A subtribo Lychnophorinae ocorre na região do Cerrado do Brasil e contém cerca de 120 espécies. Recentemente, a filogenia da subtribo Lychnophorinae, baseada em sequências de DNA e dados morfológicos foi capaz de fornecer novas informações sobre a subtribo e seus gêneros. Além disso, o Cerrado brasileiro possivelmente abriga uma parcela considerável da entomofauna neotropical. Os objetivos gerais deste projeto de pesquisa são obter perfis metabólicos de plantas da subtribo Lychnophorinae e utilizá-los como ferramenta quimiotaxonômica para auxiliar na resolução da classificação taxonômica dessa subtribo e ainda, obter perfis metabólicos de insetos que se alimentem dessas plantas, visando a identificação de possíveis interações inseto-planta. Foram analisadas 78 espécies de plantas por GC-MS e UHPLC-UV(DAD)-MS(ESI-Orbitrap) nos modos positivo e negativo de ionização. As coletas de insetos foram feitas em intervalos trimestrais e, em seguida, esses insetos foram analisados utilizando a mesma metodologia analítica das plantas. As \"impressões digitais\" metabólicas das plantas e dos insetos foram precessadas no MetAlignTM e no MSClust, e as matrizes geradas foram submetidas a análises multivariadas no SIMCA. As plantas foram submetidas a análise de componentes principais (PCA), análise de cluster hierárquico (HCA) e análise discriminante ortogonal por mínimos quadrados parciais (OPLS-DA), entretanto os insetos, juntamente com suas plantas hospedeiras, foram analisados por PCA com o intuito de determinar a correlação entre seus metabólitos secundários. Os resultados das análises metabolômicas apresentaram proximidade com a filogenia principalmente para os dois maiors gêneros, Eremanthus e Lychnophora, analisados separadamente. Portanto, os resultados sugerem que os dados gerados a partir das análises metabolômicas podem ser utilizados em estudos quimiotaxonômicos da subtribo Lychnophorinae, sobretudo como dados primários para a reconstrução filogenética de gêneros. No que diz respeito às análises de possíveis interações inseto-planta, foi possível observar que alguns espécimens apresentaram correlação significativa com as plantas hospedeiras, evidenciando que a abordagem metabolômica pode ser utilizada como ferramenta na investigação de interações inseto-planta. Nestas amostras, pôde-se observar a presença de triterpenos, flavonoides e lactonas sesquiterpênicas adquiridas nas plantas por meio da herbivoria. / The subtribe Lychnophorinae occurs in the Cerrado domain of the Brazilian Central Plateau. The relationships among its recognized genera, as well as the relationships between Lychnophorinae and other subtribes belonging in tribe Vernonieae have been recently investigated upon a phylogeny based on molecular and morphological data. In addition, a preliminar overview of insect diversity in Brazilian Cerrado suggests that it may harbor a considerable fraction of the neotropical. We here report the use of a comprehensive untargeted metabolomics approach, combining LC-MS and GC-MS data together, followed by multivariate analyses aiming to assess the congruence between metabolomics data and the phylogenetic hypothesis, as well as its potential as a chemotaxonomic tool. Also we report the use of untargeted metabolomics approach aiming to assess insect-plant interactions. We analyzed 78 species by GC-MS and LC-MS in both positive and negative ionization modes. The metabolic profiles obtained for these species were treated in MetAlign and in MSClust and the matrices generated were combined and used in SIMCA for hierarchical cluster analyses (HCA), principal component analyses (PCA) and orthogonal partial least square discriminant analysis (OPLS-DA). The insects were collected quarterly and analyzed by the same analytical methods as plants. Results show that metabolomics analyses are mostly congruent with the phylogenetic hypothesis especially at lower taxonomic levels. Therefore, our results suggest that data generated by metabolomics approaches provide valuable evidence for chemotaxonomical studies of Lychnophorinae subtribe, in particular as primary data for phylogenetic reconstruction of lineages as genera. Regarding to insects, it was possible to observe significative correlations between some insects and their host plants. In these samples, were able to identify triterpenes, flavonoids and sesquiterpene lactones.

Rice is socially and economically important because it is the main component of the diet of more than half of the world s population. Brazil is one of the main rice producers in the world and Rio Grande do Sul state yields about 60% of the national production. This grain has been recently recognized by its functional characteristics; therefore, the demand for special cultivars, such as cateto rice, black rice and red rice, has increased lately, due to their complex nutritional composition and higher value, even though the number of consumers is still low. The quality of these grains must be monitored to guarantee food safety. A fundamental parameter of the quality is the investigation of pesticide residue. This study has developed and validated a method for pesticide multiresidue determination in different types of rice by Liquid Chromatography coupled with tandem Mass Spectrometry (LC-MS/MS) employing the acetate QuEChERS method in sample preparation. In the cleanup step, the type and the quantity of sorbent were optimized. Besides the sorbents which are frequently used, like PSA and C18, others that had never been tested so far, such as chitosan and diatomaceous earth, were also evaluated. After the optimization, 150 mg of C18 as sorbent was chosen. The efficiency and the reliability of the method were evaluated according to some parameters, such as linearity, limits of detection and quantification, accuracy and precision (repeatability and intermediate precision), matrix effect and robustness. Analytical curves showed adequate linearity (r2 ≥ 0.99) for all analytes. Recovery ranges were 54-112; 50-112; 48-116 and 51-122% for red, whole, cateto and white rice, respectively, with RSD lower than 20%. The limits of quantification ranged from 5 to 25 μg kg-1. The study of the matrix effect has shown low values (< 20%) for most of analytes. Supression was the predominant effect for 80% of the analytes. This effect was compensated by matrix-matched calibration. The method analytes has shown the robustness of the different types of rice with accuracy and precision. Therefore, the proposed method can be efficiently applied to determine pesticides in different types of rice because all validation parameters comply with the limits recommended for the validation of chromatographic methods. / O arroz tem grande importância social e econômica, pois é um dos componentes principais da alimentação de mais da metade da população mundial. O Brasil está entre os principais produtores mundiais de arroz, sendo que o estado do Rio Grande do Sul contribui com cerca de 60% da produção nacional. Atualmente, este cereal vem sendo reconhecido pelas suas características funcionais, neste sentindo, apesar de um grupo ainda restrito de consumidores, cresce a demanda por cultivares especiais, como arroz cateto, arroz negro e arroz vermelho, pois este tem uma composição nutricional bastante complexa e maior valor agregado. É de extrema importância para a segurança alimentar que seja monitorado a qualidade desses grãos. Um parâmetro determinante nessa qualidade é a investigação de resíduos de agrotóxicos. Nesse trabalho foi desenvolvido e validado um método multirresíduos para determinação de agrotóxicos em diferentes tipos de arroz por Cromatografia Líquida acoplada à Espectrometria de Massas em série (LC-MS/MS) empregando o método QuEChERS acetato no preparo de amostra. Na etapa de limpeza do extrato, foram otimizados o tipo e a quantidade de sorvente, sendo avaliados além dos sorventes geralmente empregados, como PSA e C18, outros materiais, até então nunca investigados para este fim, como a quitosana e terra diatomácea. Para esse estudo após otimização foi escolhido 150 mg de C18 como sorvente. A eficiência e confiabilidade do método foram avaliadas através de alguns parâmetros de validação como linearidade, limites de detecção e quantificação, exatidão e precisão (repetibilidade e precisão intermediária), efeito matriz e robustez. As curvas analíticas apresentaram linearidade (r2 ≥ 0,99) para todos analitos no solvente e para 80% dos analitos considerando a curva trabalho. As recuperações foram entre 54-112; 50-112; 48-116 e 51-122% para o arroz vermelho, integral, cateto e branco, respectivamente, com RSD menor que 20%. Os limites de quantificação do método foram na faixa de 5 a 25 μg kg-1. Em relação ao efeito matriz, o efeito foi baixo (< 20%) para maioria dos analitos, ocorrendo supressão para 80% dos analitos. Esse efeito foi compensado com calibração por superposição na matriz. O método apresentou robustez para a variação de matriz, uma vez que pode ser aplicado em diferentes tipos de arroz mantendo a exatidão e precisão. Portanto, concluiu-se que o método proposto pode ser aplicado de forma eficiente para a determinação de resíduos de agrotóxicos em diferentes tipos de arroz, uma vez que todos os parâmetros de validação encontram-se de acordo com os limites sugeridos para validação de métodos cromatográficos.

Les nucléotides, terme regroupant les nucléosides monophosphate, diphosphate et triphosphate, sont impliqués dans de nombreux processus cellulaires. Ils représentent les éléments constitutifs des acides nucléiques, fournissent de l'énergie à des réactions métaboliques, jouent le rôle de transporteurs et seconds messagers. L'exploration des pools nucléotidiques apparaît indispensable afin de connaître leur rôle précis dans des situations physiologiques ou pathologiques. Nous avons développé une méthode de dosage des nucléotides endogènes (formes mono-, di- et triphosphate) par extraction en ligne sur colonne WAX couplée à la LC-MS/MS. La séparation analytique est réalisée sur colonne Hypercarb, sans agent de paire d'ions dans la phase mobile. Grâce à l'utilisation d'un triple quadripôle et une ionisation en mode positif, les nucléotides endogènes sont identifiés sans équivoque, y compris ceux possédant la même masse molaire. L'extraction et la séparation des nucléotides sont réalisées en 20 min. L'ensemble de la méthode, en comptant la ré- équilibration des colonnes, dure 37 min. La méthode de dosage a été validée pour les formes mono- et triphosphate et est applicable à des séries d'une vingtaine d'échantillons biologiques. D'autre part, dans une étude pré-analytique basée sur les plans d'expériences, nous avons comparé les conditions de préparation d'échantillons en vue du dosage de nucléotides intracellulaires dans 4 lignées cellulaires : 2 adhérentes (Messa et NCI-H292) et 2 en suspension (RL et L1210). Nous avons montré que les conditions pré-analytiques optimales dépendent de la lignée cellulaire soulignant ainsi l'importance de cette phase dans l'analyse des nucléotides. Enfin, l'expérience et les connaissances acquises lors du développement de la méthode de dosage des nucléotides ainsi que la large palette de molécules analysables avec cette méthode (nucléosides, nucléotides sucrés, autres métabolites), nous ont permis de développer des collaborations dans le domaine de la cancérologie avec différentes équipes de recherche. Par exemple, nous avons étudié l'implication des pools nucléotidiques dans le stress réplicatif induit par le stress oxydant et dans la reprogrammation cellulaire observée dans les cellules cancéreuses. Ainsi, les informations apportées par notre approche analytique, complémentaires des autres approches utilisées, ont montré l'implication des pools nucléotidiques dans la tumorigénèse. En conclusion, ce travail a permis de développer une technique analytique et de mettre en place une méthode de travail pour le dosage des nucléotides endogènes dans différents milieux biologiques / Nucleotides, term including nucleoside mono-, di- and triphosphates, are endogenous compounds playing various roles in biology. They are components of nucleic acids, provide energy to metabolic reactions and act as carriers or second messenger. The study of endogenous nucleotides has become of great interest in physiological and pathological conditions. We developed a method for the quantification of endogenous nucleotides, using an on-line extraction on a WAX column coupled with LC-MS/MS. Analytical separation is performed on a Hypercarb column, without ion pairing agent in the mobile phase. The use of a triple quadrupole mass spectrometer following positive mode ionization allows the unambiguous identification of nucleotides presenting the same mass. Extraction and separation of nucleotides are achieved within 20 min and the method including re-equilibration of the two columns within 37 min. The method was validated for the quantification of nucleoside mono- and triphosphates, and could be applied to series of more than twenty biological samples. Secondly, in a study based on design of experiments, pre-analytical parameters influencing results of intracellular nucleotides were compared in four cell lines. We demonstrated that optimal pre-analytical parameters depend on cell lines. This clearly highlights the importance of pre- analytical conditions for the quantification of intracellular nucleotides to be as representative as possible of the real levels in cells. Then, thanks to experience acquired during the development and the validation of the analytical method, scientific collaborations have been established with several cancer research teams. For example, implication of nucleotide metabolism in replicative stress induced by oxidative stress or in the metabolic reprogramming in cancer cells was studied. Results obtained by our analytical approach were complementary to those obtained by other techniques. To conclude, our work consisted on the study of the entire workflow for the analysis of endogenous nucleotides in various biological samples

De nombreux produits chimiques se retrouvent dispersés dans l'environnement avec des conséquences parfois néfastes pour les hommes et les écosystèmes. Parmi ces substances figurent les antibiotiques et les stéroïdes hormonaux. Peu de données sont disponibles quant à la présence et le devenir de ces substances dans l'environnement notamment pour le sol, par manque de méthodologies. Nous avons donc mis au point des procédures d'analyse de traces de ces contaminants émergents, dans l'eau et le sol. Nous avons développé des méthodes multi-résidus et inter-familles basées sur des analyses LCMS/ MS. Pour les échantillons aqueux, l'extraction est menée sur phase solide (SPE, OASIS HLB). Les MLQs sont comprises entre 0.09 et 34 ng/L. Pour la matrice solide, la procédure d'extraction est inspirée de la méthode appelée QuEChERS suivie d'une purification SPE. Elle a été validée et des MLQs entre 0.013 et 3 ng/g ont été atteintes. Les tétracyclines et les fluoroquinolones, ont été étudiées séparément car elles ont des propriétés physico-chimiques bien spécifiques les rendant difficiles à extraire correctement du sol avec une méthode inter-familles. La méthode développée sur la matrice sol a permis une étude statistique mettant en exergue l'impact de certains paramètres du sol sur les rendements d'extraction et les effets matrice. Les méthodes ont été appliquées à une étude en colonnes de sol pour obtenir des données sur le transfert, l'accumulation et la dégradation des composés dans le sol / Several chemical products are dispersed in the environment and the consequences can be sometimes harmful for humans and the ecosystems. Among these substances appear the antibiotics and the hormonal steroids. Nowadays, only few data are available on the presence and the fate of these substances in the environment in particular for solid matrices, mainly due to a lack of methodologies. Consequently, methods to analyze traces of « emergent » contaminants in water and soil were carried out. Therefore, multi-residues and inter-families procedures based on LC-MS/MS analysis were established. Concerning the aqueous samples, 23 analytes are extracted with the SPE technique (OASIS HLB). MLQs are between 0.09 and 34 ng/L. For the solid matrix, the extraction procedure of 31 compounds is inspired from the method called QuEChERS and followed by a purification step. This methodology was validated and MLQs between 0.013 and 3 ng/g were obtained. Two antibiotics families, tetracyclines and fluoroquinolones, were studied separately as they have specific physical/chemical properties and are therefore difficult to extract from soil with an inter-families method. Then, the method developed for the soil matrix allowed a statistic study which showed the impact of the soil parameters on the recoveries and matrix effects. Finally, the methodologies were applied to a soil column study which allowed obtaining data on the transfer, accumulation and degradation of the substances in soil

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