Little is known about the enzyme-substrate interactions occurring during the dechlorination of chloroform (CF) and 1,1,1-trichloroethane (1,1,1-TCA) by the enrichment culture containing Dehalobacters, hereafter called DHB-CF/MEL. Compound specific isotope analysis (CSIA) is used to investigate the factors which may affect the isotope fractionation observed for CF and 1,1,1-TCA dechlorination. This thesis reports the first isotope enrichment factors observed for CF biodegradation at -27.5‰ ± 0.9‰, thus providing fundamental information for comparing isotope enrichment factors observed during trichlorinated alkane degradation by DHB-CF/MEL. The thesis also reports how the presence of CF and 1,1,1-TCA influences isotope fractionation and explores the possible influence of substrate inhibition on isotope fractionation during 1,1,1-TCA dechlorination. The data suggests that substrate inhibition during 1,1,1-TCA dechlorination by DHB-CF/MEL may not affect carbon isotope fractionation. The results suggest that CSIA is a promising monitoring tool even for the simultaneous biodegradation of CF and 1,1,1-TCA at different 1,1,1-TCA starting concentration.

Little is known about the enzyme-substrate interactions occurring during the dechlorination of chloroform (CF) and 1,1,1-trichloroethane (1,1,1-TCA) by the enrichment culture containing Dehalobacters, hereafter called DHB-CF/MEL. Compound specific isotope analysis (CSIA) is used to investigate the factors which may affect the isotope fractionation observed for CF and 1,1,1-TCA dechlorination. This thesis reports the first isotope enrichment factors observed for CF biodegradation at -27.5‰ ± 0.9‰, thus providing fundamental information for comparing isotope enrichment factors observed during trichlorinated alkane degradation by DHB-CF/MEL. The thesis also reports how the presence of CF and 1,1,1-TCA influences isotope fractionation and explores the possible influence of substrate inhibition on isotope fractionation during 1,1,1-TCA dechlorination. The data suggests that substrate inhibition during 1,1,1-TCA dechlorination by DHB-CF/MEL may not affect carbon isotope fractionation. The results suggest that CSIA is a promising monitoring tool even for the simultaneous biodegradation of CF and 1,1,1-TCA at different 1,1,1-TCA starting concentration.

This thesis consists of two major chapters, each with its own introduction, experimental section and discussion. The TG4T/daunomycin and G4/daunomycin complexes described in Chapter One are two out of only five crystallographic quadruplex/ligand structures reported to date. In both structures daunomycin molecules stack onto a terminal G quartet preventing the G4 quadruplex from destacking and unwinding. The number of interacting ligand molecules depends on the quadruplex structure itself. The G4 quadruplex can accommodate four daunomycin molecules within one layer, while the TG4T tetraplex only accommodates three. In both structures daunosamine moieties form hydrogen bonds with the quadruplex but only daunosamine moieties from the TG4T/daunomycin structure make slight incursions into the quadruplex grooves. Both structures are stabilised by π-π interactions, hydrogen bonds, Van der Waals contacts and electrostatic interactions. The daunomycin/TG4T complex is the first ever reported and the only structure where a ligand interacts directly with the quadruplex groove. Chapter Two describes nine crystal structures of Hoechst 33258 analogues with d(CGCAAATTTGCG)2 and d(CGCGAATTCGCG)2 oligonucleotides, and is divided into two sections. Section A includes seven structures with Halogenated Hoechst 33258 analogues that are potential agents in radiotherapy, phototherapy, radioimmunotherapy or photoimmunotherapy, and the structure of the precursor. In all of the examined complexes the ligand binds to the minor groove but not all halogen substituents refine to 100% occupancy. The refined occupancies of the halogen atoms reveal that the degree of carbon-halogen cleavage is highest for ortho and lowest for para substitution. Among meta substituents pointing outside the minor groove, bromine atoms had a higher occupancy than the larger iodines. The position of the halogen atom in the minor groove is influenced by additional substituents on the phenyl ring. In most cases the bulky halogen atom is facing outside of the minor groove. Only in the 3-iodo-5-isopropylHoechst complex is iodine positioned towards the floor of the groove allowing the big isopropyl group to face outside. Section B describes the structure of a carborane-containing ligand (JW-B) bound to the minor groove of d(CGCAAATTTGCG)2. The analysis shows that is possible to position boron-rich moieties close to the cell nucleus, and JW-B may have potential in Boron Neutron Capture Therapy. / Data file restricted at the request of the author, but available by individual request, use the feedback form to request access.

This thesis consists of two major chapters, each with its own introduction, experimental section and discussion. The TG4T/daunomycin and G4/daunomycin complexes described in Chapter One are two out of only five crystallographic quadruplex/ligand structures reported to date. In both structures daunomycin molecules stack onto a terminal G quartet preventing the G4 quadruplex from destacking and unwinding. The number of interacting ligand molecules depends on the quadruplex structure itself. The G4 quadruplex can accommodate four daunomycin molecules within one layer, while the TG4T tetraplex only accommodates three. In both structures daunosamine moieties form hydrogen bonds with the quadruplex but only daunosamine moieties from the TG4T/daunomycin structure make slight incursions into the quadruplex grooves. Both structures are stabilised by π-π interactions, hydrogen bonds, Van der Waals contacts and electrostatic interactions. The daunomycin/TG4T complex is the first ever reported and the only structure where a ligand interacts directly with the quadruplex groove. Chapter Two describes nine crystal structures of Hoechst 33258 analogues with d(CGCAAATTTGCG)2 and d(CGCGAATTCGCG)2 oligonucleotides, and is divided into two sections. Section A includes seven structures with Halogenated Hoechst 33258 analogues that are potential agents in radiotherapy, phototherapy, radioimmunotherapy or photoimmunotherapy, and the structure of the precursor. In all of the examined complexes the ligand binds to the minor groove but not all halogen substituents refine to 100% occupancy. The refined occupancies of the halogen atoms reveal that the degree of carbon-halogen cleavage is highest for ortho and lowest for para substitution. Among meta substituents pointing outside the minor groove, bromine atoms had a higher occupancy than the larger iodines. The position of the halogen atom in the minor groove is influenced by additional substituents on the phenyl ring. In most cases the bulky halogen atom is facing outside of the minor groove. Only in the 3-iodo-5-isopropylHoechst complex is iodine positioned towards the floor of the groove allowing the big isopropyl group to face outside. Section B describes the structure of a carborane-containing ligand (JW-B) bound to the minor groove of d(CGCAAATTTGCG)2. The analysis shows that is possible to position boron-rich moieties close to the cell nucleus, and JW-B may have potential in Boron Neutron Capture Therapy. / Data file restricted at the request of the author, but available by individual request, use the feedback form to request access.

This thesis consists of two major chapters, each with its own introduction, experimental section and discussion. The TG4T/daunomycin and G4/daunomycin complexes described in Chapter One are two out of only five crystallographic quadruplex/ligand structures reported to date. In both structures daunomycin molecules stack onto a terminal G quartet preventing the G4 quadruplex from destacking and unwinding. The number of interacting ligand molecules depends on the quadruplex structure itself. The G4 quadruplex can accommodate four daunomycin molecules within one layer, while the TG4T tetraplex only accommodates three. In both structures daunosamine moieties form hydrogen bonds with the quadruplex but only daunosamine moieties from the TG4T/daunomycin structure make slight incursions into the quadruplex grooves. Both structures are stabilised by π-π interactions, hydrogen bonds, Van der Waals contacts and electrostatic interactions. The daunomycin/TG4T complex is the first ever reported and the only structure where a ligand interacts directly with the quadruplex groove. Chapter Two describes nine crystal structures of Hoechst 33258 analogues with d(CGCAAATTTGCG)2 and d(CGCGAATTCGCG)2 oligonucleotides, and is divided into two sections. Section A includes seven structures with Halogenated Hoechst 33258 analogues that are potential agents in radiotherapy, phototherapy, radioimmunotherapy or photoimmunotherapy, and the structure of the precursor. In all of the examined complexes the ligand binds to the minor groove but not all halogen substituents refine to 100% occupancy. The refined occupancies of the halogen atoms reveal that the degree of carbon-halogen cleavage is highest for ortho and lowest for para substitution. Among meta substituents pointing outside the minor groove, bromine atoms had a higher occupancy than the larger iodines. The position of the halogen atom in the minor groove is influenced by additional substituents on the phenyl ring. In most cases the bulky halogen atom is facing outside of the minor groove. Only in the 3-iodo-5-isopropylHoechst complex is iodine positioned towards the floor of the groove allowing the big isopropyl group to face outside. Section B describes the structure of a carborane-containing ligand (JW-B) bound to the minor groove of d(CGCAAATTTGCG)2. The analysis shows that is possible to position boron-rich moieties close to the cell nucleus, and JW-B may have potential in Boron Neutron Capture Therapy. / Data file restricted at the request of the author, but available by individual request, use the feedback form to request access.

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

No description available.

For centuries chemists have sought to improve humankind’s quality of life and address many of society’s most pressing needs through the development of chemical processes and synthesis of new compounds, often with phenomenal results. Unfortunately, there also are many examples where these chemicals have had unintended, detrimental consequences that are not apparent until years or decades after their initial use. There are numerous halogenated molecules in this category that are globally dispersed, resistant to natural degradation processes, bioaccumulative, and toxic to living organisms. Chemicals such as these are classified as persistent organic pollutants (POPs), and due to their negative environmental and health effects, they require safe, effective, and inexpensive means of remediation. This research focuses on the development and optimization of a reaction matrix capable of reductively dehalogenating several POPs. Initial experiments determined that powdered magnesium and 1% V/V acetic acid in absolute ethanol was the most effective system for degrading polychlorinated biphenyl (PCB), an extraordinarily recalcitrant environmental contaminant. Further studies showed that this matrix also was capable of degrading polychlorinated dibenzo-p-dioxins (PCDDs), polybrominated diphenyl ethers (PBDEs), and four organochlorine pesticides (OCPs); dieldrin, heptachlor, heptachlor epoxide, and chlordane. During this phase of testing, field samples contaminated with chlordane were washed with ethanol and this ethanol/chlordane solution was degraded using the same reaction matrix, thereby demonstrating this technology’s potential for “real-world” remediation projects. Finally, a set of experiments designed to provide some insight into the mechanism of dechlorination seems to indicate that two distinct processes are necessary for degradation to occur. First, the passivated iv outer layer of the magnesium must be removed in order to expose the zero-valent magnesium core. Next, an electron is transferred from the magnesium to the target molecule, causing the cleavage of the halide bond and the subsequent abstraction of either a hydrogen or proton from a solvent molecule. It is anticipated that an understanding of these fundamental chemical processes will allow this system to be tailored to a wide range of complex environmental media

No description available.

A nearly stoichiometric, (1×1) Cr₂O₃ (101̲2) surface was prepared from a single crystal of α-Cr₂O₃. The five-coordinate cations exposed at the stoichiometric surface dissociatively adsorb molecular oxygen to form a (1×1), terminating chromyl (Cr=O) layer that is stable to >1100 K. TDS and AES were used to investigate the reactivity of the halo-alkanes CFCl₂CH₂Cl, CF₂ClCH₂Cl, CF₃CH₂Cl, and CF₂CH₂F, in addition to the halo-alkenes CFCl=CH₂ and CF₂=CH₂. The halo-alkanes CFCl₂CH₂Cl, CF₂ClCH₂Cl, and CF₃CH₂Cl undergo 1,2-dihalo elimination similar to the Zn-catalyzed dehalogenation of vicinal dihalides to form alkenes. Some acetylene is also formed. The halo-alkenes CFCl=CH₂ and CF₂=CH₂ decompose to yield acetylene. Halogen removed from the molecules remains bound to the surface following TDS experiments and eventually terminates the surface chemistry due to site blocking of the cations. Reactivity is directly related to the chlorine content of the molecules investigated. Only CFCl₂CH₂Cl was reactive on a chromyl-terminated surface. / Ph. D.