Ce travail de thèse concerne le développement de catalyseurs Fe/Faujasite afin de dégrader un cocktail de micropolluants provenant d’une eau réelle de rivière dans la Meurthe (France) avec le procédé photo-Fenton hétérogène. Les micropolluants étudiés appartiennent à 4 grandes familles : des principes actifs pharmaceutiques, des produits de soins cosmétiques, des perturbateurs endocriniens, et des composés perfluorés. Pour la première fois, dans cette thèse, la dégradation d’un mélange de 21 micropolluants à l’état de traces (2 – 80 ng/L) provenant d’une réelle eau de rivière non-dopée a été effectuée. Cependant, le fait de travailler avec des concentrations très faibles en polluants reste un challenge notamment en ce qui concerne les aspects de chimie analytiques. Ce travail de thèse est donc divisé en deux grandes parties. Dans un premier temps, il a été nécessaire de trouver la meilleure eau réelle contenant un nombre élevé de micropolluants de différentes natures à des concentrations de l’ordre de quelques ng/L mais inférieures à 100 ng/L. Pour cela différentes campagnes de prélèvement ont été effectuées dans les deux principales rivières de la région Lorraine, la Meurthe et la Moselle, en différents lieux et différentes périodes de l’année. L’eau provenant du site de Moulin Noir a été choisie. Dans une deuxième partie, nous avons développé des catalyseurs fer/Faujasite pour pouvoir tester leur efficacité pour la dégradation des micropolluants provenant du site de Moulin Noir par le procédé photo-Fenton hétérogène. Dans une étape intermédiaire, l’optimisation des paramètres opératoires du procédé photo-Fenton avec nos catalyseurs a été effectuée en utilisant deux macropolluants modèles, phénol et diclofenac / Twenty one 21 micropollutants including pharmaceuticals, personal cares product, endocrine disruptors and perfluorinated compounds presenting at ng/L in the real water of Meurthe river, were successfully quantified and removed using heterogeneous photo-Fenton process. To achieve this goal, an analytical-catalytic methodology was developed and the work steps were performed linked together in a cycle-like manner. The use of the sensitive and efficient multi-residual SPE-LC-MS/MS analytical method allowed us to analyze and quantify the mixture of micropollutants present in a complex matrix during 3 periods of the year with different weather conditions, from 5 sampling sites. Results showed that the highest concentrations of most of the present micropollutants are observed in October at ng/L, Moulin Noir sampling site found to contain the largest number and type of these pollutants, the WWTP was not efficient in the removal of the micropollutants present in water and the drinking water used from tab was totally safe from micropollutants. The calculation of the fluxes and estimation of the mass balance at the rivers confluence confirmed the good precision and reliability of our measurement methodology, and specify the most suitable site for water to be taken from to be used in the removal tests which was Moulin Noir. Having the appropriate water sample, an efficient iron impregnated Faujasite catalyst was developed and used in a photo-Fenton process for the micropollutant removal tests. After characterization and optimization of the different experimental factors using the 2 model macropollutants, phenol and diclofenac, the real tests were performed on real water samples from Moulin Noir. The results demonstrated the good efficiency of the photo-Fenton process with the cocktail of 21 micropollutants. Except for sulfamethoxazole and PFOA, the concentrations of all the other micro-contaminants became lower than the limit of quantification of the LC-MS/MS after 30 minutes or 6 hours of photo-Fenton treatment depending on their initial concentrations under the effect of both adsorption and Fenton mechanisms. Comparing the photo-Fenton process to heterogeneous photocatalytic degradation over TiO2, Faster micropollutants removal occurred with the zeolite

La population générale est exposée à un grand nombre de pesticides, principalement via son alimentation. Alors que ces matières actives ont reçu une autorisation de mise sur le marché individuelle, l’effet sur l’organisme humain d’un mélange de tels composés est en revanche très peu connu. Afin de contribuer à mieux appréhender cet « effet mélange », notre étude s’est attachée à étudier le métabolisme hépatique humain in vitro de deux mélanges de pesticides communément retrouvés dans l’alimentation française / General population is exposed to several pesticides, mainly via the diet. Whereas these active ingredients obtained their marketing authorization individually, the available data regarding their impact as a mixture on the human body are scarce. In order to help better understanding this “mixture effect”, we aimed at studying the human hepatic in vitro metabolism of two pesticides mixtures commonly found in the diet

Den presenterade studien hade som syfte att utveckla en extraktions-, provpreparerings- och analysmetod för jorden, i vilken gräset Cyperus rotundus växer, i ett försök att identifiera möjliga äggläggningsattraherande ämnen för myggan Anopheles gambiae. Högupplösande vätskekromatografi med hydrofob stationär fas (RP-HPLC, en.) med Masspektrometri med Elektrosprejjonisering (ESI-MS, en.) användes för separation och detektion av de extraherade proverna. Ett flertal extraktionssatser gjordes, där parametrar och tekniker ändrades under utvecklingen av arbetet. Ultraljudsassisterad extraktion (UAE) av jord i en 50:50 (v/v) blandning av metanol och vatten visades vara mest effektiv av de undersökta metoderna. Upprening av jordextraktioner med fastfasextraktion (SPE, en.) visades ha en positiv inverkan på signalintensiteten i kromatogrammen såväl som att reducera intensiteten av de systemtoppar som eluerar vid dödtiden. Detta indikerar att de poläraste ämnena har renats bort. En jämförelse gjordes mellan en extraktion av jord som legat i blöt i fem dagar och jord som extraherats utan att blötläggas. En skillnad kunde ses och visades genom att jämföra kromatogram och masspektra från de två proverna. Tandem-MS-experiment gjordes för ett flertal prekursorjoner med avsikt att identifiera ämnena genom jämförelse med databaserna Human Metabolome Database (HMDB), Metlin och Massbank, men inga överrensstämmande jämförelser kunde göras. Tandem-MS-resultaten användes även för att jämföra efter varandra följande kromatografiska toppar med liknande MS1-spektrum. En identifieringsmetod bör utvecklas innan metoden som presenterats i detta arbete valideras och vidareutvecklas. / The present study aimed to develop an extraction, sample preparation and analysis method for the rhizosphere soil of the grass Cyperus rotundus in an attempt to identify possible oviposition attractants of the Anopheles gambiae mosquito. Reversed Phase High Performance Liquid Chromatography (RP-HPLC) coupled online with Electrospray Ionization Mass spectrometry (ESI-MS) was used for the separation and detection of the extracted samples. Multiple extraction batches were done, altering parameters and techniques along the way. Ultrasound-Assisted Extraction (UAE) of the soil with a 50:50 (v/v) mixture of methanol and water was determined to be the most effective of the attempted methods. Purifying soil extracts with Solid Phase Extraction (SPE) showed to have an impact on the signal intensities in the chromatogram as well as reducing the intensity of system peaks eluting at the dead-time. This indicates that more polar compounds were removed during the SPE purification. A comparison was done between an extraction of soil soaked in water for five days and soil extracted without prior wetting. A difference could be seen and was shown by comparing chromatograms and mass spectra from the two samples. Tandem MS experiments were done for multiple precursor ions in order to identify the compounds by comparison in databases Human Metabolome Database (HMDB), Metlin and Massbank, but no matches in the databases were found. The tandem MS results were also used for comparison of consecutive chromatographic peaks with similar MS1-spectra. An identification method should be developed before the method presented in this work is validated and optimized further.

Infections in critically ill patients are a problem for the healthcare system and at any one time, 70 % of all intensive care unit (ICU) patients are treated with antibiotics. Antibiotics bind toproteins in the blood, but only unbound drug can diffuse over capillary membranes and bindto the targeted receptor. Standard protein binding percentages for antibiotics have been developed from studies on healthy volunteers and dosing regimens for patients are adapted accordingly. The determination of the total concentration of antibiotics in patients’ bloodsamples is, based on the standard percentages, ordinarily representative for the pharmacological effect of the antibiotic. However, certain conditions that are common incritically ill patients can alter protein binding percentages, resulting in a larger or smaller unbound fraction. This in turn can result in toxicity or therapeutic failure. The aim of this project was to develop an analytical method for the determination of the unbound concentration of the Beta-Lactam antibiotics cefotaxime, flucloxacillin, cloxacillin and piperacillin in plasma. A method was successfully developed using ultrafiltration for the extraction of unbound analytes and ultra high performance liquid chromatography tandem mass spectrometry, UHPLC-MS/MS, for their quantification. The method was partly validated according to the European Medicines Agency’s guidelines on bioanalytical method validation. / Kritiskt sjuka patienter med infektioner är en börda för sjukvården och 70 % av alla patienter på intensivvårdsavdelningar är ordinerade antibiotika. Antibiotika binder till proteiner i blodet, men enbart den icke-proteinbundna (fria) fraktionen kan diffundera över kapillära membran och binda till receptorer. Standardproteinbindningsgrad för olika antibiotika har utvecklats från studier på friska frivilliga och doseringen av läkemedlen är anpassade därefter. Den totala koncentrationen av antibiotika i patienters blod är vanligen representativ för den farmakologiska effekten. Dock kan vissa sjukdomar påverka proteinbindningsgraden vilket resulterar i en större eller mindre mängd fria antibiotika i blodcirkulationen. Det här kan i sintur resultera i toxicitet eller otillräcklig effekt av läkemedlet. Syftet med det här projektet var att utveckla en analytisk metod för att bestämma den fria koncentrationen av Beta-Lactam antibiotikan cefotaxim, flukloxacillin, kloxacillin och piperacillin i plasma. En metod utvecklades med ultrafiltrering för extraktion av den fria fraktionen och högupplösande vätskekromatografi och tandem masspektrometri, UHPLCMS/MS, för kvantifiering av analyterna. Metoden validerades delvis enligt den Europeiska Läkemedelsmyndighetens riktlinjer för bioanalytisk metodvalidering.

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Cocaine abuse is a world-wide public health and social problem without a U.S. Food and Drug Administration (FDA)-approved medication. An ideal anti-cocaine medication would accelerate cocaine metabolism producing biologically inactive metabolites by administration of an efficient cocaine-specific exogenous enzyme. Recent studies in our lab have led to discovery of the desirable, highly efficient human cocaine hydrolases (hCocHs) that can efficiently detoxify and inactivate cocaine without affecting normal functions of central nervous system (CNS). Preclinical and clinical data have demonstrated that these hCocHs are safe for use in humans and effective for accelerating cocaine metabolism. However, the actual therapeutic use of a hCocH in cocaine addiction treatment is limited by the short biological half-life (e.g. 8 hours or shorter in rats) of the hCocH. In the investigation described in this thesis, we have demonstrated that mCocH and hCocH have improved the catalytic efficiency of mBChE and hBChE against cocaine by ~8- and ~2000-fold, respectively, although the catalytic efficiencies of mCocH and hCocH against other substrates, including acetylcholine (ACh) and butyrylthiocholine (BTC), are close to those of the corresponding wild-type enzymes mBChE and hBChE. In addition, we have identified the first benzoylecgonine-metabolizing enzymes that can hydrolyze benzoylecgonine and accelerate its clearance in rats. The developed LC-MS/MS method has enabled us to simultaneously determine cocaine and nine cocaine-related metabolites in whole blood samples. In development of the long-acting hCocHs, we have designed and discovered a novel hCocH form, catalytic antibody analog, which is an Fc-fused hCocH dimer (hCocH-Fc). The hCocH-Fc has not only a high catalytic efficiency against cocaine, but also a considerably longer biological half-life. A single dose of hCocH-Fc was able to accelerate cocaine metabolism in rats even after 20 days and, thus, block cocaine-induced hyperactivity for a long period of time. In consideration of the general observation that the biological half-life of a protein drug in humans is significantly longer than that in rodents, the hCocH-Fc could allow dosing once every 2-4 weeks, or longer for cocaine addiction treatment in humans.

Vascular risk factors inducing a state of chronic cerebral hypoperfusion and metabolic stress are thought to influence the onset and progression of Alzheimer’s disease (AD). To investigate the complex molecular changes underpinning cellular adaptation to metabolic stress, the first aim of this thesis was to define the proteomic response of the SH-SY5Y human neuroblastoma cell line after exposure to the metabolic challenge of oxygen glucose deprivation (OGD). 958 proteins across multiple subcellular compartments were detected and quantified by label-free liquid chromatography mass spectrometry (LC-MS). The levels of 130 proteins were significantly increased (P<0.01) after OGD and the levels of 63 proteins were significantly decreased (P<0.01) while expression of the majority of proteins (765) was not altered. Ingenuity Pathway Analysis identified novel protein-protein interactomes involved with mitochondrial energy production, protein folding, and protein degradation, indicative of coherent and integrated proteomic responses to the metabolic challenge. Approximately one third (61) of the differentially expressed proteins were associated with the endoplasmic reticulum and mitochondria. Electron microscopic analysis of these subcellular structures showed morphologic changes consistent with the identified proteomic alterations. Pertinent to AD research, amyloid binding alcohol dehydrogenase (ABAD) was found to be significantly increased in response to OGD. ABAD is emerging as a key player in mitochondrial dysfunction in AD, yet full understanding of the biochemical pathways in which this protein is involved remain elusive. Using immunoprecipitation coupled to LC-MS (IP-MS), the second aim of the thesis was to characterise the ABAD protein interactome in SH-SY5Y cells and its response to metabolic stress. 67 proteins were identified as potential ABAD interactors under control conditions, and 69 proteins were identified as potential ABAD interactors under OGD conditions. The Database for Annotation, Visualization and Integrated Discovery (DAVID) was used to determine the subcellular locations and biological functions of the ABAD interacting proteins in control and OGD conditions. DAVID identified the nuclei and mitochondria to contain the greatest number of changes in ABAD interacting proteins following OGD. “Glucose Metabolic Process” (GO:0006006) was the top functional cluster for ABAD interacting proteins in both control and OGD conditions. Independent immunoprecipitations, western blotting, immunohistochemistry and electron microscopy were used to validate specific protein interactions. OGD was found to initiate a novel interaction between ABAD and glucose-regulated protein 75 (GRP75), a finding confirmed in human AD tissue. GRP75 is a mitochondrial protein and marker of the mitochondrial associated membrane (MAM), a specialised region between the mitochondria and the ER. The MAM is known to be enriched with presenilin proteins, involved in the proteolytic cleavage of amyloid precursor protein (APP). These data were used to generate an “ABAD-GRP75-MAM hypothesis of mitochondrial dysfunction in AD”, which might provide a novel link between chronic metabolic stress, ABAD, mitochondrial dysfunction and the onset / progression of AD. The third aim of the thesis was to test this novel hypothesis. Western blotting revealed APP to be significantly decreased following OGD, concurrent with an increase in ABAD protein levels. Over-expression of ABAD protein in SH-SY5Y cells was used to test whether the increased levels of ABAD following OGD were the driving force behind APP down-regulation. ABAD over-expression in SH-SY5Y cells was found to have no detectable effect on APP. Conversely, electron microscopy revealed a dynamic response of the MAM to metabolic stress. This result, along with the interaction of ABAD with GRP75, and the enrichment of presenilins at the MAM, suggests that this specialised membrane region may have an important role to play in AD.

Simvastatin is a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor that is used for the treatment of hyperlipidemia. Simvastatin has recently been studied for its potential use in cancer therapy. In-vitro studies have shown that simvastatin displays anticancer activity, but at concentrations unlikely to be achieved in patients being receiving typical antihyperlipidemic treatment doses. Thus, several clinical trials were conducted to study the tolerability of high dose statins in cancer patients. The maximum tolerated dose of simvastatin was determined to be 15 mg/kg/day, 25-fold higher than a typical dose. However, it is not known if simvastatin plasma concentrations can reach those found to be effective in-vitro. In this context, we initiated a clinical study to determine the pharmacokinetics of high dose simvastatin in patients with chronic lymphocytic leukemia. For this purpose, an LC-MS/MS method was developed and validated for the quantitation of simvastatin and its acid form in plasma and peripheral blood mononuclear cells obtained from CLL patients. Results show that simvastatin concentrations were dose proportional relative to the antihyperlipidemic doses, but lower than those required for in-vitro cytotoxicity against cancer cells. These findings demonstrate that the in-vitro effective concentrations of simvastatin are not achievable clinically, which might explain the limited effectiveness of high dose simvastatin in this study and in previous clinical trials. In view of these data, the use of simvastatin as a sole therapy in cancer treatment was not encouraging and led us to examine the use in combination with other anticancer drugs. After screening several chemotherapeutic agents in combination with simvastatin, we showed that tipifarnib (a farnesyltransferase inhibitor) interacts synergistically in several leukemia cell lines. Mechanistically we showed that simvastatin augments the cytotoxicity of tipifarnib by disrupting the localization of RAS in the cell membrane and by subsequent deactivation of the ERK pathway. Consistent with this observation, drug treatment led to the induction of apoptosis through the caspase cascade activation and the cleaved PARP upregulation. Notably, this synergistic effect was observed at clinically achievable concentrations of simvastatin and tipifarnib. Thus, the effectiveness of this combination should be explored further in future clinical studies.

<p>Trombin är ett trypsinliknande serinproteas som har stor del i kroppens reglering av blodets fluiditet och koagulation genom att det klyver faktorer som slutligen leder till att blodet kan koagulera och fibrin bildas. Syftet med det här projektet har varit att syntetisera fragment som designats med datorgrafiska metoder på Astra Zeneca och sedan analysera fragmenten med affinitetskromatografi med trypsin immobiliserad på kiselgelspartiklar. I projektet har även ett nytt koncept prövats, nämligen att analysera reaktionsblandningarna direkt med hjälp av trypsinkolonnen utan att först isolera och rena föreningarna. De designade fragmenten har syntetiserats med standardmetoder, bl.a. har N,N’dicyklohexylkarbodiimid (DCC) använts. DCC är ett kopplingsreagens som kopplar ihop aminer med karboxylsyror genom skapande av en amidbindning, peptidbindning. Ibland då lite mer komplicerade peptider skall göras, kan aminoänden eller karboxyländen skyddas med en skyddsgrupp, för att dessa inte ska reagera under reaktionssteget och för att man ska få den peptiden man är ute efter. Vätskekromatografi-masspektrometri, LC-MS har använts för identifiering av reaktionblandningarna (substanserna). Resultaten visade att två av de framställda föreningarna retarderades på trypsinkolonnen, vilket innebär att de växelverkar med trypsin och att s.k. hits, träffar erhållits. Synteserna verkar ha gått bra, då LC-MS har visat att det är rätt produkt som finns i kolven. Projektet har visat att det går att designa fragment som man syntetiserar och sen analyserar med AFC-MS. AFC-MS har dessutom visat vara en lämplig metod för screening av svagt bindande fragment.</p> / <p>Through different, intricate mechanisms, the body regulates the coagulation and the fluidity of the blood. This is an important part of the hemostasis if for example bleeding occurs, or to provide the body with oxygen and nutrition.</p><p>Thrombin is a trypsin like serine protease that plays an important role in this process since it is cleaving factors that eventually lead to coagulation of the blood and production of fibrin.</p><p>The aim of this project has been to synthesize fragments that have been designed with computer graphical methods by Astra Zeneca and then to analyze the fragments with affinity chromatography that has trypsin immobilized on silica particles. The project also introduces a new concept i.e. to analyze reaction mixtures on the trypsin column without first isolating and purifying the compounds.</p><p>The design fragments are synthesized by earlier reported standard methods. N, N'-dicyclohexylcarbodiimide (DCC) was used as coupling reagents to form amide or peptide bonds between carboxylic acids and amines.</p><p>In some of the reactions the amino group or the carboxylic groups of the amino acid were protected, to prevent these from interfering in the reaction and avoid to formation of unwanted substances. After the reactions the protecting groups were removed in different ways. If it is attached to the amine as a Boc-group (the most common protecting group to protect amino groups) it would be removed with trifluoracetic acid, and if the carboxyl group was protected, the protecting group would be removed with catalytical hydrogenolysis, for example by adding sodium hydroxide. To analyze if the right product has been acquired, analyzes with liquid chromatography-masspectrometry has been performed.</p><p>The results showed that two of the prepared fragments were retarded on the trypsin column meaning that they interact with trypsin i.e. hits were obtained.</p><p>The results showed that the two fragments that were analyzed with the trypsin column did retard a bit, which implies that they interact with trypsin. The synthesis seems to have been successful since the liquid-chromatography has shown that the right product has been made. This project has proven that it is possible to design fragments with computer graphical methods before synthesizing and analyzing with AFC-MS. AFC-MS has also been shown to be a suitable method for screening of weak binding fragments.</p>

The project has demonstrated the use of both poly(styrene-divinylbenzene) PS-DVB and silica monolithic columns for the separation of nitro-compounds. Methods were developed with PS-DVB and ODS silica packed columns for the separation of these compounds. The first part of the project was the preparation of the monolithic stationary phases prepared from PS-DVB of (250 μm I.D. × 70 mm) functionalized with methacrylate by in-situ polymerisation. The alkylated PS-DVB then was used successfully for the first time in the separation of three aryl nitro-compounds (2-NA, 1,4-DNB and 4-NT) on micro-HPLC. However, the efficiency of this column was poor N = 318 (4675/m).The second part of the thesis used a commercial column (Chromolith Performance from Merck), with different diameters for the separation of nitro-compounds. Nitrocompounds were analysed on both Chromolith Performance 3 mm I.D. column and Chromolith Performance 4 mm I.D. column by HPLC/UV. Van Deemter plots showed that the 3 mm I.D. column gave higher efficiencies at higher flow rates than the 4.6 mm I.D. column. The plate number was 8216 (H = 0.0121 mm) at a flow rate of 0.4 ml/min (1.0206 mm/sec) and for Chromolith 4.6 mm I.D. it was 9436 (H = 0.0105 mm) at a flow rate of 0.8 ml/min (0.8577 mm/sec). The nitro-compounds analysed in this study were nitroaromatic, nitramines and nitrate esters which are used in the manufacture of explosives. These compounds were analysed for the first time using a Chromolith Performance 3 mm I.D. column on LCMS using both ESI and APCI in negative ionization modes. The sensitivity was higher in the APCI than the ESI mode in terms of higher intensity and lower background noise especially for nitroaromatic compounds. The LC-ESI-MS method was evaluated by injection of samples of pentaerythritol tetranitrate (PETN) in different concentrations. Calibration curves were constructed over the range of 1-1000 pg/μl with a correlation coefficient of (R2 = 0.9986) and with a concentration range between 1-200 ng/μl with a correlation coefficient of (R2 = 0.9971) and were found to be linear. The limit of detection (LOT) for pentaerythritol tetranitrate (PETN) was 5 pg/μl at a signal-to-noise ratio (S/N) of 3:1 and the limit of quantification (LOQ) was 10 pg/μl at a signal-to-noise ratio of 10:1. The applicability of the monolithic column for the LC-ESI-MS method was evaluated by injection of samples of the commercial explosives, P9 and Semtex 1H. The results showed that Semtex 1H contains 35% PETN using calibration curve 1-200 ng/μl and was much higher than in P9 0.0082% using calibration curve 1-1000 pg/μl.