Doctor Pharmaceuticae - DPharm / Natural products have been an important source of drugs and novel lead compounds in drug discovery. Their unique scaffolds have led to the synthesis of derivatives that continue to give rise to medicinally relevant agents. Thus, natural product-inspired drugs represent a significant proportion of drugs in the market and with several more in development. Cancer is among the leading public health problems and a prominent cause of death globally. Chemotherapy has been important in the management of this disease even though side effects that arise due to lack of selectivity is still an issue.

In this study, two regenerative thermal oxidizers (RTO) were used to test the thermal destruction, thermal recovery efficiency and the gas pressure drop over the beds characteristics when burning, respectively, airborne chlorine- and nitrogen-containing volatile organic compounds (VOCs). First, an electrically-heated RTO containing two 0.5 m ¡Ñ 0.5 m ¡Ñ 2.0 m (L ¡Ñ W ¡Ñ H) beds, both packed with gravel particles with an average diameter of around 0.0116 m and a height of up to 1.48 m with a void fraction of 0.41 in the packed section was used to study the destruction characteristics of chlorine-containing VOCs (trichloroethane, TCE and dichloromethane, DCM). With a valve switch time (ts) of 1.5 min, preset maximum destruction temperatures (TS) of 500-800 oC and superficial gas velocity (Ug) of 0.17-0.33 m/s (evaluated at an influent air temperature of around 27 ¢J), tests on the thermal recovery efficiency (TRE) and the pressure drop for the air stream without VOC in the influent air stream have been performed. With a ts of 1.5 min, Ts of 500-800 oC and Ug of 0.17-0.24 m/s (evaluated at an influent air temperature of around 27 ¢J), tests on the degree thermal destruction of VOCs with influent air streams containing one of the two VOCs: trichloroethylene (TCE) and dichloromethane (DCM) have been done. Second, an electrically-heated RTO containing two 0.152 m ¡Ñ 0.14 m ¡Ñ 1.0 m (L ¡Ñ W ¡Ñ H) beds, both packed with gravel particles with an average diameter of around 0.0111 m and a height of up to 1.0 m with a void fraction of 0.42 in the packed section was used to study the destruction and NOx formation characteristics of DMF (N, N-dimethylformamide). With a ts of 1.5 min, Ts of 750-850 ¢Jand Ug of 0.39-0.78 m/s (evaluated at an influent air temperature of around 30 ¢J), TRE and the pressure drop for the air stream without VOC in the influent air stream have been tested. With a ts of 1.5 min, a Ug of 0.39 m/s (evaluated at an influent air temperature of around 30 ¢J), and Ts of 750-950 ¢J and, thermal destruction efficiencies and nitrogen oxides (NOx) formation characteristics in burning air streams containing either DMF or DMF mixed with methyl ethyl ketone (MEK) were performed. Results demonstrate that: (1) a RTO is suitable for destruction of low concentrations (<1,000 ppm as methane) of airborne highly chlorinated VOCs such as TCE and DCM and the destructed products contain no chlorine and only trace of COCl2 (< 1% of the influent VOC); (2) for TS = 800 oC and Ug = 0.17-0.24 m/s, complete oxidation products of TCE and DCM are HCl, CO2, and H2O, and the main intermediates are CO and COCl2; (3) with ts of 1.5 min, a Ug of 0.39 m/s (evaluated at an influent air temperature of around 30 oC) and TS of 750-950 ¢J, no NOx was present in the effluent gas from the RTO when it was loaded with DMF-free air; (4) when only DMF was present in the influent air, the average destruction efficiencies exceeded 96%, and increased with the influent DMF concentration from 300 to 750 mg/Nm3. The ¡§NOx-N formation/DMF-N destruction¡¨ mass ratios were in the range 0.76-1.05, and decreased as the influent DMF concentration increased within the experimental range; (5) when both DMF and MEK were present in the influent gas, the NOx formation ratio was almost the same and the DMF destruction efficiency increased with the influent MEK/DMF ratio from 150/300 to 4500/300 (mg/mg) and in the preset temperature range. The NOx formation ratios were in the range 0.75-0.96; (6) the TRE decreased as Ug increased but was invariant with Ts; and (7) the Ergun equation was found to suffice in the estimation of the pressure drop when the gas flowed over the packing beds.

The thesis describes the synthesis and application of various alkynones derived from the bis-Weinreb amide of tartaric acid in the total synthesis of macrolactone natural products. The thesis is divided into three sections. First section of the thesis describes the optimization and generalization of the procedure for the addition of alkynyl Grignard / lithium reagents to the bis-Weinreb amide derived from tartaric acid to yield the mono alkynyl ketones was developed. Application of the formed γ-oxo amides was demonstrated in the synthesis of polyols with varied substitutions particularly the synthesis of 1,2,4-triols was accomplished using Ley’s dithianylation as the key step. Scheme 1: Synthesis of polyols from the bis-weinreb amide of tartaric acid. Application of the strategy to the total synthesis of decanolactone natural products achaetolide and (Z)-isomer of (6S,7R,9R)-6,7-dihydroxy-9-propylnon-4-eno-9-lactone was featured. Section B of the thesis deals with the enantiospecific total synthesis of 14-membered macrolactone Sch 725674. Key reactions in the synthesis include the synthesis of the 1,2,4-triol unit from tartaric acid, olefin cross metathesis and ring closing metathesis. Scheme 4: Enantiospecific total synthesis of Sch 725674. Section C of the thesis describes the enantiospecific synthesis of the C9-C22 fragment of the 28-membered polyene polyol macrolide pentamycin. Although isolated in 1958, total synthesis of this antifungal compound was not reported. In application of the methodology developed, the alkynone prepared from the bis-Weinreb amide was elaborated to the required fragment.

Bioprocesses for air pollution control can generally be categorized as bioscrubber, biofilter, and biotrickling filter systems. These processes have been proven to be economical and effective for control of volatile organic compounds (VOCs) with concentrations of <1,000 mg C m-3 in gas streams. First, an activated sludge aeration tank (W x L x H = 40 x 40 x 300 cm) with a set of 2 mm orifice air spargers was utilized to treat gas-borne VOCs (toluene, p-xylene, and dichloromethane) in air streams. The effects of liquid depth (Z), aeration intensity (G/A), the overall mass transfer rate of oxygen in clean water (KLaO2), the Henry¡¦s law constant of the tested VOC (H), and the influent gaseous VOC concentration (C0) on the efficiency of removal of VOCs were examined and compared with a literature-cited model. Results show that the measured VOC removal efficiencies and those predicted by the model were comparable at a G/A of 3.75 ¡V 11.25 m3 m-2 hr-1 and C0 of around 1,000 ¡V 6,000 mg m-3. Experimental data also indicate that the designed gas treatment reactor with KLaO2 = 5 ¡V 15 hr-1, could achieve > 85% removal of VOCs with H = 0.24 ¡V 0.25 at an aerated liquid depth of 1 m, and > 95% removal of dichloromethane with H = 0.13 at a 1 m liquid depth. The model predicts that, for gas treatment in common activated sludge tanks, with KLaO2 = 5 ¡V 10 hr-1, depth = 3 ¡V 4.5 m, G/A = 9 ¡V 18 m3 m-2 hr-1, > 92% VOC removal can be achieved with operating parameters of Z of 3.0 m and KLaVOC/(G/A) of about 0.28 m-1, for VOCs with H < 0.3, such as most oxygen-containing hydrocarbons with low molecular weights, and benzene, toluene, ethylbenzene, and dichloromethane. Second, an activated sludge aeration tank and a sieve-plate column with six sieve plates were utilized to treat gas-borne VOCs in air streams. The tank was used for the biodegradation of the absorbed VOCs from the column which utilized the activated mixed liquor drawn from the tank as a scrubbing liquor. This research proposed a model for VOC absorption to a down-flow activated sludge liquor in a sieve-plate column. The experimental setup consisted of a pilot-scale activated-sludge tank and a sieve-plate tower, as demonstrated. The sieve-plate tower was constructed from a 25 x 25 x 162 cm (W x L x H) acrylic column with six custom-made sieve plates. Each plate has 382 holes which are 3 mm in diameter arranged on a square pitch. The holes give an open area of 3.82% of the whole plate area for gas flow. Two 25 mm-i.d. down-comer pipes were also equipped to allow for the downflow of the activated sludge liquor. Ports were provided at the column inlet, outlet, and each plate for gas and liquid sampling. Experiments were conducted and the model verified based on the results of tests on the removal efficiencies of isopropyl alcohol (IPA), toluene and p-xylene in the system operated at a range of influent VOC concentrations, air application rates, and liquid/gas flow ratios (L/G). The model developed by a material balance for the gaseous- and liquid-VOC over each plate of the column was developed and experimentally verified in this study. Superficial gas velocity over the column plate (U), number of plates (N), volumetric liquid-phase VOC-transfer coefficient (KLaVOC), aerated liquid depth over the plate (Z), volumetric liquid/gas flow-rate ratio (L/G), dimensionless Henry¡¦s law coefficient of the VOC to be absorbed (H), VOC content of the influent scrubbing liquor (xN+1), and the biodegradation rate constant of the VOC in the activated sludge mixed liquor (k) are among the affecting parameters to the effectiveness of the VOC removal. Model application by the model for effects of affecting parameters on the VOC removal effectiveness indicates that L/G, plate number N, biodegradation rate constant k, Henry¡¦s law constant of VOC H are among the important ones. A L/GH of greater than 2 and N of around 6 are enough for the effective (>90%) removal of the influent VOCs with H < 0.01 if no biodegradation occurred in the column. However, a N of over 16 is required for the influent VOCs with H of around 0.2. Biodegradation with a rate constant of around 100 hr-1 in the column greatly improves the column performance.

In risk assessment of risk chemicals, variability in susceptibility in the population is an important aspect. The health hazard of a pollutant is related to the internal exposure to the chemical, i.e. the target dose, rather than the external exposure. The target dose may be calculated by physiologically based pharmacokinetic (PBPK) modeling. Furthermore, variability in target dose may be estimated by introducing variability in the physiological, anatomical, and biochemical parameters of the model. Data on these toxicokinetic model parameters may be found in the scientific literature. Since the early seventies, a large number of experimental inhalation studies of the kinetics of several volatiles in human volunteers have been performed at the National Institute for Working Life in Solna. To this day, only very limited analyses of these extensive data have been performed. A Bayesian analysis makes it possible to merge a priori knowledge from the literature with the information in experimental data. If combined with population PBPK modeling, the Bayesian approach may yield posterior estimates of the toxicokinetic parameters for each subject, as well as for the population. One way of producing these estimates is by so-called Markov-chain Monte Carlo (MCMC) simulation. The aim of the thesis was to apply the MCMC technique on previously published experimental data. Another objective was to assess the reliability of PBPK models in general by the combination of the extensive data and Bayesian population techniques. The population kinetics of methyl chloride, dichloromethane, toluene and styrene were assessed. The calibrated model for dichloromethane was used to predict cancer risk in a simulated Swedish population. In some cases, the respiratory uptake of volatiles was found to be lower than predicted from reference values on alveolar ventilation. The perfusion of fat tissue was found to be a complex process that needs special attention in PBPK modeling. These results provide a significant contribution to the field of PBPK modeling of risk chemicals. Appropriate statistical treatment of uncertainty and variability may increase confidence in model results and ultimately contribute to an improved scientific basis for the estimation of occupational health risks.

Abstract The harmful emissions of chlorinated volatile organic compounds (CVOCs) originate only from man-made sources. CVOCs are used in a variety of applications from pharmaceuticals production to decaffeination of coffee. Currently, CVOC emissions are limited by strict legislation. For these reasons, efficient CVOC abatement technologies are required. Catalytic oxidation is very promising option for this purpose, since catalysts can be tailored to each case to maximize the efficiency and minimize the formation of unwanted products, such as dioxins or Cl2. The goal of this thesis was to study the role of the physico-chemical properties of catalysts in dichloromethane (DCM) oxidation. To reach the aim, several catalytic materials were prepared and characterized, and their performance was tested in total oxidation of DCM. The catalytic materials used were powders of four single metal oxides (&#947;-Al2O3, TiO2, CeO2, MgO), three mixed oxides (Al2O3-xSiO2) washcoated on a cordierite monolith and four active phases (Pt, Cu, V, Mn). At first, support properties were studied. It was found that the DCM conversion and HCl production are dependent on support acidity when the studied single oxides are considered. The best DCM conversions and HCl yields were observed with the support having the highest total acidity (&#947;-Al2O3). Further, the quality of the by-products formed was dependent on the type of the acid sites present on the support surface. Secondly, the impregnation of the active compound was observed to improve the selectivity of the material. From the tested active phases, Pt presented the best performance, but also V2O5 and CuO showed almost equal performances. Especially CuO supported on &#947;-Al2O3, that had less formation of by-products and is less toxic than V-containing oxides, seems to be a promising alternative to Pt. Concerning stability, no deactivation was observed after 55h of testing of Pt/Al2O3. Furthermore, in the used reaction conditions, the formation of CuCl2 is not thermodynamically favoured. Finally, the good characteristics of the powder form catalysts were successfully transferred to the monolith. The performance of the Pt/90Al2O3-10SiO2 catalyst in DCM oxidation was improved when the channel density was increased due to an increase in geometric surface area and mechanical integrity factor, and a decrease in open fraction area and thermal integrity factor. / Tiivistelmä Haitallisten kloorattujen orgaanisten yhdisteiden (CVOC) päästöt ovat ihmisten aiheuttamia. CVOC-yhdisteitä käytetään mm. liuottimina lääkeaineiden valmistuksessa ja kofeiinin poistossa. Nykyisin CVOC-päästöjä rajoitetaan tiukalla lainsäädännöllä. Näistä syistä tehokas CVOC-yhdisteiden käsittelymenetelmä on tarpeen. Katalyyttinen hapetus on hyvä vaihtoehto tähän tarkoitukseen, koska katalyytit voidaan räätälöidä niin, että puhdistuksen tehokkuus saadaan maksimoitua samalla kun ei-haluttujen tuotteiden, kuten dioksiinit ja kloorikaasu, muodostuminen voidaan minimoida. Tämän väitöskirjatyön tavoitteena oli selvittää katalyyttien fysikaalis-kemiallisten ominaisuuksien yhteyksiä dikloorimetaanin (DCM) hapetukseen. Tavoitteen saavuttamiseksi valmistettiin useita katalyyttejä, jotka karakterisoitiin ja testattiin DCM:n kokonaishapetuksessa. Työssä tutkittiin neljää jauhemaista metallioksidia (&#947;-Al2O3, TiO2, CeO2 ja MgO), kolmea metallioksidiseosta (Al2O3-xSiO2), jotka pinnoitettiin kordieriittimonoliitille, sekä neljää aktiivista ainetta: Pt, Cu, V and Mn. Aluksi työssä keskityttiin tukiaineiden ominaisuuksiin. Työn tulokset osoittivat, että DCM:n konversio ja HCl:n tuotanto ovat riippuvaisia tukiaineen happamuudesta. Paras tulos saavutettiin alumiinioksidilla, jolla oli korkein kokonaishappamuus. Lisäksi havaittiin, että sivutuotteiden laatu riippuu tukiaineen pinnalla olevien happopaikkojen tyypistä. Aktiivisen aineen impregnointi tukiaineeseen paransi materiaalin selektiivisyyttä. Tutkituista aineista Pt osoittautui parhaimmaksi, mutta myös V2O5 ja CuO olivat lähes yhtä hyviä. Erityisesti CuO-katalyytti, joka tuotti vähemmän sivutuotteita ja joka on materiaalina vähemmän haitallinen kuin V2O5, osoittautui lupaavaksi jalometallikatalyyttien korvaajaksi. Materiaalien stabiilisuuteen liittyen Pt/Al2O3-katalyytin toiminnassa ei havaittu muutoksia 55 tunnin testauksen jälkeen. Lisäksi CuCl2:n muodostuminen ei mallinnuksen mukaan ole termodynaamisesti todennäköistä tutkituissa reaktio-olosuhteissa. Jauhemaisen katalyytin hyvät ominaisuudet pystyttiin pinnoituksessa siirtämään monoliittirakenteiseen katalyyttiin. Pt/90Al2O3-10SiO2 -katalyytin aktiivisuus DCM:n hapetuksessa tehostui, kun monoliitin aukkoluku kasvoi aiheutuen suuremmasta geometrisestä pinta-alasta ja mekaanisesta eheystekijästä sekä pienemmästä avoimen pinnan osuudesta ja termisestä eheystekijästä.

Abstract Volatile Organic Compounds (VOCs) in wastewaters from the chemical industry are of major concern because of their environmental and health impacts. The reclamation of VOCs from wastewaters would not only reduce the hazard to the environment but also contribute to an efficient use of resources. The thesis explores the reclamation of n-butanol and dichloromethane from sodium chloride containing mixtures by pervaporation. Another aim was to gain understanding of mass transport phenomena during the pervaporation of multicomponent systems, and the effect of sodium chloride on the pervaporation performance. In this work, the reclamation of n-butanol and dichloromethane was conducted as a sequence of pervaporation stages which utilised first hydrophobic and then hydrophilic membranes. The objective was to segregate the mixture of n-butanol/dichloromethane/sodium chloride/water into three different streams: a re-use quality concentrate of VOCs, brine, and discharge quality purified water. The effect of the experimental variables, VOCs feed concentration, feed temperature and sodium chloride content on the performance of the pervaporation stages was studied. A statistical design, response surface methodology, was used to further resource efficiency. The results indicate the potential of pervaporation for the reclamation of n-butanol and dichloromethane from aqueous mixtures. A single step of pervaporation of n-butanol/dichloromethane/sodium chloride/water systems using the CMX-GF-010-D (Celfa) and PERTHESE® 500-1 (P 500-1) membranes does not sufficiently concentrate the VOCs for direct re-use. It is also demonstrated that the electrolyte does not permeate through the membranes and does not affect their separation effectiveness significantly. The pervaporation of the water/dichloromethane/n-butanol system using the hydrophilic poly(vinyl alcohol)-titanium dioxide/polyacrylonitrile/polyphenylene sulfide (PVA-TiO2/PAN/PPS) membrane is effective for dewatering purposes. The membrane shows impermeable features towards dichloromethane in the studied conditions. The analysis of the mass transport phenomena demonstrates that, under the experimental conditions studied, the resistance towards the mass transport of the compounds through the membrane is mainly exhibited by the membrane itself. This study also shows the advantage of analysing the effect of temperature on membrane permeation by the permeation activation energy instead of by the apparent activation energy.

Non-thermal plasma generated in a dielectric barrier packed-bed reactor has been used for the remediation of chlorinated volatile organic compounds. Chlorinated VOCs are important air pollutant gases which affect both the environment and human health. This thesis uses non-thermal plasma generated in single and multiple packed-bed plasma reactors for the decomposition of dichloromethane (CH2Cl2, DCM) and methyl chloride (CH3Cl). The overall aim of this thesis is to optimize the removal efficiency of DCM and CH3Cl in air plasma by investigating the influence of key process parameters. This thesis starts by investigating the influence of process parameters such as oxygen concentration, initial VOC concentration, energy density, and plasma residence time and background gas on the removal efficiency of both DCM and CH3Cl. Results of these investigations showed maximum removal efficiency with the addition of 2 to 4 % oxygen to nitrogen plasma. Oxygen concentrations in excess of 4 % decreased the decomposition of chlorinated VOCs as a result of ozone and NOx formation. This was improved by adding an alkene, propylene (C3H6), to the gas stream. With propylene additives, the maximum remediation of DCM was achieved in air plasma. It is thought that adding propylene resulted in the generation of more active radicals that play an important role in the decomposition process of DCM as well as a further oxidation of NO to NO2. Results in the single bed also showed that increasing the residence time increased the removal efficiency of chlorinated VOCs in plasma. This was optimized by designing a multiple packed-bed reactor consisting of three packed-bed cells in series, giving a total residence time of 4.2 seconds in the plasma region of the reactor. This reactor was used for both the removal of DCM, and a mixture of DCM and C3H6 in a nitrogen-oxygen gas mixture. A maximum removal efficiency of about 85 % for DCM was achieved in air plasma with the use of three plasma cells and the addition of C3H6 to the gas stream. Nitrogen oxides are air pollutants which are formed as by-products during the decomposition of chlorinated VOCs in plasmas containing nitrogen and oxygen. Results illustrate that the addition of a mixture of DCM and C3H6 resulted in the formation of the lowest concentration of nitric oxide, whilst the total nitrogen oxides concentrations did not increase. A summary of the findings of this work is presented in chapter eight as well as further work. To conclude, the maximum removal efficiency of dichloromethane was achieved in air plasma with the addition of 1000 ppm of propylene and the use of three packed-bed plasma cells in series. The lowest concentration of nitric oxide was formed in this situation.

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.

Die Unsicherheit analytischer Ergebnisse lässt Rückschlüsse auf die Zuverlässigkeit der angewandten Analyseverfahren zu. Entsprechend den Qualitätsanforderungen an akkreditierte Prüf- und Kalibrierlaboratorien (siehe u.a. Norm ISO/IEC 17025) ist daher die Angabe der Messunsicherheit erforderlich. Zur Ermittlung der Messunsicherheit typischer FT-IR-spektroskopischer Analysen von Feststoffen und Flüssigkeiten wurde eine systematische Untersuchung des Einflusses zahlreicher FT-, proben- und gerätespezifischer Parameter auf den Messwert durchgeführt. Die Überprüfung der Ordinatenrichtigkeit bei FT-IR-spektroskopischen Messungen an Gläsern erfolgte mit Hilfe eines Transmissionsstandards des National Physical Laboratory (NPL), der aus einem Schott NG11 Neutralglasfilter besteht, und bei Messungen an Flüssigkeiten durch Vergleich der nach einem Absolutverfahren bestimmten Extinktionskoeffizienten des Dichlormethans mit von der IUPAC publizierten Standardwerten. Nach Optimierung der Messbedingungen stimmen die Transmissionswerte des NPL-Standards und die Extinktionskoeffizienten des Dichlormethans mit den entsprechenden Standardwerten überein. Die für die Bestimmung dieser Messgrößen aufgestellten Unsicherheitsbilanzen gestatten die quantitative Abschätzung des Einflusses einzelner Parameter auf die Gesamtunsicherheit. Bei Anwendung der KBr-Presstechnik für die quantitative Analyse von unlöslichen Feststoffen haben die Partikelgröße des Analyten und die Homogenität der Presslinge einen dominierenden Einfluss auf die Qualität der Spektren. Um die Unsicherheit zu minimieren, die auf dem Einfluss der Partikelgröße beruht, muss es ein Ziel der Probenvorbereitung sein, dass die Partikeldurchmesser der Kalibriersubstanz und des Analyten einen Wert von ca. 2 µm nicht überschreiten und möglichst gleich groß sind. Bei Untersuchungen mit Calciumcarbonat-Proben unterschiedlicher Partikeldurchmesser hat sich die Halbwertsbreite der auszuwertenden Bande, die mit der Partikelgröße korreliert, als ein geeignetes Maß für die Beurteilung erwiesen, wie gut letztere Bedingung erfüllt ist. Eine inhomogene Verteilung der absorbierenden Partikel im Pressling kann durch Durchstrahlen der gesamten Oberfläche des Presslings oder durch Mitteln der an verschiedenen Positionen des Presslings gemessenen Spektren berücksichtigt werden. Diese Arbeiten zeigen, dass die Anwendung der KBr-Presstechnik bei sorgfältiger Probenvorbereitung und Einhaltung optimaler Messbedingungen im Rahmen der Messunsicherheit zu richtigen Ergebnissen führt. / The uncertainty of analytical results allows to assess the reliability of the applied analytical methods. According to quality requirements for accredited laboratories (see e.g. ISO/IEC 17025) it is necessary to report the uncertainty of measurement. In order to evaluate the uncertainty of typical quantitative FT-IR spectroscopic analysis of solids and liquids a detailed study of the influence of numerous FT-, sample- and instrument-specific parameters on the measurand was performed. The trueness of FT-IR spectroscopic measurements on glasses was tested with a transmittance standard of the National Physical Laboratory (NPL) that consists of a Schott NG11 neutral density filter. The trueness of FT-IR spectroscopic measurements on liquids was checked by the comparison of the molar absorptivities of dichloromethane determined by transmission measurements with the standard values published by IUPAC. After optimisation of the measurement conditions the transmittance values of the NPL standard and the molar absorptivities of dichloromethane are in accordance with the corresponding standard values. The uncertainty budgets evaluated for these measurends makes it possible to assess the contribution of a single parameter on the combined uncertainty. By using the KBr pressed pellet technique for the quantitative analysis of insoluble solids the particle size of the analyte and the homogeneity of the pellets are the major contributions to the quality of spectra. In order to minimize the uncertainty due to the particle size a purpose of the sample preparation must be to reduce the particle sizes of the calibration sample and the analyte to a maximum size of 2 µm and both particle sizes should agree. A detailed study with calcium carbonate samples with different mean particle sizes has shown that the half-width of the analysed band depends on the particle size and is suitable to test how far the last mentioned condition is fulfilled. In order to minimize the uncertainty due to an inhomogeneous distribution of the absorbing particles in the pellets the infrared beam must pass through the whole surface area of a pellet or the mean value of the spectra measured at different positions on the surface area has to be evaluated. This work proves that the use of the pressed pellet technique can lead to correct results within the limits of the uncertainty if the sample preparation is carried out carefully and optimal measurement conditions are kept.