Conselho Nacional de Desenvolvimento Científico e Tecnológico / Enamino carbonylic compounds are important synthetic intermediates used in the synthesis of heterocycles and natural products. Our group of research has been dedicating much of our efforts to the development and implementation of simple and efficient methodologies to obtain β-enamino carbonylic compounds, mainly due to their functionality and ability to react both as ambident nucleophiles and as ambident electrophiles. In this dissertation, we study the synthesis and reactivity of new α-chloro functionalized N-substituted β-enamino acyclic compounds. In the synthesis of α-chlorinated β-enamino esters (2a-g) was employed the condensation of ethyl 2-chloro-acetoacetate with primary amines. This convenient and safe procedure uses the clean chemistry methodology of microwave-assisted solid support (K-10) synthesis in the absence of solvent. The reactivity and regiochemistry of the α-chlorinated β-enamino compounds was assessed through the study of cyclization reactions involving the coupling of these substrates with dinucleophiles such as hydrazine and semicarbazide derivatives. The obtained heterocycles showed the influence of the chloro substituent at the α position, which led to the inversion of reactivity of this center, allowing the preparation of heterocyclic compounds having four and six members. These functionalized heterocycle derivatives are attractive targets for the discovery of new active molecules as they have high potential to present a broad range of biological properties. Therefore, evaluation of the antimicrobial activity of the newly synthesized compounds was carried out using the in vitro Bioautography assay. / Compostos enamino carbonílicos são importantes intermediários sintéticos usados na síntese de heterociclos e produtos naturais. Nosso grupo de pesquisa tem se dedicado ao desenvolvimento e implementação de metodologias simples e eficientes para obter compostos β-enamino carbonílicos, principalmente devido à sua funcionalidade, atuando como sistema ambidentado, tanto com caráter nucleofílico como eletrofílico. Neste trabalho, nós estudamos a síntese e a reatividade de novos β-enamino compostos acíclicos α-cloro funcionalizados Nsubstituídos. Na síntese de β-enamino ésteres α-clorados (2a-g) foi empregada a condensação do 2-cloro acetoacetato de etila com aminas primárias. Este procedimento simples e seguro, emprega a metodologia de suporte sólido (K-10) associada ao uso de energia de microondas (MO) na ausência de solvente, sendo considerado como um protocolo de Química Limpa. A reatividade dos centros eletrofílicos dos β-enamino compostos α-clorados foi estudada em reações de ciclização, reagindo-se estes compostos frente a dinucleófilos como hidrazinas e semicarbazida, avaliando-se o padrão de reatividade destes compostos e a regioquímica dos sistemas heterocíclicos polifuncionalizados 3 e 4 obtidos. Os heterociclos obtidos mostraram a influência do substituinte cloro na posição α, o qual levou a uma inversão na reatividade deste centro, possibilitando a obtenção de compostos heterocíclicos de quatro e seis membros. Os sistemas heterocíclicos funcionalizados obtidos são alvos atrativos para a descoberta de novas moléculas ativas, devido as suas características estruturais com elevado potencial para apresentar propriedades biológicas. Portanto, estudos foram conduzidos para determinar a atividade antimicrobiana de uma série de compostos sintetizados empregando o ensaio in vitro através do método de Bioautografia.

Este estudo teve como objetivo avaliar o desempenho de 2 reatores anaeróbios de fluxo ascendente e manta de lodo (UASB), em escala de bancada, tratando licor negro diluído, durante 635 dias de operação contínua. Durante a Fase I os reatores foram alimentados com licor negro diluído, enriquecido com nutrientes essenciais, simulando os efluentes de uma indústria de pasta celulósica não branqueada. Durante a Fase II, um dos reatores foi alimentado com o mesmo substrato mais uma mistura de cinco compostos organoclorados, simulando os efluentes de uma indústria de pasta celulósica branqueada. O aumento da taxa de carregamento orgânico, na Fase I, de 0,40 kg DQO/dia m3 para 0,70 kg DQO/dia m3 não provocou diminuição da eficiência de remoção de DQO. Por outro lado, o aumento da taxa de carregamento hidráulico de 0,50 m3/dia m3 para 1,00 m3/dia m3 provocou diminuição de aproximadamente 10% na eficiência média de remoção de DQO no reator 1 e em torno de 12% no reator 2. A adição de 15,00 mg/L dos compostos organoclorados, não influenciou significativamente o desempenho do reator. A eficiência média de remoção de DQO na Fase II foi de 82% no reator de controle e de 79% no reator de tratamento. As eficiências médias de remoção dos compostos organoclorados foram de: 99,9% para o 2-clorofenol, 97,5% para o 2,4- diclorofenol, 95,8% para o 2,6-diclorofenol, 98,9% para o 2,4,6-triclorofenol e 92,8% para o tetraclorocatecol. / This research was conducted to assess the performance of two bench scale upflow anaerobic sludge blanket (UASB) reactors used for the treatment of diluted black liquor, over 635 days of continuous operation. During Phase I of the study both reactors were fed with the diluted black liquor enriched with essential nutrients. This substrate simulated the wastewater from unbleached cellulose pulp plant. Progressing in the research, during Phase II, one of the reactors was fed with the same substrate plus a mixture of five chlorinated organic compounds to simulate the wastewater from a bleached pulp plant. The increase in the organic loading rate, during Phase I, from 0.40 kg COD/m3.day to 0.70 kg COD/m3.day did not result in a decrease in the COD removal efficiency. On the other hand, the increase in the hydraulic loading rate from 0.50 m3/day.m3 to 1.00 m3/day.m3 resulted in a decrease of approximately 12 percent points in the average COD removal efficiency in the reactor 1 and approximately 12 percent points in the reactor 2. The addition of 15 mg/L of chlorinated organic compounds did not have a significant influence in the reactor performance in this research. The removal efficiency of COD during the second phase reached 82% in the control reactor and 79% in the treatment reactor. The average removal efficiencies for the chlorinated organic compounds were: 99.9% for 2-chlorophenol; 97.5% for the 2,4-dichlorophenol; 95.8% for the 2,6- dichlorophenol; 98.9% for the 2,4,6-trichlorophenol and 92.9% for the tetrachlorocatechol.

A proposta desta pesquisa foi avaliar a aplicação do processo oxidativo avançado (POA), peróxido de hidrogênio (\'H IND.2\'O IND.2\') e radiação ultravioleta (UV), como pós-tratamento de um reator anaeróbio utilizado para tratar efluentes de branqueamento de indústrias de celulose kraft. O principal objetivo do pós-tratamento químico foi aumentar a biodegradabilidade e a remoção de compostos recalcitrantes nos efluentes. O tratamento biológico foi realizado por meio de reator anaeróbio horizontal de leito fixo (RAHLF), em escala de bancada, seguido pelo tratamento \'H IND.2\'O IND.2\'/UV dos efluentes. As doses do agente oxidante variaram de 50 a 500 mg \'H IND.2\'O IND.2\'/L e as aplicações de energia pela lâmpada de radiação ultravioleta foram variadas, principalmente, de 3,8 a 15,0 kWh por \'M POT.3\' de efluente tratado. Desta forma, amostras de efluentes foram coletadas de duas indústrias brasileiras de celulose kraft branqueada, originando respectivamente duas fases experimentais. Na primeira fase, o RAHLF apresentou boa estabilidade de operação, obtendo adequadas eficiências de remoção de DQO (61 ± 3%), COT (69 ± 9%), \'DBO IND.5\' (90 ± 5%) e AOX (55 ± 14%). Todavia, não ocorreu boa remoção dos compostos de absorbância na \'UV IND.254\'. Além disso, houve aumento de constituintes da lignina do afluente para o efluente do RAHLF. Por sua vez, o pós-tratamento com \'H IND.2\'O IND.2\'/UV no efluente anaeróbio proporcionou eficiência de remoção variada nos parâmetros DQO (0 a 11%), \'UV IND.254\' (16 a 35%), lignina (0 a 29%) e AOX (23 a 54%). Em relação a melhoria da biodegradabilidade, todas as dosagens de \'H IND.2\'O IND.2\' estudadas promoveram aumento na relação \'DBO IND.5\'/DQO. Para uma relação \'DBO IND.5\'/DQO média inicial igual a 0,09, correspondente ao efluente do RAHLF, o maior aumento obtido foi de 131% aplicando 500 mg \'H IND.2\'O IND.2\'/L e 7,5 kWh/\'M POT.3\'. O tratamento conjugado biológico e químico foi necessário para atingir ao padrão europeu de emissão de AOX (< 0,4 kg AOX por tonelada de polpa de celulose seca produzida). Na segunda fase, o RAHLF apresentou alguns problemas operacionais, tais como entupimento nas conexões e quebras do reator de vidro, pelo possível aumento da carga orgânica volumétrica aplicada presente nos efluentes coletados na segunda indústria de celulose. O pós-tratamento com \'H IND.2\'O IND.2\'/UV mostrou menor desempenho nessa fase experimental, o que pode ser explicado também pelo maior teor de matéria orgânica residual, lignina, alcalinidade e íons cloretos ainda remanescentes nos efluentes tratados biologicamente. Visando complementar os estudos, testes em reatores aeróbios nos efluentes do RAHLF, tratados e não tratados pelos processos \'H IND.2\'O IND.2\'/UV, foram avaliados. Os resultados demonstraram que os melhores resultados (menor tempo de aeração necessário para atingir a mesma concentração dos parâmetros avaliados) foram alcançados pelos reatores que receberam efluentes previamente tratados pelo POA. Uma avaliação simplificada do custo operacional associado com a aplicação do tratamento \'H IND.2\'O IND.2\'/UV, em escala plena, indicou um custo de R$ 1,87 por \'M POT.3\' de efluente tratado. / The purpose of this research was to assess the application of the advanced oxidation processes (AOP), hydrogen peroxide (\'H IND.2\'O IND.2\') and ultraviolet radiation (UV), as post-treatment for an anaerobic reactor used to treat bleaching effluents from kraft pulp mills. The main objective of the chemical post-treatment was to increase the biodegradability and remove recalcitrant compounds from the wastewater. The biological treatment was performed in a fixed bed anaerobic horizontal reactor (FBAHR), in bench scale, followed by \'H IND.2\'O IND.2\'/UV treatment of the effluents. The oxidizer dosage varied from 50 to 500 mg \'H IND.2\'O IND.2\'/L while the energy applied by the UV lamp ranged, mainly, from 3.8 to 15.0 kWh per \'M POT.3\' of treated effluent. This way, wastewater samples were collected from two brazilian bleached kraft pulp mills thus the results were reported as two different experimental phases. In the first phase the FBAHR showed good operational stability and reached the expected removal efficiencies for COD (61 ± 3%), TOC (69 ± 9%), \'BOD IND.5\' (90 ± 5%) and AOX (55 ± 14%). However, the anaerobic treatment did not provide good removal of compounds indicated by \'UV IND.254\' absorbency. Furthermore, there was an increase of lignin from the affluent to effluent of the FBAHR. In its turn the \'H IND.2\'O IND.2\'/UV post-treatment provided a wide range of removal depending upon the dosage: COD (0 to 11%), \'UV IND.254\' (16 to 35%), lignin (0 to 29%) and AOX (23 to 54%). Regarding to the improvement in biodegradability, all dosages of \'H IND.2\'O IND.2\' applied in this work promoted an increase in the \'BOD IND.5\'/COD ratio. For an average initial \'BOD IND.5\'/COD ratio equal to 0.09, which corresponds to the FBAHR effluent, the greatest increase was 131% with 500 mg \'H IND.2\'O IND.2\'/L and 7.5 kWh/\'M POT.3\'. It was necessary to couple the anaerobic and the oxidative treatment to reach the current European allowable AOX emission load (< 0.4 kg AOX per ton of produced dried cellulose pulp). During the second phase of the experiments, the FBAHR had operational problems as plugging of the tubes and cracking of the reactor wall that may be partially attributed to the higher organic load applied, as the effluents from the second mill were stronger. The AOP post-treatment also had lower performance during this experimental phase, which may be explained by the higher concentration of the organic matter, lignin, alkalinity and chloride ions, still present in the biologically treated effluents. To complete the experiments samples from effluents of the FBAHR and AOP process were submitted to aerobic assays. By tests done in aerobic reactors in the FBAHR effluents, treated and non-treated by the processes \'H IND.2\'O IND.2\'/UV, it was verified that the best results, shorter aeration time requirement to reach the same concentration of the evaluated parameters, were obtained from reactors that received previously treated by AOP effluents. A simplified evaluation of the operational cost associated with the use of the tested AOP, in full scale, indicated a cost of R$ 1.87 per \'M POT.3\' of treated effluent.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-05-05T19:17:02Z No. of bitstreams: 1 joaovitordeassis.pdf: 9015544 bytes, checksum: 493b436d8f7e8eb4f99f7775b557aba2 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-05-17T13:42:03Z (GMT) No. of bitstreams: 1 joaovitordeassis.pdf: 9015544 bytes, checksum: 493b436d8f7e8eb4f99f7775b557aba2 (MD5) / Made available in DSpace on 2017-05-17T13:42:03Z (GMT). No. of bitstreams: 1 joaovitordeassis.pdf: 9015544 bytes, checksum: 493b436d8f7e8eb4f99f7775b557aba2 (MD5) Previous issue date: 2009-07-17 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Nesse trabalho descreve-se, num primeiro momento, a síntese de derivados inositóis condensados com o 2-mercaptobenzotiazol ou com o 2mercaptobenzimidazol, potenciais agentes antibacterianos. A seguir é descrita a preparação de derivados clorados de inositol. No primeiro capítulo relata-se a obtenção e a avaliação das propriedades antituberculose de derivados do 2mercaptobenzotiazol e do 2-mercaptobenzimidazol condensados com um inositol. Tais compostos foram preparados via abertura de intermediário epóxido, o qual foi obtido a partir do L-quebrachitol, um produto natural isolável do látex da seringueira. Foram sintetizados 8 compostos inéditos, sendo quatro derivados do 2mercaptobenzotiazol e quatro derivados do 2-mercaptobenzimidazol. Na obtenção de alguns desses compostos foi empregada irradiação de microondas como fonte de aquecimento. Utilizou-se nestas reações um aparelho de microondas doméstico adaptado em nosso laboratório. Alguns desses compostos foram testados in vitro contra o Mycobacterium tuberculosis, no laboratório de Bacteriologia (IPEC) da FioCruz-RJ. Todavia, tais compostos não se mostraram ativos contra esse microorganismo. Alguns compostos também foram enviados para avaliação da atividade antibacteriana no National Institute of Allergy and Infectious Diseases (NIAID-EUA). O segundo capítulo desse trabalho relata-se a preparação de análogos clorados nas posições C2 ou C3 do quiro-inositol, usando-se também como material de partida o L-quebrachitol. Tais derivados halogenados são candidatos a possíveis compostos bioativos, assim como precursores interessantes para a preparação de outras moléculas. As estruturas dos produtos obtidos foram elucidadas por espectroscopia na região do infravermelho, RMN de 1H e RMN de 13C. Algumas dessas estruturas foram também evidenciadas por experimentos de COSY, HMQC, HMBC e espectrometria de massas. / In this work we describe, in a first moment, the synthesis of inositol derivatives condensed with 2-mercaptobenzothiazole or 2-mercaptobenzimidazole, potential antibacterial agents. In a second part, the preparation of chlorinated inositol derivatives is reported. In the first chapter, we describe the synthesis and antitubercular evaluation of 2-mercaptobenzothiazole and 2-mercaptobenzimidazole derivatives condensed with inositol. These compounds were prepared by opening an epoxide intermediate, which was obtained from L-quebrachitol, a natural product isolated from Hevea brasiliensis. Eight new compounds were synthesized: four 2mercaptobenzothiazole and four 2-mercaptobenzimidazole derivatives. For the preparation of some compounds, microwave irradiation was used as the heating source. A domestic microwave apparatus was adapted for these reactions in our laboratory. Some compounds were evaluated in vitro against Mycobacterium tuberculosis at the laboratory of bacteriology (IPEC) of FioCruz-RJ. However, such compounds did not display biological activity against this microorganism. Some compounds were also submitted for antibacterial activity assay at National Institute of Allergy and Infectious Diseases (NIAID-USA). Chapter 2 of this work reports the preparation of inositols chlorinated at the C2 or C3 position, using as starting material L-quebrachitol. These halogenated derivatives are potential new bioactive compounds, as well as interesting precursors for the preparation of other molecules. The compounds were characterized by infrared spectroscopy, 1H and 13C NMR. Some structures were also characterized by COSY, HMQC, HMBC and mass spectrometry.

La désinfection de l’eau de piscine est essentielle pour prévenir la propagation de maladies infectieuses. Cependant, les désinfectants réagissent avec les composés organiques présents dans l’eau y compris ceux introduits par les baigneurs, tels que les fluides corporels et les crèmes solaires, conduisant à la formation de sous-produits de désinfection (SPD) associés à des effets néfastes sur la santé. Le devenir des filtres UV organiques, présents dans les crèmes solaires et les produits de soins personnels, dans les piscines d'eau de mer chlorées est peu connu. Les objectifs de la présente étude étaient d'étudier la réactivité des filtres UV organiques dans l'eau de mer chlorée, d’identifier les SPD générées par les filtres UV, d'analyser l’occurrence des SPD dans les piscines d'eau de mer, et d'examiner leur génotoxicité. Des expériences de chloration ont été menées dans l'eau de mer reconstituée sur cinq filtres UV couramment utilisés : l’oxybenzone (OXY), le dioxybenzone (DIOXY), l’avobenzone (AVO), l’octyl méthoxycinnamate (OMC), et l’octocrylène (OC). Tous les composés étudiés sauf l’OC ont été dégradés conduisant à la formation de SPD bromés pour lesquels des schémas réactionnels ont été proposés. L'occurrence de ces SPD a été étudiée dans les piscines d'eau de mer où des SPD bromés ont été quantifiés. La génotoxicité de l’hydrate de bromal (BH), l’un des SPD généré par OXY et DIOXY et détecté dans les piscines d'eau de mer, a été évaluée. BH a induit une activité génotoxique dans le test d'Ames et l'essai de comète. Cette étude montre que les filtres UV peuvent agir comme précurseurs pour la formation de SPD génotoxiques dans l’eau de mer chlorée. / Disinfection of swimming pool water is critical to prevent outbreaks of infectious diseases. However, disinfectants react with organic compounds present in water, including anthropogenic inputs (e.g. body fluids, sunscreens), leading to the formation of disinfection byproducts (DBPs) that have been linked to adverse health effects. Little is known about the fate of organic UV filters, present in sunscreens and personal care products, when introduced into chlorinated seawater swimming pools. The aims of the present study were to investigate the reactivity of five commonly used organic UV filters in chlorinated seawater, identify DBPs generated from the UV filters, analyze the occurrence of these DBPs in seawater swimming pools, and examine their genotoxicity. Chlorination experiments were conducted to analyze the reactivity of the UV filters oxybenzone (OXY), dioxybenzone (DIOXY), avobenzone (AVO), octyl methoxycinnamate (OMC), and octocrylene (OC) in reconstituted seawater. All the studied UV filters except OC were degraded in chlorinated seawater resulting in the formation of brominated DBPs. Based on the identified byproducts, transformation pathways were proposed. The occurrence of the identified DBPs was investigated in seawater pools. Several brominated DBPs were identified in seawater pool samples. The genotoxicity of bromal hydrate (BH), which was generated by the benzophenone UV filters OXY and DIOXY and detected in the investigated seawater pools, was assessed. BH induced genotoxic activity in the Ames test and in the comet assay. Overall, this study shows that UV filters can act as precursors for the formation of genotoxic DBPs in chlorinated seawater pools.

O presente trabalho foi desenvolvido com o objetivo de verificar a eficiência de um reator anaeróbio híbrido (RAH) alimentado com uma mistura de ácidos orgânicos acético, propiônico, butírico, e láctico, bem como do álcool metanol, perfazendo uma DQO de 6,88 g/L, e avaliar nessas condições, a degradação do pentaclorofenol (PCP) na faixa de 2,0 a 21,0 mg/L. O RAH apresentou adequada performance na ausência de PCP, tendo sido inoculado com um lodo com AME cerca de 0,57 g DQO-CH4/g SV.d. Durante os 21 meses de operação do RAH na ausência de PCP verificou-se uma remoção média de DQO de 93% produção média de metano de 84%. Através de testes de toxicidade realizadas em batelada com o lodo granulado do RAH, antes da adição de PCP no reator, calculou-se o IC50, cujos valores foram 10, 12 e 13,69 mg/L. Na presença de PCP na faixa de 2,0 a 2,1 mg/L, o RAH apresentou remoção média de DQO de 96,7%, produção média de metano de 85,5% e remoção dos ácidos voláteis próxima a 74% do acético, 93% do butírico e 64% do propiônico. Individualmente, na presença da maior concentração de PCP adicionada, ocorreu decréscimo na remoção dos ácidos voláteis, principalmente do ácido propiônico e na taxa de conversão DQO/biogás. O PCP foi removido do sistema na ordem de 99% pela ação do lodo granulado com predominância do grupo das Archaea metanogênicas, verificada por exames microscópicos e hibridação \"in situ\". Valores da ordem de grandeza microbiana para os microrganismos metanogênicos nos períodos anteriores e posteriores a adição de PCP permaneceram na faixa de 105 e 106 céls./mL, quando cultivados em metanol e lactato mais sulfato, respectivamente. Os resultados sugerem que as Archaea metanogênicas podem estar envolvidas na degradação do PCP. A velocidade de remoção do organoclorado foi igual a 1,07 mg PCP/g SV.d quando a maior concentração de PCP foi estudada (21,0 mg/L). / The present research aimed to verify the efficiency of an Anaerobic Hybrid Reactor (AHR) supplied with a mixture of fatty acids, acetic, propionic, butyric and lactic and methanol as well. The total amount of COD was 6.88 g/L. The performance of the reactor was remarkably stable and efficient during PCP additions at range from 2.0 to 21.0 mg/L. The AHR showed a great performance in the PCP absence, inoculated with sludge with an specific methanogenic (SMA) activity of 0,057 g.COD-CH4/g. VS.d. During the 21 months of operation without PCP, the reduction of COD was around 93% and methane was up to 84% in the biogas. Before PCP addiction, two toxicity batch tests conducted with the granular sludge presented IC50 values around 10.12 mg/L and 13.69 mg/L. In the presence of PCP, at the range of 2.0 to 21.0 mg/L, the efficiency of volatile fatty acids breakdown was 93%, 64% and 74% respectively for butyric, propionic and acetic acids. Individually, at the presence of the higher PCP concentration studied, a decrease in the conversion of COD to biogas and organic acids removal occurred, mainly with propionic acid. PCP total removal of more than 99% was reached by granular sludge activities formed by the total time of reactor operation with a prevalence of methanogenic Archaea, verified under direct microscopy exams and in situ hybridization. Methanogenic cells predominance was noticed with 105 to 106 cells/mL during enumeration on methanol and lactate plus sulfate culture media, respectively. The results suggest that methanogenic Archaea can be involved in PCP degradation. The organochlorine removal rate was 1.07 mg PCP.g-1 VS.d-1 during the highest PCP (21.0 mg/L) concentration addition.

Le dichlorométhane (DCM ; CH2Cl2) est un polluant chloré toxique émis dans l’environnement principalement par les activités industrielles. Ce polluant peut être dégradé par des bactéries méthylotrophes qui utilisent des composés en C1 réduits comme seule source de carbone et d’énergie. La protéobactérie Methylorubrum extorquens DM4 porte quatre gènes dcm au sein du transposon catabolique dcm très conservé chez les bactéries dégradant le DCM. Le gène dcmA code la DCM déshalogénase de la famille des glutathion-S transférases essentielle à la croissance avec le DCM. Son activité est modulée par DcmR, un facteur de transcription qui régule l’expression de dcmA ainsi que son propre gène, orienté de manière divergente par rapport à dcmA. DcmR porte un domaine hélice-tour-hélice de fixation à l'ADN et un second domaine appelé methanogen / methylotroph, DcmR sensory (MEDS) potentiellement impliqué dans la fixation d’un composé hydrocarboné ligand. Les objectifs de ma thèse étaient de répondre à plusieurs questions : i) quel est le niveau d’expression des transcrits dcm et des protéines correspondantes par rapport à d’autres gènes et protéines dont l’abondance est modifiée en réponse à la croissance sur le DCM ? ii) Comment le facteur de transcription DcmR intervient-il dans la régulation des gènes dcm ? iii) Quelle est la variabilité du gène dcmR et de son environnement génétique in situ ? Des méthodes globales « -omiques » de transcriptomique et de protéomique ont permis d’inventorier les ARN et les protéines dont l’abondance varie chez la souche sauvage DM4 cultivée soit avec le DCM ou le méthanol, le substrat de référence de la méthylotrophie. Les gènes dcm sont parmi les plus exprimés en présence de DCM, ce qui confirme leur régulation en présence de DCM. Deux approches complémentaires ciblant la détermination des sites d’initiation de la transcription (TSS-Seq) et de la traduction (N-terminome) ont permis la recherche de motifs de régulation dans les régions 5’UTR (5’-untranslated terminal region) et les promoteurs de gènes régulés en réponse à la croissance avec le DCM. Le rôle régulateur de dcmR a été étudié en comparant les phénotypes de croissance, l’activité promotrice par fusion transcriptionnelle, la quantification des ARN et des protéines des gènes dcm chez la souche sauvage par rapport à des mutants du gène dcmR seul ou combiné à d’autres gènes dcm mutés. Ces travaux ont permis de confirmer qu’en absence de DCM, DcmR inhibe la transcription de son propre gène ainsi que celle de dcmA. Outre DcmR, la répression nécessite aussi l’expression d’au moins un des autres gènes dcm et ceci par un mécanisme indépendant des boîtes de 12 pb conservées dans les promoteurs des gènes dcmR et dcmA. Lors de la croissance en condition DCM, l’absence du gène dcmR confère une vitesse de croissance ralentie, qui ne résulte pas d’une différence de production des ARN et des protéines codées par le transposon dcm. Pour que l’expression du gène dcmA soit activée, l’ensemble de la région intergénique entre dcmR et dcmA doit être présente, ce qui suggère la présence de sites de régulation pour la fixation d’un facteur de transcription indépendant de DcmR. L’ensemble de ces résultats a permis de proposer un nouveau modèle de régulation des gènes dcm. Alors que le gène dcmR a été détecté en quantité similaire à celle du gène dcmA par qPCR dans des échantillons de sites contaminés par le DCM, une analyse bioinformatique à partir des données de séquences indique que des gènes dcmR-like sont trouvés dans d’autres contextes génétiques que celui du transposon catabolique dcm. Ainsi, DcmR pourrait exercer un rôle de régulateur dans d’autres contextes ouvrant de nouvelles pistes pour l’identification des ligands du domaine MEDS. / Dichloromethane (DCM; CH2Cl2) is a toxic chlorinated pollutant mainly emitted in the environment through industrial activities. Some methylotrophic bacteria that utilize reduced one carbon compounds as sole source of carbon and energy can degrade DCM. The four dcm genes found in the catabolic dcm transposon are highly conserved among DCM-degrading bacteria, including in the Proteobacterium Methylorubrum extorquens DM4. The dcmA gene encodes a DCM dehalogenase of the glutathione-S transferase family which is essential for growth with DCM. The transcriptional factor DcmR regulates the expression of dcmA and its own gene, two genes which are divergently oriented. DcmR consists of a helix-turn-helix domain for DNA fixation and of a domain, called MEDS for ‘methanogen / methylotroph, DcmR sensory’, proposed to bind a hydrocarbon ligand. The aim of my PhD was to address the following questions: i) What is the expression level of dcm transcripts and their corresponding proteins compared to other genes and proteins whose abundance is modulated in response to growth with DCM? ii) How does DcmR regulate the expression of the dcm genes? iii) How variable is dcmR and its genetic environment in situ? Global transcriptomic and proteomic approaches were followed to inventory transcripts and proteins whose abundance varied in the wild-type DM4 strain grown with DCM compared to with methanol, the reference substrate for methylotrophy. The dcm genes were among the most expressed in cultures grown with DCM, confirming their regulation in response to DCM. The identification of transcription start sites (TSS-Seq) and translation starts (N-terminome) allowed the subsequent search for regulation motifs in the promoter and 5’-untranslated terminal regions (5’UTR) of regulated genes in response to DCM utilization. The role of dcmR was studied using growth phenotypes, promoter activities in transcription fusion assays, dcm transcript and corresponding protein product quantification, comparing the wild-type strain with strains mutated only in dcmR, or also in other dcm genes. In absence of DCM, DcmR inhibited the transcription of its own gene and of dcmA. In addition to DcmR, repression requires the expression of one of the other dcm genes by a mechanism that does not involve previously predicted DcmR-binding sites (a 12 bp conserved box shared by dcmR and dcmA promoters). Mutants with impaired dcmR show slower growth with DCM, although no difference in transcript or protein abundance encoded by the dcm transposon was observed. The complete dcmR-dcmA intergenic region was shown to be required to activate dcmA expression. This suggests that regulatory sites present in the intergenic region may be involved in DcmR-independent transcription activation. Taken together, these data allowed to propose a new model of dcm gene regulation. The dcmR gene was detected and quantified in similar amounts as dcmA in DCM-contaminated environmental samples, despite the fact that bioinformatics analysis of sequence databases suggests that dcmR-like genes are not necessary associated with the catabolic dcm transposon. Thus, DcmR may play a role in other contexts. This may provide new leads for future investigations of potential ligands of the MEDS domain.

Biogeochemical cycle of chlorine, particularly the formation of organically bound chlorine is still not well understood. In continental ecosystems chlorides act as source of chlorine, and also as a stress factor. Chlorides originate from precipitation of marine cloud masses. Organically bound chlorine in the environment is formed naturally by biotical and abiotical way. The biotical factors are microorganisms, plants, soil enzymes and animals. Volatile chlorinated hydrocarbons (VOCl) represent one group of organically bound chlorines. Several volatile chlorinated hydrocarbons reacts with atmospheric ozone, consequently causing depletion of the ozone layer. The most important known terrestrial source of volatile chlorinated hydrocarbons is the spruce forest ecosystem. Chlorine in the soil can be transformed by microorganisms into organically bound chlorine or translocated by transpiration stream in plants, where they are also transformed enzymatically into organically bound chlorine, and both of them can be emitted into the atmosphere. Too large amounts of chloride can affect the physiological functions of plants. In this thesis experiments were designed for measuring the natural emissions of volatile halogenated hydrocarbons from plants and fungi, with various periods of incubation, and also to...

This master thesis is focused on groundwater remediation of chlorinated ethylenes and methanes in the area of chemical factory Spolchemie in the Czech Republic, Ústí nad Labem city. For these purposes nano zero valent iron particles were used. For the remediation two separate applications of different types of particles together in suspension with tracer (lithium chloride), were carried out. The results from the first application were evaluated by the supervisor of this thesis and are briefly summarized and discussed together with the second application which was evaluated by the author of this thesis. Second application of particles was carried out in October 2015 and observed for 424 days. Observation consisted of monitoring of groundwater level, physical-chemical parameters and collection of water samples for the analysis of concentrations of chlorinated hydrocarbons, their degradation products and several chosen ions. Nanoiron particles had the assumed effect onto the physical-chemical parameters and reduction of contamination. The application of tracer had proven the expected groundwater flow and made a possibility to distinguish between the process of reductive dehalogenation and dilution caused by the injected water, the dilution did not exceeded 5 % in most monitored points. Other...

Die elektrokinetische Bodensanierung nutzt Transporteffekte aus, die entstehen, wenn ein elektrisches Feld an einen Bodenkörper angelegt wird. Die Sanierungsmethode versagt bei immobilen Schadstoffen. Allerdings scheint unter bestimmten Bedingungen ein im Boden induzierter Abbau der Schadstoffe möglich. Dafür müssen so genannte &amp;quot;Mikroleiter&amp;quot; und Redox-Systeme im Boden vorhanden sein. Hierzu werden theoretische Modelle vorgestellt. An einem konkreten Beispiel (mit aliphatischen Chlorkohlenwasserstoffen kontaminierter Boden) wurde getestet, ob direkt im Boden ein Abbau der Schadstoffe mit der Technik der elektrochemischen Bodensanierung erzielt werden kann. Die Analyse der CKW wurde per GC-MS durchgeführt. Theoretisch ist ein reduktiver Abbau oder eine Dechlorierung durch Eliminierungsreaktionen möglich. Die Ergebnisse der Laborversuche (Einwaage 100-3.000 g) erbrachten unter Einhaltung bestimmter Faktoren einen erfolgreichen Abbau der CKW. Bei ständiger Bewässerung des Bodens und einer Feldstärke von mindestens 600 V/m konnte ein Abbau festgestellt werden. Die Erhöhung des Chlorid-Gehaltes im Abwasser zeigt die erfolgreiche Abbaureaktion an. Potentielle Abbauprodukte mit weniger Chlor-Atomen im Molekülaufbau konnten nachgewiesen werden. Diese Stoffe lassen sich mit den ursprünglichen Kontaminationssubstanzen verknüpfen. Da eine Migration der Schadstoffe im elektrischen Feld nicht beobachtet wurde, wird eine im Boden induzierte Abbaureaktion angenommen. In Bodenzonen mit hohem pH-Wert (Eliminierung) und in Kathodennähe (Reduktion) waren die stärksten Abbauraten zu verzeichnen. Bei Versuchen ohne ständige Bewässerung war der CKW-Abbau schwach. Die Widerstände stiegen stark an. Ein Versuch in einem Container (Einwaage Boden: ca. 2 t) erbrachte nur einen punktuellen Abbau der CKW. Die Struktur des Originalbodens verhinderte den Aufbau eines homogenen elektrischen Feldes. In einem Fassversuch (Einwaage Boden: ca. 33 kg) wurden die Versuchsbedingungen modifiziert. Anlagerungen an allen Elektroden verhinderten eine ausreichend hohe effektive Feldstärke im Bodenkörper. Sanierungseffekte fanden nur lokal begrenzt statt. Im Vergleich von Labor zu Großversuchen zeigte sich, dass die eingetragene Ladungsmenge eine entscheidende Rolle spielt.