A Esclerose Lateral Amiotrófica (ELA) é uma doença crônica, progressiva e neurodegenerativa causada pela morte dos neurônios motores. O diagnóstico destes pacientes pode levar até 12 meses para acontecer, sendo que estes vão à óbito entre 3-5 anos do início dos sintomas. Há, porém, grande variabilidade de quadro clínico, com alguns pacientes falecendo com menos de 1 ano do início dos primeiros sinais, e outros que sobrevivem por décadas. A identificação da ELA8, causada por uma mutação missense no gene VAPB (c.C166T, p.P56S), tem contribuído significativamente com o conhecimento dos mecanismos moleculares por trás da ELA. A literatura recente tem evidenciado que a diminuição dos níveis de VAPB está presente em modelos celulares e murinos da doença, e também em amostras de pacientes, sugerindo que esta proteína teria papel central na doença e uma contribuição significativa para a morte dos neurônios motores. O presente trabalho buscou três objetivos principais: (1) o diagnóstico molecular através de um painel de sequenciamento de nova geração que inclui os genes SOD1, FUS, TARDBP, SETX, SPG11, FIG4 e VAPB; (2) a avaliação dos níveis de RNAm de VAPA, VAPB e EPHA4 em pacientes de ELA8, controles familiares e outros pacientes de ELA, com o intuito de investigar possíveis papéis destes genes na doença; e por fim, (3) o desenvolvimento de um ensaio quantitativo para as proteínas VAPA, VAPB e VAPC baseado em cromatografia líquida acoplada à espectrometria de massas em tandem (LC-MS/MS), para a posterior avaliação de VAPB como possível biomarcador em ELA, e de suas isoformas VAPA e VAPC como modificadores da doença. Para a análise genômica, foram avaliados 67 pacientes, sendo que 31 (ou 46%) apresentaram a mutação c.C166T em VAPB; 4 pacientes (6%) em SOD1, sendo que um destes apresentou uma mutação também em FIG4; 1 paciente (1.5%) foi identificado uma mutação patogênica em FUS; outro, duas mutações deletérias em trans em SPG11. Os níveis de RNAm de VAPB, VAPA e EPHA4 não são estatisticamente distintos entre pacientes e controles; porém, os níveis de EPHA4 estavam significativamente elevados em dois pacientes de início bulbar da doença. Para o desenvolvimento do método quantitativo por LC-MS/MS, foram escolhidos 8 peptídeos inequívocos para análise, estabelecidos dos parâmetros de corrida, e desenvolvidos dois padrões internos (linhagens SILAC e VAPB recombinante) para a quantificação. Esta ferramenta desenvolvida poderá auxiliar não apenas os estudos moleculares que envolvem os mecanismos por trás ELA8, responsável por uma elevada taxa dos casos familiais brasileiros, mas também poderá determinar o potencial de VAPB como biomarcador para Esclerose Lateral Amiotrófica / Amyotrophic Lateral Sclerosis is a chronic, progressive neurodegenerative disorder caused by the death of motor neurons. Diagnosis can take up to 12 months, with no molecular marker to expedite this process. In this scenario, patients die within 3 to 5 years of symptom onset, although a large clinical variability is seen, with severe patients dying less than one year after onset, and others surviving for decades. The identification of ALS8, caused by a missense mutation in the VAPB gene (c.C166T; p.P56S), has contributed significantly to the knowledge of molecular mechanisms behind ALS. Recent literature has evidenced that the decrease of VAPB levels is present in cellular and murine models, and also in patient samples, suggesting a central role in motor neuron death in ALS. The present work sought three main objectives: (1) a molecular diagnosis through a NGS sequencing panel including the SOD1, FUS, TARDBP, SETX, SPG11, FIG4 and VAPB genes; (2) analyze the expression levels of VAPA, VAPB and EPHA4 in patients, family controls and other forms of ALS, in order to investigate their possible roles in ALS8; and (3) the development of a targeted quantitative mass spectrometry based assay, gold standard in protein quantification due to its precision and sensitivity, for the VAPA, VAPB and VAPC proteins, seeking the analysis of VAPB as a potential biomarker in ALS and of its isoform\'s potential roles as modifiers in the disease. The genomic analyses revealed that out of 67 patients, 31 presented the ALS8 mutation in VAPB, 4 patients (6%) presented a mutation in SOD1, with one patient carrying a second mutation in FIG4; 1 (1.5%) patient was identified with a pathogenic mutation in FUS; and another presented two pathogenic mutations in trans in the SPG11 gene. Thus, we were able to diagnose over half of the patients included in this study with a panel of only 7 genes. VAPB, VAPA and EPHA4 mRNA levels are not statistically different between patients and controls; however, EPHA4 was shown to be highly elevated in two bulbar-onset non-ALS8 patients. For the development of the LC-MS/MS targeted assay, 8 surrogate peptides were chosen for analysis, run parameters were established, and two internal standards for quantification were developed (SILAC cell lines and recombinant VAPB). This tool will prove to be useful not only towards elucidating the molecular mechanisms behind ALS8, one of the most prevalent forms of familial ALS in Brazil, but also to determine VAPB\'s potential as a biomarker for ALS

Cette thèse initie et développe un programme ayant pour but de définir une stratégie de priorisation efficace pour accélérer la découverte de molécules bioactives au sein d’extraits végétaux. Dans le cadre de ce projet, plusieurs criblages biologiques ont été menés sur une collection de 292 extraits d'Euphorbiaceae.Afin d’identifier et cibler au sein de ces mélanges complexes les structures d’intérêt biologique tout en écartant les molécules connues ou présentant un intérêt structural limité, les profils métabolomiques des extraits ont été acquis par spectrométrie de masse tandem. Pour exploiter au mieux la quantité d'information générée par ces analyses, les spectres MS2 ont ensuite été organisés sous forme de réseaux moléculaires. Ces réseaux permettent de lier les ions détectés en fonction de la similarité de leurs voies de fragmentation et donc de leur proximité structurale. Les informations taxonomiques et d’activités biologiques ont été croisées avec les données spectrales au sein de cette carte moléculaire multi-informative, offrant ainsi une approche nouvelle pour accélérer la découverte de nouveaux ligands des cibles biologiques étudiées et pour une sélection plus pertinente des extraits à forte diversité structurale.Si l’outil des réseaux moléculaires représente une méthode innovante et particulièrement instructive pour le phytochimiste, il présente cependant quelques défauts qui limitent son spectre d'utilisation et ses capacités en métabolomique. Une deuxième partie de cette thèse a donc été consacrée à l’implémentation d’une étape de prétraitement pour améliorer la fiabilité des réseaux et au développement de MetGem, un logiciel dédié à la génération de réseaux moléculaires permettant d’optimiser la gestion et l’analyse des matrices de scores de similarité spectrale. / This thesis initiates and develops a program seeking to accelerate the discovery of new therapeutic molecules using an efficient prioritization strategy. As part of this project, a collection of 292 Euphorbiaceae extracts was screened over several biological targets.To focus on unknown bioactive chemicals and to avoid the isolation of known or inactive molecules, the acquisition of high resolution tandem mass spectrometry profiles of these extracts was performed. To highlight relevant information within these data, MS2 spectra were organized as molecular networks. It consists in visualizing tandem mass spectrometry data by detecting related MS2 spectra and representing them in a same spectral space. Taxonomical details and bioassay screening results were merged with the network visualization to generate a comprehensive multi-informative molecular map, which offers a radically novel outlook to target novel bioactive scaffolds and select extracts with high structural diversity. Although very instructive for the phytochemist, the molecular networking tool has some imperfections that limit its potential in metabolomics. Therefore, the second part of this thesis was dedicated to the introduction of a data preprocessing step to enhance the networks reliability and to the development of MetGem, a software dedicated to the generation of molecular networks to improve the way matrices of similarity scores are managed and analyzed.

Une importante proportion de formes atypiques de parkinsonismes a été rapportée en Guadeloupe en 1999. Il ressort des études épidémiologiques menées sur place, que tous les patients atteints étaient de grands consommateurs de produits alimentaires et médicinaux de la famille des Annonaceae, et plus particulièrement du genre Annona. De nombreux genres appartenant à cette famille renferment des molécules fortement cytotoxiques : les acétogénines d’Annonaceae. Leur présence dans les fruits d’Annona muricata a déjà été mise en évidence. Cependant, peu de données existent sur leur teneur en acétogénines dans ces fruits ou dans ceux d’espèces proches. Par ailleurs, bien que l’annonacine, acétogénine principale d’A. muricata soit neurotoxique in vivo, aucune donnée quantitative de son accès au cerveau n’est disponible. Nous nous sommes donc attachés au cours de ce travail, à développer des méthodes de dosage de l’annonacine par LC-MS/MS dans le plasma et le cerveau de Rat pour déterminer sa biodisponibilité et la fraction à tropisme cérébral, dans le but de comprendre pourquoi des molécules si fortement cytotoxiques n’entraînaient pas d’intoxication aigue lors de l’exposition alimentaire. Nous avons par ailleurs développé une méthode de quantification des acétogénines totales par 1H RMN dans des extraits bruts de fruits, appliquée à des lots d’A. muricata d’origines variées. Une méthode par LC-MS/MS a également été développée pour une description plus approfondie des acétogénines présentes dans des extraits bruts de fruits, appliquée à différents lots d’A. squamosa. Les fruits d’A. reticulata et d’A. glabra ont également fait l’objet d’investigations. Ces deux approches combinées ont contribué à améliorer l’estimation de l’exposition aux acétogénines dans le cadre de l’alimentation. / Abstract : A high proportion of atypical parkinsonisms was reported in French West Indies in 1999. Epidemiological studies pointed out an association with the consumption of fruits and medicinal herbs from Annonaceae of the Annona genera. Numerous Annonaceae members contain Annonaceous acetogenins (AAGs), which are highly cytotoxic molecules. They were found in the pulp fruit of Annona muricata. However, scarce only quantitative exist for this fruit and those of related species. Moreover, although annonacin, the major AAG of A. muricata proved neurotoxic in vivo, no quantitative data is available towards its distribution to the brain. We therefore developed a method for annonacin quantitation in Rat plasma and brain homogenate, in order to determine its bioavailability and the fraction reaching the brain, to understand why those highly cytotoxic molecules are not responsible for acute toxicity when fruits are ingested. We then developed a quantitation method for global estimation of AAGs in crude fruit extracts by 1H NMR, which we applied to the fruit pulp of A. muricata batches from diverse locations. An LC-MS/MS method was also developed for the qualitative study of AAGs. It was applied to different batches from A. squamosa fruits. The species A. reticulata and A. glabra were also examined. Those two approaches contributed in a better estimation of AAGs exposure by fruit consumption.

Utomhusprodukter gjorda i plast är sårbara för nedbrytning på grund av UV-energi från solljus. För att stoppa denna fotonedbrytning används UV-stabilisatorer i plastmaterial; en av huvudkategorierna är hindrade aminljusstabilisatorer (HALS, hindered amine light stabilizers). Detta projekt ämnade att utveckla analytiska metoder passande för polymera HALS-föreningar som används i polyolefin-produkter. Huvudfokuset låg på vätskekromatografi med antingen UV eller masspektrometri (MS) detektion. Identifiering lyckades till viss del, extraktion misslyckades, och det gjordes inga kvantifieringsförsök. Detta examensarbete utfördes som ett samarbete mellan KTH och företaget Norner AS. På grund av detta har detaljerna såsom vilka HALS som användes, hur sammansättningen av elueringsgradienter förändrades och masspektrum från MS analys tagits bort eller ändrats till generiska termer. Högpresterande vätskekromatografi (HPLC, high-performance liquid chromatography) med metoden normalfas (normal phase) ansågs vara ett otillfredsställande alternativ på grund av begränsningen av lämpliga lösningsmedel. Både för HPLC och LC-MS-analys användes omvänd fas (reverse phase) med en C18 50 mm kolonn. Gällande HPLC-analys visade sig en våglängd på 240 nm för UV-detektion vara lämplig. För MS-detektion användes positiv elektrosprayjonisering på grund av föreningarnas basiska pKa-värden. Elueringsgradienten med de mest tillfredsställande resultaten var gradientversion 5, med vatten som lösningsmedel A och metanol som lösningsmedel B. En variant av denna gradient användes för LC-MS; gradientversion 5 mod följer samma mönster men med vatten + Y vol% syra som lösningsmedel A, för att möjliggöra jonisering. Additiv UV 2 har låg löslighet i polära lösningsmedel, men genom förtvålning kunde additiv UV 2 lösas i metanol. Med gradientversion 5 mod har förtvålad additiv UV 2 en retentionstid på 1,2 minuter i både HPLC och LC-MS-analys. För HPLC-analys av additiv UV 3 användes gradientversion 5 som resulterade med en retentionstid på 16,8 minuter, och för LC-MS med gradientversion 5 mod 16 minuter. Analys av additiv UV 5 med HPLC gav en retentionstid på 2 minuter med gradientversion 5, och för LC-MS-analys med gradientversion 5 mod var retentionstiden 1,3 minuter. På grund av inkonsekventa resultat mellan HPLC-analyser eller mellan HPLC och LC-MS-resultat kunde inte retentionstid för identifiering av additiven UV 1, UV 4 och UV 6 bestämmas. Extraktion av polymerprover med olika HALS gjordes i toluen vid 80 °C under en timme. I HPLC var det ingen skillnad i kromatogrammen mellan de olika HALS-produkterna. Resultaten från LC-MS stödde resultaten från HPLC, så slutsatsen att extraktionsmetoden inte var framgångsrik drogs. En anledning till detta kan vara att detektionsgränsen i MS och UV överstiger den extraherade halten av HALS. / Outdoor plastic products are vulnerable to degradation due to UV energy from sunlight. To hinder this photodegradation UV stabilizers are used in plastic materials; one of the main categories is hindered amine light stabilizers (HALS). This project aimed to develop analytical methods suitable for polymeric HALS used in polyolefin products. Liquid chromatography was the primary analytical method, with either UV or mass spectrometry (MS) detection. Identification of HALS was partially successful, the attempted extraction was unsuccessful, and there was no attempt at quantification. This thesis was performed as a collaboration between KTH and the company Norner AS. Due to this, the details such as which HALS were used, how the composition of elution gradients changed, and the mass spectrum from MS analysis have been removed or changed to generic terms. The high-performance liquid chromatography (HPLC) method normal phase was deemed as an unsatisfactory option due to the limitation of appropriate solvents. For HPLC and LC-MS analysis reversed phase with a C18 50 mm column was used. In HPLC analysis, a wavelength of 240 nm for UV detection was found to be suitable. For MS detection, positive electrospray ionization was used due to the compounds' basic pKa values. The elution gradient with the most satisfactory results was gradient version 5, with water as solvent A and methanol as solvent B. A variant of this gradient was used for LC-MS; gradient version 5 mod follows the same pattern but with water + Y vol% acid as solvent A, to enable ionization. UV 2 has low solubility in polar solvents, but through saponification UV 2 could be dissolved in methanol. With gradient version 5 mod, saponified UV 2 has a retention time of 1.2 minutes in both HPLC and LC-MS analysis. For HPLC analysis of additive UV 3, gradient version 5 was used which resulted in a retention time of 16.8 minutes, and for LC-MS with gradient version 5 mod 16 minutes. Analysis of UV 5 with HPLC gave a retention time of 2 minutes with gradient version 5, and for LC-MS analysis with gradient version 5 mod the retention time was 1.3 minutes. Due to inconsistency in results between HPLC analysis or between HPLC and LC-MS results, retention time for identification of additives UV 1, UV 4, and UV 6 could not be stated.  Extraction of polymer samples with different HALS was done in toluene at 80 ॰C for one hour. In HPLC, there was no difference in the chromatograms between the different HALS compounds. The results from LC-MS supported the results from HPLC, thus it was concluded that the extraction method was not successful. A reason for this could be that the detection limit in MS and UV might exceed the extracted amounts of the compounds.

Le début du 21e siècle a vu naître un intérêt croissant pour la problématique des résidus de médicaments dans les milieux aquatiques. L’apparition de nouvelles technologies analytiques permettant la détection et la quantification de molécules à l’état d’ultra-traces a fortement contribué à cette expansion. Ces améliorations technologiques ont permis la recherche et la quantification de nombreux xénobiotiques dans les différents compartiments aquatiques. Depuis une quinzaine d’années, l’attention des équipes de recherche s’est ainsi portée sur les résidus de médicaments à usage humain ou vétérinaire. La faune et la flore aquatiques sont ainsi exposées à des mélanges de composés certes à très faibles doses (souvent de l’ordre de quelques ng à quelques centaines de ng) mais en continu et les effets de cette exposition sont très peu documentés. Ces contaminants font partie des polluants émergents et il n’existe actuellement pasde réglementation en Europe concernant leur présence dans l’environnement. La contamination des milieux aquatiques par les résidus de médicaments à usage humain est souvent le signe d’une urbanisation importante et les cours d’eau soumis à de fortes pressions domestiques sont des sitesd’étude privilégiés. Néanmoins, les données concernant les milieux ruraux sont encoreparcellaires bien qu’ils représentent les quatre cinquièmes du territoire français. Les travaux de cette thèse ont pour objectif premier d’établir un état des lieux des ressources en eau d’un territoire à dominante rurale, le département de la Dordogne. Pour répondre à cette problématique, la recherche de résidus de médicaments s'est orientée sur une liste de 40 molécules comprenant des hypolipidémiants, des antibiotiques, des béta-bloquants, des anti-inflammatoires non stéroïdiens, des anticancéreux, etc. Une méthode chromatographique multi-résidus (pré-concentration en ligne - LC-MS/MS) a été développée et validée pour les eaux de surface. En accord avec la Directive européenne Cadre sur l'Eau (DCE), des sites ont été échantillonnés tous les mois pendant un an (2011) sur six cours d'eau majeurs du département de la Dordogne de manière à déterminer leur état écologique (physico-chimique et biologique).Parallèlement, la caractérisation des résidus de médicaments a été réalisée sur ces mêmes échantillons. Les différentes campagnes de mesure ont permis d'identifier les principales zones impactées par les composés pharmaceutiques et ont également montré des corrélations linéaires avec d'autres paramètres (ions ammonium, phosphore, etc.).Suite aux premiers résultats obtenus lors de l'état des lieux du département de laDordogne, une étude complémentaire focalisée sur la présence des résidus de médicaments dansl'Isle a été menée en parallèle. Elle a permis de caractériser les voies d’introduction des résidus de médicaments et mieux comprendre l’impact de la ville de Périgueux sur l’Isle. Ainsi, un des objectifs a été de caractériser la présence de composés pharmaceutiques dans cette rivière et de comprendre l’origine de ces molécules par l’identification des effluents domestiques des stationsd’épuration présentes dans la zone urbaine. / The beginning of the 21th century saw a growing interest for pharmaceuticals in aquaticsystems. The development of new analytical technologies allowing detection and quantification ofcompounds at ultra-trace levels mostly contributed to this expansion. These technologicalimprovements allowed to evidence and quantify many xenobiotics in various aquaticcompartments. Since about fifteen years, research teams focused their attention to human orveterinary pharmaceuticals. These contaminants are among emerging pollutants and no regulationexists in Europe concerning their presence in the environment. The presence of few tenths ofpharmaceuticals has been demonstrated. Living organisms in aquatic bodies are thereforeexposed to mixtures of compounds certainly at low doses (often between few ng and fewhundreds of ng) but continuously and the effects of this exposure are few documented. Thecontamination of aquatic systems by pharmaceuticals is usually the sign of an importanturbanization and streams with strong domestic pressures are often chosen as sampling sites inscientific studies. Nevertheless, the data concerning rural areas are still scarce although theyrepresent four fifth of the French territory.This work aims to establish a current situation of water resources of a rural territory, theDordogne administrative department. The occurrence of 40 pharmaceuticals including lipidregulators, antibiotics, β-blockers, non-steroidal anti-inflammatories, anticancer drugs, etc inseveral streams has studied with a multi-residue analytical method involving an on-linepreconcentration, a chromatographic separation followed by tandem MS/MS detection. Themethod has been validated for surface waters according to COFRAC requirements.Many sites were sampled every month during one year (2011) on six major streams of theDordogne department. The various campaigns allowed identifying the main areas contaminatedby pharmaceuticals and some linear correlations with other parameters (ammonium ion,phosphorus, etc.) have been evidenced.Beside the results obtained over all the Dordogne department, a focus on the presence ofpharmaceuticals in Isle river, around Périgueux, the main urban area of Dordogne, was led. Thissurvey allowed characterizing the introduction ways of pharmaceuticals and better understandingthe impact of the city of Périgueux on the river Isle.

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

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.