly the objective to reach to a good chemical and ecological status of water bodies. In this context, the aim of our study was to develop a multibiomarker approach on juvenile marine fish in order to evaluate the biological effects of chemical pollution in coastal areas. Molecular detoxification parameters (EROD, GST) and an antioxidant enzyme (CAT) were used as early warning tools of toxicity allowing the prevention of irreversible damages. In parallel, different physiological biomarkers (somatic and recent growth, RNA:DNA ratio, morphometric and lipidic indices) were analysed as reflecting damages on juveniles health. Sensitivity and relevance of molecular and physiological biomarkers were tested experimentally on juvenile : i) sea bass exposed to acute petroleum pollution, ii) sea bass and turbot submitted to a mix of contaminants in environmental concentrations during controlled and semi-controlled (caging) conditions. Our results show the ability of EROD, and in lower degree the GST, to detect short exposure (2 and 4 days) of organisms to petroleum and to reflect their deleterious effects on fish health. This relationship between molecular and physiological biomarkers was more difficultly established under multiple pollutions. Growth and condition indices were found to be more sensitive to the different levels of chemical contamination analysed (metal, PAHs). Their analyses allowed us to evaluate the weakened condition of organisms caged in the harbour area during 38 days. This caging experiment was relevant especially for juvenile sea bass in which no physiological stress was detected in the reference station. Deleterious effects of chemical contaminant on turbot juvenile health were also observed in controlled condition after 21 days exposure to the same harbour sediments and to an estuarine sediment. In complement to these experiments, a field study was realized on juvenile flounders sampled in some estuaries along the French and Belgium coast. A decrease of morphometric and lipidic indices were found in juvenile flounders from the three anthropogenic estuaries showing the highest metal bioconcentrations compared to the reference estuary. Results from these different studies showed the potentiality of growth and condition indices to reflect biological effects of chemical contaminants on juvenile marine fish. However, their use could be limited by their lower specificity to pollutant than parameters involved in detoxification. These works show therefore the need to use biomarkers at different level of biological organization in biomonitoring programs.

Polimorfismos nos genes da n-acetiltransferase 2 (NAT2), CYP2E1 e glutationa S-transferase (GST) têm sido associados a diferenças na resposta ao tratamento da tuberculose. O papel de variantes dos genes NAT2, CYP2E1 e GSTM1/GSTT1, no perfil de segurança do tratamento da tuberculose, foi avaliado em 99 pacientes com tuberculose, sem co-infecção por HIV ou vírus da hepatite, tratados por 6 meses. Amostras de sangue foram colhidas antes e durante o tratamento para avaliação de marcadores de lesão hepatocelular (ASLT e AST), colestase (ALP, GGT e bilirrubinas) e função renal (creatinina). O DNA genômico foi extraído de sangue colhido em EDTA pelo método precipitação salina. Os polimorfismos NAT2 foram analisados por PCR-RFLP e seqüenciamento de DNA. Os polimorfismos da região promotora do CYP2E1 foram detectados por PCR-RFLP e para a análise dos genótipos nulos de GSTM1 (GSTM1*0) e GSTT1 (GSTT1*0) foi utilizada a PCR multiplex. Durante o tratamento, 59,6% dos pacientes apresentaram reações adversas aos medicamentos (RAM) e alterações nos marcadores de lesão hepatocelular e colestase, com aumento de 1 a 4 vezes o limite superior de referência. Foi observada forte relação entre RAM e alterações nos marcadores séricos (p< 0,05) e também com o uso de medicação concomitante (p< 0,001). As freqüências dos alelos NAT2*4 e NAT2*6 foram maiores e menores, respectivamente, quando comparadas com outros estudos na população brasileira. O perfil de acetilador lento (alelos NAT2*5, NAT2*6 e NAT2*7) foi associado com manifestação de RAM e hepatotoxicidade. Os portadores dos genótipos NAT2*4/*5 e NAT2*5/*5 apresentaram, respectivamente, risco 2,4 e 5,0 vezes maior de RAM que os portadores dos demais genótipos NAT2 (p< 0,05). O genótipo funcional GSTM1*1/GSTT1*1 foi associado com alterações acentuadas de ALT, AST e ALP (p< 0,05). Enquanto que as variantes da CYP2E1 não foram associadas a alterações no perfil bioquímico ou com risco de RAM ou hepatotoxicidade. Em conclusão, o perfil de acetilação lenta de NAT2 e o genótipo funcional de GSTM1/GSTT1 aumentam a susceptibilidade de lesão hepatocelular e outras RAM induzidas pelos antimicobacterianos utilizados no tratamento da tuberculose. / Polymorphisms in N-acetiltransferase 2 (NAT2), CYP2E1 and glutatione S-transferase (GST) have been associated with differences in response to antituberculosis drugs. The role of the NAT2, CYP2E1 and GSTM1/GSTT1 variants on safety profile of the anti-tuberculosis therapy was evaluated in 99 tuberculosis patients, without co-infection by HIV or hepatitis virus, treated during 6 months. Blood samples were collected before and after the therapy to evaluate serum markers for hepatocelullar damage (ASLT and AST), cholestasis (ALP, GGT and bilirrubin) and kidney function (creatinine). Genomic DNA was extracted from EDTA-blood samples by salting-out method. NAT2 polymorphisms were analyzed by PCR-RFLP and DNA sequencing. CYP2E1 promoter region polymorphisms were detected by PCR-RFLP and for analysis of the null genotypes GSTM1 (GSTM1*0) e GSTT1 (GSTT1*0) PCR multiplex technique was used. During the therapy, 59.6% of the patients had adverse drug reactions (ADR) and alterations on hepatocellular damage and cholestasis serum markers, with increase of 1 to 4 times the upper limit reference level. There was a significant relationship between ADR and serum markers alterations (p< 0,05), as well as, the concomitant medicine (p< 0,001). The frequencies of the NAT2*4 and NAT2*6 alleles were higher and lower, respectively, when compared to other studies in the Brazilian population. The slow acetilator profile (NAT2*5, NAT2*6 and NAT2*7 alleles) was associated with ADR and hepatotoxicity manifestations. The NAT2*4/*5 and NAT2*5/*5 genotypes carriers had, respectively, 2.4 and 5.0 times higher risk for ADR than those carrying the other NAT2 genotypes (p< 0,05). The functional genotype GSTM1*1/GSTT1*1 was associated with enhanced variations on ALT, AST and ALP (p< 0.05). No relationship was found between CYP2E1 variants and variations on biochemical profile or risk for ADR or hepatotoxicity. In conclusion, the NAT2 slow acetilator profile and the GSTM1/GSTT1 functional genotype increase the susceptibility to hepatocellular damage and other ADR induced by antibiotics used in tuberculosis therapy.

Polimorfismos nos genes da n-acetiltransferase 2 (NAT2), CYP2E1 e glutationa S-transferase (GST) têm sido associados a diferenças na resposta ao tratamento da tuberculose. O papel de variantes dos genes NAT2, CYP2E1 e GSTM1/GSTT1, no perfil de segurança do tratamento da tuberculose, foi avaliado em 99 pacientes com tuberculose, sem co-infecção por HIV ou vírus da hepatite, tratados por 6 meses. Amostras de sangue foram colhidas antes e durante o tratamento para avaliação de marcadores de lesão hepatocelular (ASLT e AST), colestase (ALP, GGT e bilirrubinas) e função renal (creatinina). O DNA genômico foi extraído de sangue colhido em EDTA pelo método precipitação salina. Os polimorfismos NAT2 foram analisados por PCR-RFLP e seqüenciamento de DNA. Os polimorfismos da região promotora do CYP2E1 foram detectados por PCR-RFLP e para a análise dos genótipos nulos de GSTM1 (GSTM1*0) e GSTT1 (GSTT1*0) foi utilizada a PCR multiplex. Durante o tratamento, 59,6% dos pacientes apresentaram reações adversas aos medicamentos (RAM) e alterações nos marcadores de lesão hepatocelular e colestase, com aumento de 1 a 4 vezes o limite superior de referência. Foi observada forte relação entre RAM e alterações nos marcadores séricos (p< 0,05) e também com o uso de medicação concomitante (p< 0,001). As freqüências dos alelos NAT2*4 e NAT2*6 foram maiores e menores, respectivamente, quando comparadas com outros estudos na população brasileira. O perfil de acetilador lento (alelos NAT2*5, NAT2*6 e NAT2*7) foi associado com manifestação de RAM e hepatotoxicidade. Os portadores dos genótipos NAT2*4/*5 e NAT2*5/*5 apresentaram, respectivamente, risco 2,4 e 5,0 vezes maior de RAM que os portadores dos demais genótipos NAT2 (p< 0,05). O genótipo funcional GSTM1*1/GSTT1*1 foi associado com alterações acentuadas de ALT, AST e ALP (p< 0,05). Enquanto que as variantes da CYP2E1 não foram associadas a alterações no perfil bioquímico ou com risco de RAM ou hepatotoxicidade. Em conclusão, o perfil de acetilação lenta de NAT2 e o genótipo funcional de GSTM1/GSTT1 aumentam a susceptibilidade de lesão hepatocelular e outras RAM induzidas pelos antimicobacterianos utilizados no tratamento da tuberculose. / Polymorphisms in N-acetiltransferase 2 (NAT2), CYP2E1 and glutatione S-transferase (GST) have been associated with differences in response to antituberculosis drugs. The role of the NAT2, CYP2E1 and GSTM1/GSTT1 variants on safety profile of the anti-tuberculosis therapy was evaluated in 99 tuberculosis patients, without co-infection by HIV or hepatitis virus, treated during 6 months. Blood samples were collected before and after the therapy to evaluate serum markers for hepatocelullar damage (ASLT and AST), cholestasis (ALP, GGT and bilirrubin) and kidney function (creatinine). Genomic DNA was extracted from EDTA-blood samples by salting-out method. NAT2 polymorphisms were analyzed by PCR-RFLP and DNA sequencing. CYP2E1 promoter region polymorphisms were detected by PCR-RFLP and for analysis of the null genotypes GSTM1 (GSTM1*0) e GSTT1 (GSTT1*0) PCR multiplex technique was used. During the therapy, 59.6% of the patients had adverse drug reactions (ADR) and alterations on hepatocellular damage and cholestasis serum markers, with increase of 1 to 4 times the upper limit reference level. There was a significant relationship between ADR and serum markers alterations (p< 0,05), as well as, the concomitant medicine (p< 0,001). The frequencies of the NAT2*4 and NAT2*6 alleles were higher and lower, respectively, when compared to other studies in the Brazilian population. The slow acetilator profile (NAT2*5, NAT2*6 and NAT2*7 alleles) was associated with ADR and hepatotoxicity manifestations. The NAT2*4/*5 and NAT2*5/*5 genotypes carriers had, respectively, 2.4 and 5.0 times higher risk for ADR than those carrying the other NAT2 genotypes (p< 0,05). The functional genotype GSTM1*1/GSTT1*1 was associated with enhanced variations on ALT, AST and ALP (p< 0.05). No relationship was found between CYP2E1 variants and variations on biochemical profile or risk for ADR or hepatotoxicity. In conclusion, the NAT2 slow acetilator profile and the GSTM1/GSTT1 functional genotype increase the susceptibility to hepatocellular damage and other ADR induced by antibiotics used in tuberculosis therapy.

Submitted by Cássia Santos (cassia.bcufg@gmail.com) on 2017-09-01T13:05:01Z No. of bitstreams: 2 Dissertação - Wanderson Lucas da Costa - 2017.pdf: 3043089 bytes, checksum: 32c1b9c81dae778ab37d939fdba41eb5 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-09-15T13:46:23Z (GMT) No. of bitstreams: 2 Dissertação - Wanderson Lucas da Costa - 2017.pdf: 3043089 bytes, checksum: 32c1b9c81dae778ab37d939fdba41eb5 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-09-15T13:46:23Z (GMT). No. of bitstreams: 2 Dissertação - Wanderson Lucas da Costa - 2017.pdf: 3043089 bytes, checksum: 32c1b9c81dae778ab37d939fdba41eb5 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-08-07 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Paracoccidioides spp. are thermodymorphic fungi that when inhaled by humans, these conidia find a favorable environment, changing to the yeast phase and becoming pathogenic causing paracoccidioidomycosis (PCM), one of the most prevalent systemic mycoses in Brazil. Some antifungals are used in the treatment of PCM. Treatment depends on the patient's progression and tolerability of each drug, but their treatment may be for long periods and cause various side effects in the patient. The chiquimate pathway is coordinated by 7 enzymes that perform consecutive steps to convert erythrose-4-phosphate and phosphoenol pyruvate (PEP) into chorismate. In microorganisms, this pathway is involved in the production of the amino acids phenylalanine, tyrosine and tryptophan; These amino acids are essential to the maintenance of these organisms. In this work, pGEX4T3 vector cloning and heterologous expression of Pb18 EPSP synthase belonging to the chiquimate pathway were performed. This protein was expressed in E. coli (DE3) strain and purified. Antibodies were produced for expression analysis of the protein in Western blot. The modeling of EPSP synthase was performed aiming to identify the amino acids involved in the active site. The pull down-GST assay with soluble Pb18 proteins allowed the identification of 40 proteins that interact with EPSP synthase. These proteins belong to different functional categories, which are involved with the availability of phosphoenol pyruvate, the substrate necessary for the functioning of the chiquimate pathway. / Paracoccidioides spp. são fungos termodimórficos que ao serem inalados pelo ser humano, esses conídios encontram um ambiente propício, mudando para a fase de levedura e tornando-se patogênico causando a paracoccidioidomicose (PCM), umas das micoses sistêmicas de maior prevalência no Brasil. Alguns antifúngicos são empregados no tratamento da PCM. O tratamento depende do avanço da doença e da capacidade de tolerância do paciente a cada medicamento, mas o seu tratamento pode ser por longos períodos e causando diversos efeitos colaterais no paciente. A via do chiquimato é coordenada pela ação de 7 enzimas que realizam passos consecutivos para transformar a eritrose-4-fosfato e fosfoenol piruvato (PEP) em corismato. Em micro-organismos, esta via está envolvida com a produção dos aminoácidos fenilalanina, tirosina e triptofano; estes aminoácidos são essenciais à manutenção desses organismos. Neste trabalho foi realizado a clonagem em vetor pGEX4T3 e expressão heteróloga da EPSP–sintase de Pb18 pertencente à via do chiquimato. Essa proteína foi expressa em linhagem E. coli (DE3) e purificada. Os anticorpos foram produzidos para análise da expressão da proteína em Western blot. A modelagem da EPSP-sintase foi realizada visando identificar os aminoácidos envolvidos no sítio ativo. O ensaio de pull down-GST com proteínas solúveis de Pb18 possibilitou a identificação de 40 proteínas que interagem com EPSP-sintase. Essas proteínas pertencem a diferentes categorias funcionais, as quais estão envolvidas com a disponibilidade de fosfoenol piruvato, substrato necessário para o funcionamento da via do chiquimato.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2017-04-24T13:45:43Z No. of bitstreams: 2 Dissertação - Andrea Leite Camargo Santana - 2017.pdf: 3114758 bytes, checksum: 4fa4c3e9a2e7062f2940b053f1250619 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-04-24T13:46:06Z (GMT) No. of bitstreams: 2 Dissertação - Andrea Leite Camargo Santana - 2017.pdf: 3114758 bytes, checksum: 4fa4c3e9a2e7062f2940b053f1250619 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-04-24T13:46:06Z (GMT). No. of bitstreams: 2 Dissertação - Andrea Leite Camargo Santana - 2017.pdf: 3114758 bytes, checksum: 4fa4c3e9a2e7062f2940b053f1250619 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-03-29 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Paracoccidioides fungi are the causative agents of paracoccidioidomycosis, an endemic disease in Latin America with great socioeconomic importance. Inhalation of spores, the infectious particles of the fungus, is a common way to get the infection. Its treatment is slow and generates long-term side effects making it necessary to study new metabolic pathways that can be potential targets for antifungal development. In this context, stands out the chiquimate pathway in Paracoccidioides spp., related to the production of chorismate and production of aromatic amino acids, and the chorismate synthase involved in the last stage of this pathway. The chorismate synthase from Pb18 was cloned into pGEX-4T3 vector, expressed in pLysS cells and purified on a glutathione-sepharose column. Antibodies were produced from the immunization of mice with the recombinant protein obtained and used in Western blot assay to confirm protein expression. For characterization of the enzyme, its modeling was carried out. Pull-down-GST assay was performed to identify interactions between the chorismate synthase and soluble proteins present in total protein extract of Pb18. The interactions found included proteins of different functional categories related to protein synthesis, related with metabolism of common intermediates such as folate and quinolinate or related with the availability of ATP, phosphoenolpyruvate and erythrose-4-phosphate required in the chiquimate pathway. Unexpected interactions with proteins of the cell cycle and also with regulating proteins of the cell division process were observed. / Fungos do gênero Paracoccidioides são os agentes causadores da paracoccidioidomicose, uma doença endêmica na América Latina de grande importância socioeconômica. A inalação de esporos, partículas infecciosas do fungo, é uma forma comum de adquirir a infecção. O seu tratamento é lento e gera efeitos colaterais tornando necessário o estudo de novas vias metabólicas que sejam potenciais alvos para o desenvolvimento de antifúngicos. Nesse contexto, destaca-se a via do chiquimato em Paracoccidioides spp., relacionada à obtenção de corismato e produção de aminoácidos aromáticos, sendo corismato sintase a enzima envolvida na última etapa dessa via. A corismato sintase proveniente de Pb18 foi clonada em vetor pGEX-4T3, expressa em células pLysS e purificada em coluna de glutationa-sefarose. Anticorpos foram produzidos a partir da imunização de camundongos com a proteína recombinante obtida e utilizados em ensaio de Western blot a fim de confirmar a expressão proteica. A modelagem da corismato sintase foi realizada e os aminoácidos envolvidos no sítio ativo foram identificados. Ensaios de pull-down-GST foram realizados a fim de identificar os complexos de interações entre a corismato sintase e proteínas solúveis presentes em extrato proteico total de Pb18. As interações encontradas incluíram proteínas de diferentes categorias funcionais relacionadas à síntese proteica, ao metabolismo de intermediários comuns como folato e quinolinato ou ainda com a disponibilidade de ATP, fosfoenolpiruvato e eritrose-4-fosfato necessários na via do chiquimato. Interações inesperadas com proteínas do ciclo celular e também com proteínas reguladoras do processo de divisão celular foram observadas.

Made available in DSpace on 2016-08-17T14:53:06Z (GMT). No. of bitstreams: 1 Luciana Fortes Farias.pdf: 24712291 bytes, checksum: d8760f57e945d0cde298c31a44b38539 (MD5) Previous issue date: 2009-11-04 / This work is discussed the conceptual model of a multi-agent system for environmental monitoring with the use of biomarkers of aquatic organisms present in the port complex of São Luís-Maranhão-Brasil, second most important in the country in cargo handling. Located in the São Marcos Bay, this complex have an estuarine ecosystems which have suffered attacks in the current process of economic development, caused by intense port handling, dumping of ballast water and washing the vessels, overfishing, introduction of exotic species in the middle pollution in urban and industrial effluents, subject to severe environmental impacts that should be monitored. Methodologically, the modeling of the monitoring solution, we used the existing environmental conditions and aquatic life caught in two different sites of the port complex, the first in a potentially contaminated area and the second in a contamination-free (control), proposing the creation of a network of sensors in these locations. Invest conceded data by Carvalho-Neta (2007) whose research includes to catch fish in these perimeters, then submitting them for laboratory analysis to measure the enzyme activity of glutathione S-transferase (GST) and Catalase (CAT), the result was processed and recorded in bio-ontology . The core of the solution of Multi-agent system is based on the results derived from the biochemical analysis of GST, inspiring the modeling software agent that simulates the behavior of this enzyme. The solution also includes an application running on mobile devices that makes the collection of environmental variables in the selected points, validates them and makes the inference of those who could not be collected. Multi-agent System for Environmental Monitoring of the Port Complex of the Island of São Luís-Maranhão- Brasil, therefore, is made up of the bio-ontology, sensor networking, mobile application collection and inference of data from environmental conditions, software agents to simulate biochemical analysis, calculation of GST activity and other staff related to the maintenance and security of the SMA. / Nesta dissertação é discutido o modelo conceitual de um sistema multiagente para monitoramento ambiental com uso de marcadores biológicos de organismos aquáticos presentes no complexo portuário de São Luís-MA, segundo mais importante do país em movimentação de carga. Situado na Baía de São Marcos, esse complexo possui um dos ecossistemas estuarinos que mais têm sofrido agressões no atual processo de desenvolvimento econômico, provocadas pela intensa movimentação portuária, despejo de água de lastro e lavagem dos navios, pesca predatória, introdução de espécies exóticas no meio, poluição por efluentes domésticos e industriais, sujeitando o ambiente a fortes impactos ambientais que devem ser monitorados. Metodologicamente, na modelagem da solução de monitoramento, utilizou-se o registro das condições ambientais e de organismos aquáticos capturados em dois pontos distintos do complexo portuário: o primeiro, em uma área potencialmente contaminada e o segundo em uma livre de contaminação (controle), propondo-se a criação de uma rede de sensores nesses locais. Empregou-se dados cedidos por Carvalho-Neta (2007) cuja pesquisa contou com a captura de peixes nesses perímetros, submetendo-os posteriormente a análise laboratorial para medição da atividade enzimática da Glutationa s- Transferase (GST) e Catalase (CAT), tendo todos os resultados processados e registrados em bio-ontologia. O núcleo da solução do Sistema Multiagente baseia-se nos resultados oriundos da análise bioquímica da GST, inspirando a modelagem de agente de software que simula o comportamento desta enzima. Todos esses dados foram registrados em bio-ontologia. A solução contempla também uma aplicação executada em dispositivos móveis que realiza a coleta das variáveis abióticas nos pontos selecionados, valida-as e realiza a inferência daquelas que não puderam ser coletadas. O Sistema Multiagente para Monitoramento Ambiental do Complexo Portuário da Ilha de São Luís, portanto, é constituído pelo conjunto da bio-ontologia, rede de sensores, aplicação móvel de coleta e inferência de dados das condições do meio ambiente, agentes de software para simulação de análise bioquímica, cálculo da atividade da GST e outros agentes relacionados à manutenção e segurança do SMA.

Formins are proteins facilitating formation of actin filaments. They affect structure of cytoskeleton and participate in cytokinesis and tip growth. There are 2 classes of formins in Arabidopsis thaliana, which include FH1 and FH2 (Formin Homology 1 and 2) domain. Formins of the class I have usually a transmembrane domain on N-terminus. Due to this fact they can interact with membranes. Some formins from the class II include PTEN domain (Phosphatase and Tensin Homolog) derived from sequences of PTEN proteins which has lost the function of phosphatase. It is assumed this domain can bind on a membrane via the phosphatase section or C2 domain. This thesis was focused on the formin AtFH13 from the class II in Arabidopsis thaliana and on its PTEN domain. There were analyzed differences between mutants and wild-types in length of roots in seedlings and in size of seeds and seed coats, and observed the effect of dexamethasone on the length of roots on AtFH13. PTEN domain of the formin was isolated from cDNA, cloned to a vector and fused with YFP. The tagged protein was visualized by the method of transient expression in epidermal cells in the leaves of Nicotiana benthamiana. No big differences were observed between plants mutant in the gene AtFH13 and wild-type in choice parameters. Dexamethasone did't influence...

Dans un contexte de lutte intégrée contre les moustiques, l'Entente Interdépartementale pour la Démoustication (E.I.D. Ain, Isère, Rhône, Savoie Rhône-Alpes s'est tournée vers une lutte totalement biologique (Bti) pour lutter contre les moustiques. Mon sujet de thèse s'inscrit dans la suite d'une collaboration scientifique constante depuis 40 ans entre l'E.I.D. et le laboratoire de recherche dans lequel j'ai effectué ma thèse. Cet organisme de gestion utilise le Bti depuis 20 ans. Et bien qu'à ce jour, aucune population de moustique ne soit apparue résistante au Bti, ce gestionnaire s'interroge sur la possibilité d'apparition de populations résistantes aux traitements insecticides. Des travaux antérieurs ont laissé supposer qu'il existait une différence de sensibilité des larves de moustiques aux insecticides en fonction de leur gîte d'origine, les larves originaires de gîtes herbacées étant moins tolérantes que celles provenant des gîtes arborescents. Il nous a semblé nécessaire de comprendre et ainsi de s'intéresser aux différents mécanismes de résistance des larves de moustiques pour permettre, demain, une lutte plus efficace contre cet insecte. Et nous nous intéresserons à la résistance à divers xénobiotiques alimentaires : du téméphos (insecticide organophosphoré) au Bacillus thuringiensis var. israelensis (Bti - bactério-insecticide) en passant par de la litière naturelle issue de la décomposition de feuilles dans les gîtes à moustiques se révélant toxique pour les larves. L'intérêt de cette thèse est double. D'un point de vue fondamental, la connaissance et la compréhension de la résistance (des enzymes impliquées aux facteurs environnementaux en passant par les gènes mis en jeu) stimulent mes recherches. Et d'un point de vue appliqué, il est nécessaire de mettre au point, enfin, un système de lutte efficace non polluant, qui passe par la compréhension globale des résistances. La démarche expérimentale utilisée dans ce travail est d'identifier les dysfonctionnements environnementaux sur le terrain, les analyser au laboratoire sur des espèces modèles (ici Aedes aegypti) les mécanismes à l'origine de ces perturbations, puis revenir sur le terrain pour confronter les résultats de laboratoire avec ceux obtenus in natura (ici Ochlerotatus cataphylla, Aedes rusticus). Ainsi cette étude va porter à la fois sur des espèces de terrain (Aedes rusticus, Ochlerotatus cataphylla, Culex pipiens ...) que sur des espèces de laboratoires (Aedes aegypti, Aedes albopictus ...). Pour comprendre les mécanismes de résistance mis en jeu par ce nuisant, nous avons travaillés à plusieurs niveaux d'études avec des études écotoxicologiques réalisées grâce à des études de terrain en collaboration avec l'E.I.D. (Entente Interdépartementale pour la Démoustication), des études biochimiques nous permettant de caractériser les enzymes de résistance mises en jeu, et des études génétiques et moléculaires pour approfondir ces mécanismes, en espérant trouver les gènes impliqués.

Student Number : 0002483R - MSc dissertation - School of Molecular and Cell Biology - Faculty of Science / Isothermal titration calorimetry was used to investigate the forces that drive ligand binding to the active site of rGST M1-1. In an attempt to gain insight into the recognition of non-substrate ligands by GSTs, this study also investigates interactions between rGST M1-1 and ANS, a non-substrate ligand. At 25 °C, complex formation between rGST M1-1 and GSH, GSO3 -, and S-hexylglutathione is characterised by a monophasic binding isotherm with Kd values of 38.5 mM, 2.1 mM and 0.2 mM, respectively. One molecule of each ligand is bound per monomer of rGST M1-1. Binding of these ligands is enthalpically favourable and entropically unfavourable with a resultant favourable Gibbs free energy, overall. The effects of temperature and buffer ionisation on the energetics of binding were studied. The enthalpic and entropic contributions for all three ligands exhibited temperature dependence over the temperature range investigated (5-30 °C). The Gibbs free energy showed negligible changes with increasing temperature due to enthalpy-entropy compensation. The temperature dependence of the binding enthalpy yielded heat capacity changes of – 2.69 kJ/mol/K and –3.68 kJ/mol/K at 25 °C for GSH and S-hexylglutathione binding and –1.86 kJ/mol/K overall for GSO3 -. The linear dependence of DH on temperature for GSO3 - binding to rGST M1-1 suggests the formation of a more constrained complex which limits the fluctuations in conformations of the mu-loop at the active site. The non-linear dependence of DH on temperature for GSH and Shexylglutathione binding to the enzyme suggests the formation of a complex that samples different bound conformations due to the mobility of the mu-loop even after ligand is bound. Calorimetric binding experiments in various buffer systems with different ionisation enthalpies suggest that the binding of GSH to rGST M1-1 is coupled to the deprotonation of the thiol of GSH while GSO3 - binding to rGST M1-1 is independent of the buffer ionisation. At 25 °C, the rGST M1-1#1;ANS association is represented by a monophasic binding isotherm with one molecule of ANS bound per monomer of rGST M1-1. The interaction is both enthalpically and entropically driven with a Kd value of 27.2 mM representing moderate affinity. The effect of temperature on the interaction was investigated over the temperature range of 5-30 °C. The linear dependence of the binding enthalpy on temperature indicates that no significant structural changes occur upon binding of ANS to the enzyme (DCp = -0.34 kJ/mol/K). The change in heat capacity associated with the interaction can be attributed to the burial of the polar sulphonate group of ANS and the exposure of the anilino and naphthyl rings to solvent as well as the possibility of weak electrostatic interactions between ANS and residues at the active site. The effect of ethacrynic acid, GSH, GSO3 - and S-hexylglutathione on the fluorescence of ANS was investigated in order to obtain some idea as to the location of the ANS binding site on rGST M1-1. ANS was displaced by GSO3 -, S-hexylglutathione and ethacrynic acid, while no displacement occurred upon binding of GSH to the active site of rGST M1-1. Displacement studies and molecular docking simulations indicate that ANS binds to the H-site of rGST M1-1 and the possibility of a second binding site for the molecule cannot be ruled out.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.