Contaminated aquatic sediments pose health risks to fish, wildlife, and humans and can limit recreational and economic uses of surface waters. Technical and cost effective in situ approaches for sediment management and remediation have been identified as a research need. Subaqueous in situ capping is a promising remedial approach; however, little is known regarding its impact on underlying sedimentary processes and the feasibility of bioaugmented caps at sites subject to contaminated groundwater seepage. This work specifically addresses (1) the impact of capping on biogeochemical processes at the sediment-water interface, (2) the ability and degree to which indigenous sediment microorganisms colonize an overlying cap, (3) the effect of advective flow direction on redox conditions within a cap, (4) natural contaminant bioattenuation processes within capped sediment, and (5) limitations toward a functional bioreactive in situ cap. Laboratory-scale experiments with capped sediment columns demonstrated that emplacement of a sand-based in situ cap induced an upward, vertical shift of terminal electron accepting processes into the overlying cap while simultaneously conserving redox stratification. Upflow conditions simulating a groundwater seep compressed anaerobic processes towards the cap-water interface. Microorganisms indigenous to the underlying sediment colonized cap material and spatial population differences generally reflected redox stratification. Downflow of oxic surface water through the cap, simulating tidally-induced recharge, created fully oxic conditions within the cap, demonstrating that flow direction strongly contributes to redox conditions. Experiments simulating capped sediment subject to contaminated groundwater seepage revealed a reduction of natural bioattenuation processes with time, stemming from the elimination of labile organic matter deposition to the sediment and a subsequent lack of electron donor. Thus, parent contaminants within groundwater seeps will be subject to minimal biotransformations within the sediment before entering a reducing cap. A bioreactive cap, inoculated with microorganisms capable of reductive dehalogenation, was established to reductively dechlorinate tetrachloroethene present in the groundwater; however electron donor amendments to sediment effluent were required to achieve complete dechlorination of tetrachloroethene to non-toxic ethene. Results from this work improve understanding of biogeochemical and bioattenuation processes within capped aquatic sediments and should aid in the development of active capping technologies.

Submitted by Jos? Henrique Henrique (jose.neves@ufvjm.edu.br) on 2017-02-15T16:41:12Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) philipe_dias_avila_lima.pdf: 4093796 bytes, checksum: 7076093f9237e97d72538450053db3ed (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2017-03-06T12:26:30Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) philipe_dias_avila_lima.pdf: 4093796 bytes, checksum: 7076093f9237e97d72538450053db3ed (MD5) / Made available in DSpace on 2017-03-06T12:26:30Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) philipe_dias_avila_lima.pdf: 4093796 bytes, checksum: 7076093f9237e97d72538450053db3ed (MD5) Previous issue date: 2016 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / Funda??o de Amparo ? Pesquisa do Estado de Minas Gerais (FAPEMIG ) / Nos ?ltimos anos, um n?mero crescente de pesquisas tem demonstrado o papel chave das esp?cies reativas (ERs) na acelera??o do processo de envelhecimento e agravamento de doen?as inflamat?rias e cr?nico-degenerativas. Tais esp?cies, altamente reativas, podem oxidar v?rias biomol?culas e provocar graves les?es no organismo. O efeito das ERs ? equilibrado no organismo por a??o de antioxidantes enzim?ticos e n?o enzim?ticos, sendo estes ?ltimos, representados em sua maioria pelos compostos fen?licos, sendo os flavon?ides os principais representantes. Os flavon?ides podem ser consumidos na dieta di?ria atrav?s de v?rios alimentos como frutas, legumes, vinhos etc., por?m, o principal alvo para busca de tais compostos s?o as plantas medicinais que s?o constantemente utilizadas pela popula??o. Tendo em vista a import?ncia de desenvolvimento e busca de novos compostos antioxidantes naturais, o presente estudo teve como principal objetivo avaliar a atividade antioxidante dos extratos etan?licos de algumas esp?cies vegetais que v?m sendo utilizadas na medicina popular, dentre outras finalidades, como antiinflamat?rias, cicatrizantes e outros usos relacionados a atividade antioxidante. As esp?cies vegetais utilizadas foram: Ageratum fastigiatum (GARDN.) R. M. KING et H. ROB., Croton antisyphiliticus MART., Kielmeyera rubriflora CAMB., Miconia ferruginata DC., e Norantea adamantium CAMB., coletadas no munic?pio de Diamantina ? MG e regi?es subjacentes, ?reas de predomin?ncia do bioma Cerrado. Avaliando a atividade antioxidante e teor de fen?licos totais (FT) nos extratos, pode-se observar que todos os extratos, exceto da A. fastigiatum (partes a?reas) apresentaram altos teores de compostos fen?licos, determinado pelo m?todo colorim?trico de Folin-Ciocalteau (FC) e eficiente poder redutor (PR), com destaque para a N. adamantium (partes a?reas), que apresentou o maior teor de FT (684,59 mg EAG/g extrato) e o menor valor para o PR (EC50 = 66,77 ?g/mL). Quanto ? captura das esp?cies radicalares DPPH? e ABTS?+, os extratos seguiram uma correla??o positiva, determinada pelo coeficiente de Pearson, ou seja, os extratos com maiores teores de FT tamb?m apresentaram as melhores atividades de captura das esp?cies radicalares com destaque para a N. adamantium (partes a?reas) e C. antisyphiliticus (caule) que apresentaram resultados de EC50 estatisticamente semelhantes ao padr?o ?cido g?lico (AG) para a captura de ambas as esp?cies radicalares utilizadas. Para a captura dos reagentes HOCl e H2O2 todos os extratos foram eficientes, por?m apresentaram melhores resultados apenas em altas concentra??es (>100?g/mL), sendo necess?rio um estudo de citotoxicidade para avaliar se tais concentra??es n?o s?o t?xicas ?s c?lulas humanas. Por fim, foi realizada uma avalia??o de interfer?ncia dos extratos no burst oxidativo produzidos por neutr?filos humanos isolados ativados com PMA e todos os extratos apresentaram atividade protetora frente ?s esp?cies produzidas pelo burst oxidativo com destaque para C. antisyphiliticus (caule) e A. fastigiatum (partes a?reas) que apresentaram menores valores de EC50, igual a 8,51 e 5,74 ?g/mL respectivamente. / Disserta??o (Mestrado) ? Programa de P?s-gradua??o em Ci?ncias Farmac?uticas, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2016. / An increasing number of studies have been lately demonstrating the key role of the reactive species (RS) in accelerating aging and worsening inflammatory and chronic-degenerative diseases. This species, highly reactive, are able to oxidize several biomolecules and cause severe injuries to the organism. The effect of the RS is balanced by the enzymatic and non-enzymatic antioxidants, the latter being represented mostly by phenolic compounds and flavonoids key players which can be daily consumed in fruits, vegetables, wine etc. However, the main samples to search for these compounds are the medicinal plants extensively used by the population. Considering the importance of the development and screening of new natural antioxidant compounds, the present study aimed to evaluate the antioxidant activity of the ethanolic extracts of some species of plants that have been used in the popular medicine for its anti-inflammatory, healing and other uses related to antioxidant activity, among other features. The plants were: Ageratum fastigiatum (GARDN.); R. M. KING and H. ROB., Croton antisyphiliticus MART., Kielmeyera rubriflora CAMB., Miconia ferruginata DC., and Norantea adamantium CAMB., colleted at the municipality of Diamantina - MG and surroundings, where the biome Cerrado is predominant. The analysis of the antioxidant activity and the total phenolic content (TP) of the extracts showed that, except for the A. fastigiatum (aerial parts), all of the others presented high contents of phenolic compounds through the colorimetric assay of Folin-Ciocalteau (FC) and the effectiveness of the reductive power (RP). Highlights here to N. adamantium (aerial parts) extract which demonstrated the highest content of TP (684,59 mg EAG/g extract) and the lowest value of RP (EC50 = 66,77 ?g/mL). Regarding the capture of the following free radicals, DPPH? and ABTS?+, the extracts demonstrated a positive correlation, determined by the Pearson factor, which means that the extracts with the highest contents of TP also displayed better capture activities of those free radicals. It is important to emphasize N. adamantium (aerial parts) and C. antisyphiliticus (stem), once they presented statistically similar EC50 to the garlic acid (GA) for the capture of both of the free radicals used. For the capture of HOCl and H2O2, all the extracts showed to be efficient, nevertheless they presented better results only in high concentrations (>100?g/mL). Therefore it is relevant to perform a citotoxicity assay on human cells. At last, the interference of the extracts was evaluated on the oxidative burst produced by human neutrophils isolated and activated with PMA and all of them showed to be protective against the free radicals produced in the assay, in which C. antisyphiliticus (stem) and A. fastigiatum (aerial parts) standout for the lowest EC50, 8,51 and 5,74 ?g/mL respectively.

abstract: DehaloR^2 is a previously characterized, trichloroethene (TCE)-dechlorinating culture and contains bacteria from the known dechlorinating genus, Dehalococcoides. DehaloR^2 was exposed to three anthropogenic contaminants, Triclocarban (TCC), tris(2-chloroethyl) phosphate (TCEP), and 1,1,1-trichloroethane (TCA) and two biogenic-like halogenated compounds, 2,6-dibromophenol (2,6-DBP) and 2,6-dichlorophenol (2,6-DCP). The effects on TCE dechlorination ability due to 2,6-DBP and 2,6-DCP exposures were also investigated. DehaloR^2 did not dechlorinate TCC or TCEP. After initial exposure to TCA, half of the initial TCA was dechlorinated to 1,1-dichloroethane (DCA), however half of the TCA remained by day 100. Subsequent TCA and TCE re-exposure showed no reductive dechlorination activity for both TCA and TCE by 120 days after the re-exposure. It has been hypothesized that the microbial TCE-dechlorinating ability was developed before TCE became abundant in groundwater. This dechlorinating ability would have existed in the microbial metabolism due to previous exposure to biogenic halogenated compounds. After observing the inability of DehaloR^2 to dechlorinate other anthropogenic compounds, DehaloR^2 was then exposed to two naturally occurring halogenated phenols, 2,6-DBP and 2,6-DCP, in the presence and absence of TCE. DehaloR^2 debrominated 2,6-DBP through the intermediate 2-bromophenol (2-BP) to the end product phenol faster in the presence of TCE. DehaloR^2 dechlorinated 2,6-DCP to 2-CP in the absence of TCE; however, 2,6-DCP dechlorination was incomplete in the presence of TCE. Additionally, when 2,6-DBP was present, complete TCE dechlorination to ethene occurred more quickly than when TCE was present without 2,6-DBP. However, when 2,6-DCP was present, TCE dechlorination to ethene had not completed by day 55. The increased dehalogenation rate of 2,6-DBP and TCE when present together compared to conditions containing only 2,6-DBP or only TCE suggests a possible synergistic relationship between 2,6-DBP and TCE, while the decreased dechlorination rate of 2,6-DCP and TCE when present together compared to conditions containing only 2,6-DCP or only TCE suggests an inhibitory effect. / Dissertation/Thesis / M.S. Civil and Environmental Engineering 2012

Submitted by JÚLIO HEBER SILVA (julioheber@yahoo.com.br) on 2017-04-12T17:27:16Z No. of bitstreams: 2 Dissertação - Kassyo Lobato Potenciano da Silva - 2017.pdf: 3631765 bytes, checksum: debcde89ec6bb553c1b37cafd56ee370 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-04-13T11:21:48Z (GMT) No. of bitstreams: 2 Dissertação - Kassyo Lobato Potenciano da Silva - 2017.pdf: 3631765 bytes, checksum: debcde89ec6bb553c1b37cafd56ee370 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-04-13T11:21:48Z (GMT). No. of bitstreams: 2 Dissertação - Kassyo Lobato Potenciano da Silva - 2017.pdf: 3631765 bytes, checksum: debcde89ec6bb553c1b37cafd56ee370 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-02-20 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The polymorphic fungi Fonsecaea pedrosoi and Cladophialophora carrionii are the main etiological agents of Chromoblastomycosis (CBM), a chronic cutaneous mycotic infection, which occurs mainly in humid tropical regions. The scarcity of effective therapeutic protocols added to the clinical polymorphism of CBM leads to prolonged therapies and a high relapse rate. There are few data on the molecular biology of F. pedrosoi and C. carrionii, although some virulence factors have already been described, revealing the need to explore the mechanisms used by these pathogens during infection. Iron is a vital micronutrient for all organisms, with the exception of some bacteria, participating in essential cellular processes, such as: DNA replication, oxidative stress, cellular respiration, oxygen transport, energy production, among others. During the infection, the host decreases the availability of iron to the pathogen to contain the advancement of the infection; while the pathogen activates mechanisms for obtain and use the metal to survive in the infected tissues, revealing a scenario of intense dispute. In order to characterize the presence of these systems in F. pedrosoi and C. carrionii, in sílico analyzes were performed, revealing the presence of orthologs for proteins involved in reductive iron assimilation, siderophore mediated uptake and regulators of capture and utilization strategies of the metal, these sequences containing conserved domains consistent with the predicted functions. The expression profile of transcripts demonstrated the induction of iron reducing scavengers, siderophore biosynthesis intermediates and the hapX positive regulator during iron deprivation, in addition to repressing the negative regulatory sreA. The production of siderophores was observed after qualitative approaches, and the siderophore ferricrocine was subsequently identified intra- and extracellularly in F. pedrosoi and C. carrionii. These siderophores were able to restore the growth of a mutant strain of ΔsidA from A. nidulans. Preferred iron sources were observed after growth assays and, in addition, some of these sources were used by F. pedrosoi and C. carrionii during infection in macrophages, demonstrating that these pathogens may have strategies for remodeling iron homeostasis for survival. / Os fungos polimórficos Fonsecaea pedrosoi e Cladophialophora carrionii são os principais agentes etiológicos da Cromoblastomicose (CBM), uma infecção micótica cutânea crônica, que ocorre principalmente em regiões tropicais úmidas. A escassez de protocolos terapêuticos eficazes somada ao polimorfismo clínico da CBM, conduzem a terapias prolongadas e elevada taxa de recidiva. Poucos são os dados a respeito da biologia molecular de F. pedrosoi e C. carrionii, embora alguns fatores de virulência já tenham sido descritos, salientando a necessidade de se explorar os mecanismos utilizados por estes patógenos durante a infecção. O ferro é um micronutriente vital para todos os organismos, com exceção de algumas bactérias, participando de processos celulares essenciais, tais como: replicação do DNA, combate ao estresse oxidativo, respiração celular, transporte de oxigênio, produção de energia, dentre outros. Durante a infecção o hospedeiro diminui a disponibilidade de ferro para o agente patogênico para conter o avanço da infecção; enquanto o patógeno ativa mecanismos de obtenção e utilização do metal para sobreviver nos tecidos infectados, revelando um cenário de intensa disputa. Com o objetivo de caracterizar a presença destes sistemas em F. pedrosoi e C. carrionii, análises in sílico foram realizadas, revelando a presença de ortólogas para proteínas envolvidas na assimilação redutiva de ferro, captação mediada por sideróforos e reguladores das estratégias de captação e utilização do metal, contendo estas sequências domínios conservados consistentes com as funções preditas. O perfil de expressão de transcritos demonstrou a indução de captadores redutivos de ferro, intermediários da biossíntese de sideróforos e do regulador positivo hapX durante a privação de ferro, além da repressão do regulador negativo sreA. A produção de sideróforos foi observada após abordagens qualitativas, sendo posteriormente identificado o sideróforo ferricrocina intra e extracelularmente em F. pedrosoi e C. carrionii. Estes sideróforos mostraram-se capazes de restaurar o crescimento de uma linhagem mutante de ∆sidA de A. nidulans. Fontes preferenciais de ferro foram observadas após ensaios de crescimento e, além disso, algumas destas fontes foram utilizadas por F. pedrosoi e C. carrionii durante infecção em macrófagos, demonstrando que estes patógenos podem possuir estratégias de remodelamento da homeostase de ferro para sobrevivência.

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(工学) / 甲第14942号 / 工博第3169号 / 新制||工||1475(附属図書館) / 27380 / UT51-2009-M856 / 京都大学大学院工学研究科物質エネルギー化学専攻 / (主査)教授 小澤 文幸, 教授 大江 浩一, 教授 中村 正治 / 学位規則第4条第1項該当

This thesis covers the development of catalytic methodologies for the mild and chemoselective hydrosilylation of amides. The first part describes the investigation of the Mo(CO)6-catalyzed reduction of carboxamides. It was found that the reduction could be controlled by tuning the reaction temperature and either amines or aldehydes could be obtained selectively. The system showed an unprecedented chemoselectivity and the amide reduction could take place in the presence of other reducible functional groups such as ketones, aldehydes, and imines. Moreover, the transformation could be performed on a preparative scale and was further employed in the synthesis of Donepezil, a pharmaceutical drug used in the treatment of Alzheimer´s disease. The third chapter concerns the development of the Mo(CO)6-mediated hydrosilylation protocol for the reduction of carboxamides containing acidic α-hydrogens. In this case, enamines were formed and a high level of chemoselectivity was observed. Enamines containing sensitive functional groups such as ketones, aldehydes and imines were generated. The enamines were not isolated but used in subsequent catalytic reductive functionalization of amides, which is described in the last part of the thesis (Chapters 4 – 7). The in situ formed enamines were reacted with a wide variety of electrophiles, generating heterocyclic compounds as triazolines, triazoles, 4,5-dihydroisoxazoles and pyrimidinediones. N-sulfonylformamidines as well as thioacrylamides could also be prepared with this approach. The protocols for the synthesis of triazolines, triazoles and N-sulfonylformamidines could additionally be performed on a preparative scale, showing the practicality of the methodology.

The use of enzymes, nature´s own catalysts, both isolated or as whole cells to perform chemical transformations is called biocatalysis. As a complement to classical chemical catalysis, biocatalysis can be an environmentally friendly and more economical option in the production and synthesis of chemicals. Research on the application of amine transaminases in synthesis of chiral amines have exploded over the last two decades and interest from the industry is increasing. Amine transaminases are promising catalysts due to their ability to perform reductive amination of ketones with excellent enantioselectivity. For a process to be efficient, high substrate specificity of the applied enzyme is an important factor. A variant of Chromobacterium violaceum amine transaminase that was obtained through rational design has an increased specific activity toward (S)-1-phenylethylamine and a set of 4´-substituted acetophenones. This result makes this variant a promising catalyst for the asymmetric synthesis of similar amines. Amine transaminase catalyzed asymmetric synthesis of amines generally suffers from unfavorable equilibrium. Two methods that include spontaneous tautomerization and biocatalytic amidation for equilibrium displacement have therefore been developed. Efficient assays and screening methods are demanded for the discovery and development of novel amine transaminases. For this purpose, a sensitive fluorescence-based assay that holds promise as a high-throughput screening method was developed. One of the major obstacles for application of enzymes in industrial processes is the instability of the enzyme toward harsh conditions. The stability of Chromobacterium violaceum amine transaminase was investigated and improved using co-solvents and other additives. Co-lyophilization with surfactants was also applied to improve the performance of the same enzyme in organic solvents. / <p>QC 20161017</p>

About 85% of the world’s energy is derived from non-renewable sources—coal, petroleum, and natural gas. Solar photocatalysis is one way to potentially generate cheap renewable fuels by harnessing energy from the sun using a photosensitizer and converting it into chemical energy. The efficiency of a photosensitizer depends on its capacity to form a prolonged triplet excited state. Zinc dipyrrin complexes have the potential to be efficient sensitizers for reductive photochemistry, but their ability to form long-lived triplet excited states still needs extensive research. The overall aim of this research is to probe the role charge separation plays in the formation of triplet state in metal complexes of dipyrrin photosensitizers. The specific objectives are to synthesize and characterize zinc and boron dipyrrin complexes, analyze their photophysical properties—such as steady state spectroscopy, low temperature emission spectroscopy—and quantify their triplet states using time-resolved transient absorption spectroscopy.

Ce document de thèse est divisé en deux parties indépendantes, la première partie décrit la synthèse et l'évaluation biologique de nouveaux analogues du composé anti-vasculaire, l'isocombretastatine A-4. Ce travail se situe à l'interface entre la chimie et la biologie.Des réactions séquentielles monotopes mettant en œuvre des réactions de couplage pallado-catalysées entre des N-tosylhydrazones et divers bromure de nitro-aryles et bromure de nitro-biaryles suivies d'une cyclisation réductrice ont conduit à la synthèse d’une chimiothèque d'indoles C2-, C3-, N-aryles et des carbazoles de vinyle avec une variété de substrats. De plus, cette méthodologie a permis d'explorer un nouveau mécanisme de synthèse inattendue d’indoles.L'étude biologique de ces nouvelles séries a montré que huit molécules sont dotées d’activités antiprolifératives intéressantes de l'ordre du nanomolaire contre les cellules humaines de cancer du côlon. Deux molécules ont présenté des activités très prometteuses, ayant des IC50 similaires à celle de l'isoCA-4.Par conséquent, cette partie a contribué à développer de nouvelles approches permettant l’accès rapide et simple à des molécules hautement fonctionnalisées, dont certaines possèdent une activité biologique prometteuse.Par ailleurs, la deuxième partie est liée à la synthèse de nouveaux analogues de thiazole et de benzothiazole de MIM-1 (molécule d'inhibition de Mcl-1) grâce à une série de réactions connues. Plusieurs analogues de benzothiazole ont été obtenus en une seule ou deux étapes, et les composés thiazole ont été obtenus en cinq étapes. La modification des structures des deux séries d'analogues est en cours, afin d’évaluer leur activité antitumorale, but principal de cette partie. / This thesis document is divided into two unrelated parts, the first part reported the synthesis and the biological evaluation of new analogs of the anti-vascular compound, isocombretastatin A-4. This work lies at the chemistry-biology interface.One-pot sequential reactions implementing palladium-catalyzed cross-coupling between N-tosylhydrazones and various nitro-aryl bromides and nitro-biaryl bromides followed by reductive cyclization led to the synthesis of a broad library of C2-, C3-, N-aryl indoles and vinyl carbazoles with vast substrate scope. Moreover, this methodology allowed to explore a new mechanism for the synthesis of indoles through unexpected pathways.Interestingly, the biological evaluation of these new series had identified eight molecules with antiproliferative activities in the nanomolar range against human colon carcinoma, with two molecules showing very promising results by having IC50 similar to that of isoCA-4.Hence, this part contributed to develop new processes for an easy access of highly substituted compounds which could possess promising biological activity.On the other hand, the second part is related to the synthesis of original thiazole and benzothiazole analogs of MIM-1 (Mcl-1 inhibition molecule) through a series of known reactions. Several benzothiazole analogs were obtained in one or two steps, and the thiazole compounds were obtained in five steps. More structural modifications of the two series of analogs is ongoing to achieve the innate goal of this part, which is to evaluate their antitumor activity.

Organohalides have been abundantly utilized as pesticides and in industrial processes for the past 100 years, with over 30 000 sites in Europe still being contaminated today. Because of their recalcitrance, large quantities have accumulated in soils, sediments, and groundwater. Many organohalides can cause multiple adverse health effects, including neurological damage, congenital malformations, and a variety of human cancers. Fortunately, bacterial genera from a diverse range of phyla are capable of detoxifying these organohalides via anaerobic respiration, i.e., by using them as their terminal electron acceptor. These metabolic pathways involve a reductive dehalogenation reaction, during which a chlorine atom dissociates and thereby either immediately reduces the toxicity of the organohalide, or enables it to be further degraded by a broader range of organisms. Thus, organohalide-respiring bacteria can be used for the bioremediation of contaminated environments. To be able to support this application, fundamental research on these reactions and the metabolism of organohalide-respiring bacteria is a prerequisite. Many aspects of the physiology of organohalide-respiring bacteria are unresolved. Organohalide-respiring bacteria harbor up to 38 reductive dehalogenase homologous genes, which putatively encode the key enzymes of reductive dehalogenation. However, the regulation, protein-coding ability, the function of these enzymes as well as their interactions with other proteins has yet to be elucidated. Organohalide-respiring bacteria are difficult to study due to their slow growth, low biomass yields, oxygen sensitivity and genetic inaccessibility. The aim of this thesis was to circumvent these obstacles by introducing new methods for studying organohalide respiration and thereby enabling the formulation of informed predictions about the functions of reductive dehalogenases and the identity of their regulators. For this, obligate and facultative organohalide-respiring bacteria were assessed. To form a basis of the current research in the field, all available genomic, transcriptomic and proteomic literature on organohalide-respiring bacteria were reviewed and compared. Through combining quantitative expression data of hundreds of orthologs and subjecting them to statistical analyses, many new aspects of the metabolism of organohalide-respiring bacteria were uncovered. Especially notable were the unclear expression patterns of reductive dehalogenases and their accessory proteins. An important conclusion from this review was that shotgun proteomics is essential to reveal how many reductive dehalogenase proteins are produced in parallel, but this approach alone cannot clarify the function of these enzymes nor their underlying regulation processes. Therefore, the next chapter of this thesis aimed to extend and refine the standard proteomics approaches. First, proteomics conducted via mass spectrometry requires optimization of sample processing and analysis. Utilizing harsher conditions for protein extraction and digestion substantially improved proteome coverage compared to previous studies, especially of membrane proteins. The combination of this approach with a highly stringent statistical filtering procedure allowed a more detailed, reliable and thus more valid view of the proteome to be obtained from the model organism Sulfurospirillum halorespirans. The quantification of the putative protein histidine kinase provided the first evidence of its involvement in controlling organohalide respiration together with the putative response regulator, forming a complete two-component regulatory system. The quantification of the putative quinol dehydrogenase membrane subunit also supported its involvement in the organohalide respiratory chain of this genus. We observed that S. halorespirans undergoes the same type of peculiar memory-effect as Sulfurospirillum multivorans, that is, continuing to produce its complete dehalogenating machinery even after prolonged cultivation on a non-halogenated electron acceptor. To reveal the underlying mechanism, protein lysine acetylation was additionally measured, which is an important post-translational modification involved in many regulatory processes across all living organisms. Lysine acetylations are, e.g., known to alter the binding properties of DNA-interacting proteins like transcription factors or response regulators but have a range of other regulatory effects. In the first ‘acetylome’ study of an organohalide-respiring bacterium and an Epsilonproteobacterium, one-third of all S. halorespirans proteins were found to be acetylated at one point over the course of a long-term cultivation experiment. Interestingly, the putative response regulator of the two-component regulatory system described earlier was acetylated during the metabolic transition phase, after short-term adaptation to a non-halogenated electron acceptor. Another advancement of shotgun proteomics was its combination with thermal proteome profiling to elucidate substrate specificities of reductive dehalogenases and their regulators. The underlying principle behind thermal proteome profiling is to identify the interaction of a protein with a binding ligand through its impact on the thermal stability of the protein. The thermal stability of hundreds of proteins can be measured in parallel by a proteomics approach. Aliquots of protein extract are first incubated at different temperatures, and the non-denatured fraction of each protein is then quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS), thus allowing the composition of melting curves of each protein to be determined. With this unbiased approach, unknown protein-ligand interactions can also be identified. In a proof-of-concept study on S. multivorans, we adapted the method to anaerobic conditions and showed that this technique is suitable for the detection of interactions between enzymes and their specific substrates. For example, a melting curve shift was detected when the tetrachloroethene reductive dehalogenase, PceA, bound to its known substrate, trichloroethene. Furthermore, the melting curve shift of the putative response regulator in the two-component regulatory system indicated at least an indirect interaction between it and trichloroethene, providing the first biochemical evidence of its role in organohalide respiration besides mere expression data. In conclusion, this work not only includes the first systematic analysis of all omics-based studies conducted to date but substantially advanced the methods for assessing organohalide-respiring bacteria by providing a more detailed picture of their physiology. Besides methodological advances, it was demonstrated that the two-component regulatory system interacts with halogenated compounds and that its post-translational modification might impact long-term downregulation of the organohalide respiratory apparatus in Sulfurospirillum spp. The insights into the involvement of the two-component regulatory system in the organohalide respiration of Sulfurospirillum spp. would not have been uncovered by using less complex standard shotgun proteomics measurements. In the future, our findings will help to further elucidate regulators and functioning of reductive dehalogenases also in other organohalide-respiring bacteria.:Summary 7 Zusammenfassung 10 1 Introduction 14 1.1 Halogenated compounds and the environment……………………...……….……. 14 1.2 Transformation of organohalides……………………..……………….…………….. 15 1.3 Reductive dehalogenation………………………..……………………………….…... 16 1.3.1 Dehalococcoides mccartyi……………………………………………….……… 18 1.3.2 Sulfurospirillum spp. …………………..………………………………..……... 20 1.4 Proteomics……………………..………………..…………………………………...….. 22 1.4.1 The principle of shotgun proteomics..………………..………………....……. 22 1.4.2 Protein lysine acetylations–an important post-translational modification…………………………………………………………...………… 24 1.4.3 Thermal proteome profiling..………………..………..……..………………... 28 1.5 Objectives..………………..……..………..………..………………..…………………. 29 2 Publications 31 2.1 Overview of publications..………..………………..………….………..…………….. 31 2.1.1 Publication 1..………..………….………..…….………..………………………. 31 2.1.2 Publication 2..………..…………..………..…….………..……………………… 31 2.1.3 Publication 3..………..…………….………..…..………..……………………… 32 2.1.4 Publication 4..………..…………..……….…..………..……………….……….. 32 2.2 Published articles..………..……………....…………..………..………………..……. 33 3 Discussion 88 3.1 The application of ‘omics’ to organohalide-respiring bacteria..………..………... 88 3.2 Parallel proteome and acetylome analysis..………..………………..…………….. 91 3.2.1 Specific challenges for the analysis of protein lysine acetylations………. 92 3.2.2 Insights into the metabolism of S. halorespirans..………..………………... 93 3.3 Protein interaction analysis by thermal proteome profiling..………..……......... 97 3.3.1 Other potential approaches to study protein-ligand-interactions..…….... 98 3.3.2 Potential of using thermal proteome profiling for organohalide- respiring bacteria..………..……….………..………….………..……………… 99 3.4 Conclusions and future perspectives..………..……………..………..…..………… 101 4 References 104 5 Appendix 118 5.1 Declaration of authorship..………..……………..………..……………………..…… 118 5.2 Author contribution of published articles..………..……………..……………….... 118 5.3 Curriculum vitae..………..………………..…………….………..…………………… 124 5.4 List of publications and conference contributions..………..……………...………. 124 5.5 Acknowledgements..………..………… ………..…………………..…………..…….. 127 5.6 Supplementary material..…………………..………..………………………….……. 128 5.6.1 Supplementary material for Publication 3..………..……..………..……….. 128 / Während der letzten einhundert Jahre wurden halogenierte organische Verbindungen großflächig in Industrie und Landwirtschaft eingesetzt, wodurch heute mehr als 30 000 Flächen in Europa kontaminiert sind. Aufgrund ihrer eingeschränkten Abbaubarkeit konnten sich riesige Mengen in Böden, Sedimenten und Grundwasser ausbreiten. Viele halogenierte organische Verbindungen können erhebliche nachteilige Auswirkungen auf die Gesundheit des Menschen haben, u.a. neurologische Schäden, Fehlbildungen und eine Vielzahl von Krebserkrankungen. Glücklicherweise sind bestimmte Bakterientypen unterschiedlicher Phyla in der Lage, diese Stoffe mittels anaerober Atmung, d.h. über deren Nutzung als terminalen Elektronenakzeptor, umzuwandeln. Diese reduktive Dehalogenierung, bei der ein Chlor-Rest abgespalten wird, vermindert die Toxizität der meisten Organohalide bzw. macht sie zugänglich für den Abbau durch ein breiteres Organismenspektrum. Demgemäß können Organohalid-atmende Bakterien für die Bioremediation kontaminierter Flächen genutzt werden. Voraussetzung für deren Einsatz ist jedoch das Verständnis der zugrundeliegenden biochemischen Reaktionen und des Metabolismus der Organohalid-Atmer. Viele Aspekte der Physiologie Organohalid-atmender Bakterien sind noch ungeklärt. Die Organismen besitzen bis zu 38 unterschiedliche Gene, die reduktive Dehalogenasen, die Schlüsselenzyme der Organohalid-Atmung, kodieren. Allerdings sind deren Regulation, Proteinkodierung, die Funktion der einzelnen Enzyme sowie deren Interaktionen mit anderen Proteinen noch unbekannt. Die Forschung an Organohalid-atmenden Bakterien wird durch deren langsames Wachstum, die geringen Zelldichten, die hohe Sensitivität gegenüber Sauerstoff und fehlende gentechnische Methoden erschwert. Ziel dieser Arbeit war es, die genannten Hindernisse mittels neuartiger Methoden an Organohalid-Atmern zu umgehen und damit Regulatoren und Funktionsweise der reduktiven Dehalogenasen zu bestimmen. Hierfür wurden sowohl obligate als auch fakultative Organohalid-atmende Bakterien herangezogen. Als Grundlage führte ich zunächst alle bisher durchgeführten Genomik-, Transkriptomik- und Proteomikstudien zu Organohalid-atmenden Bakterien zusammen. Hunderte zu Orthologen kombinierte und statistisch analysierte quantitative Expressionsdaten lieferten dabei ein umfassendes Bild vom Metabolismus der Organohalid-Atmer. Insbesondere die unklaren Expressionsmuster der reduktiven Dehalogenasen und ihrer akzessorischen Proteine wurden offenbar. Eine wichtige Erkenntnis des Review-Prozesses war, dass Standard-Proteomikansätze zwar unerlässlich sind, um beispielsweise die gleichzeitige Produktion mehrerer reduktiver Dehalogenasen offenzulegen, aber weder deren Funktionen noch Regulation aufklären können. Aus diesem Grund sollten im weiteren Verlauf dieser Arbeit die bisher genutzten Shotgun-Proteomikmethoden weiterentwickelt werden. Für eine umfassende Proteinanalyse mittels Massenspektrometrie müssen zunächst Probenaufarbeitung und Analyse optimiert werden. Durch die Verwendung harscherer Bedingungen bei Proteinextraktion und -verdau konnten wir die Proteomabdeckung, insbesondere unter Membranproteinen, im Vergleich zu früheren Studien erheblich verbessern. In Kombination mit einem sehr stringenten statistischen Filterprozess erlaubte dies einen detaillierten und validen Blick auf das Proteom des Modellorganismus Sulfurospirillum halorespirans. Die Quantifizierung der mutmaßlichen Protein-Histidinkinase ist der erste Beleg dafür, dass diese zusammen mit dem Regulationsprotein im Zweikomponentensystem an der Kontrolle der Organohalid-Atmung in Sulfurospirillum spp. beteiligt ist. Die quantifizierte Membranuntereinheit der Quinoldehydrogenase stützt die Annahme zu deren Beteiligung an der Atmungskette dieses Organismus. Wir konnten weiterhin zeigen, dass in S. halorespirans die gleiche außergewöhnliche Langzeitregulation wie in Sulfurospirillum multivorans wirksam ist, sodass auch nach langanhaltender Kultivierung auf nicht-halogenierten Substraten der komplette Organohalid-Atmungsapparat synthetisiert wird. Zur Aufklärung der zugrundeliegenden Regulation erweiterten wir unsere Analyse um Protein-Lysin-Acetylierungen, wichtige posttranslationale Modifikationen, die an verschiedensten regulatorischen Prozessen in allen Lebewesen beteiligt sind. Protein-Lysin-Acetylierungen beeinflussen z.B. die Wechselwirkungen zwischen Transkriptionsfaktoren oder Regulationsproteinen und der DNA, aber haben noch viele weitere regulatorische Effekte. In dieser ersten „Acetylom“-Studie an einem Organohalid-atmenden Bakterium bzw. einem Epsilonproteobacterium, konnten wir zeigen, dass ein Drittel aller S. halorespirans-Proteine im Verlauf der Langzeitkultivierung mindestens einmal acetyliert wurden. Interessanterweise war auch das mutmaßliche Regulatorprotein des oben erwähnten Zweikomponentensystems während der metabolischen Umstellungsphase, d.h. nach Kurzzeitanpassung an den nicht-halogenierten Elektronenakzeptor, acetyliert. Eine zusätzliche Weiterentwicklung der klassischen proteomischen Messungen war deren Kombination mit Thermal Proteome Profiling, um Substratspezifitäten und Regulatoren von reduktiven Dehalogenasen zu bestimmen. Zugrundeliegendes Prinzip des Thermal Proteome Profiling ist die Identifikation eines Proteinbindungspartners über dessen Einfluss auf die Thermostabilität der Faltung eines Proteins. Die Thermostabilität tausender Proteine kann mit Hilfe eines Proteomikansatzes bestimmt werden. Hierfür werden extrahierte Proteine zunächst aufgeteilt und unterschiedlichen Temperaturen ausgesetzt. Die nicht-denaturierte Fraktion jedes Proteins kann mittels Flüssigchromatographie mit Tandemmassenspektrometrie-Kopplung (LC-MS/MS) quantifiziert und zu Schmelzkurven zusammengesetzt werden. Mit dieser Methode können auch unbekannte Protein-Liganden-Interaktionen identifiziert werden. In unserer Machbarkeitsstudie an S. multivorans konnten wir zeigen, dass die von uns modifizierte Technik auch zur Aufklärung von Enzym-Substrat-Interaktionen und sogar unter anaeroben Bedigungen eingesetzt werden kann. So konnte nachgewiesen werden, dass die Schmelzkurve der reduktiven Tetrachlorethen-Dehalogenase PceA durch Bindung ihres bekannten Substrates Trichlorethen signifikant verschoben wurde. Außerdem deutet die Verschiebung der Schmelzkurve des mutmaßlichen Regulatorproteins des Zweikomponentensystems zumindest auf eine indirekte Interaktion mit Trichlorethen hin und ist damit, abgesehen von bloßen Expressionsdaten, der erste biochemische Beleg für dessen Rolle bei der Organohalid-Atmung. Zusammenfassend beinhaltet diese Arbeit nicht nur die erste systematische Analyse und Kombination aller bisher verfügbaren „Omics“-Studien, sondern auch deren Weiterenwiclung für die Untersuchung organohalid-atmender Bakterien, wodurch ein detailliertes Bild von deren Physiologie geschaffen werden konnte. Neben den technischen Neuerungen konnte gezeigt werden, dass das Zweikomponentensystem von Sulfurospirillum sp. mit halogenierten organischen Verbindungen interagiert und dass dessen posttranslationale Modifikation die Langzeitreulation des Organohalid-Atmungsapparates beeinflussen könnte. Die Einblicke in die Beteiligung des Zweikomponentensystems an der Organohalidatmung in Sulfurospirillum sp. wären durch Nutzung von weniger komplexen Standard-Proteomikmethoden unentdeckt geblieben. In Zukunft können uns diese neu entwickelten Methoden dabei unterstützen, Funktionalität und Regulation von reduktiven Dehalogenasen in anderen Organohalid-Atmern aufzuklären.:Summary 7 Zusammenfassung 10 1 Introduction 14 1.1 Halogenated compounds and the environment……………………...……….……. 14 1.2 Transformation of organohalides……………………..……………….…………….. 15 1.3 Reductive dehalogenation………………………..……………………………….…... 16 1.3.1 Dehalococcoides mccartyi……………………………………………….……… 18 1.3.2 Sulfurospirillum spp. …………………..………………………………..……... 20 1.4 Proteomics……………………..………………..…………………………………...….. 22 1.4.1 The principle of shotgun proteomics..………………..………………....……. 22 1.4.2 Protein lysine acetylations–an important post-translational modification…………………………………………………………...………… 24 1.4.3 Thermal proteome profiling..………………..………..……..………………... 28 1.5 Objectives..………………..……..………..………..………………..…………………. 29 2 Publications 31 2.1 Overview of publications..………..………………..………….………..…………….. 31 2.1.1 Publication 1..………..………….………..…….………..………………………. 31 2.1.2 Publication 2..………..…………..………..…….………..……………………… 31 2.1.3 Publication 3..………..…………….………..…..………..……………………… 32 2.1.4 Publication 4..………..…………..……….…..………..……………….……….. 32 2.2 Published articles..………..……………....…………..………..………………..……. 33 3 Discussion 88 3.1 The application of ‘omics’ to organohalide-respiring bacteria..………..………... 88 3.2 Parallel proteome and acetylome analysis..………..………………..…………….. 91 3.2.1 Specific challenges for the analysis of protein lysine acetylations………. 92 3.2.2 Insights into the metabolism of S. halorespirans..………..………………... 93 3.3 Protein interaction analysis by thermal proteome profiling..………..……......... 97 3.3.1 Other potential approaches to study protein-ligand-interactions..…….... 98 3.3.2 Potential of using thermal proteome profiling for organohalide- respiring bacteria..………..……….………..………….………..……………… 99 3.4 Conclusions and future perspectives..………..……………..………..…..………… 101 4 References 104 5 Appendix 118 5.1 Declaration of authorship..………..……………..………..……………………..…… 118 5.2 Author contribution of published articles..………..……………..……………….... 118 5.3 Curriculum vitae..………..………………..…………….………..…………………… 124 5.4 List of publications and conference contributions..………..……………...………. 124 5.5 Acknowledgements..………..………… ………..…………………..…………..…….. 127 5.6 Supplementary material..…………………..………..………………………….……. 128 5.6.1 Supplementary material for Publication 3..………..……..………..……….. 128