Les hydrocarbures aromatiques monocycliques (BTEX) et polycycliques (HAP) sont pour la plupart reconnus pour leur toxicité via ingestion. Le suivi de leur contenu dans les matrices alimentaires est donc indispensable. Plusieurs approches sensibles et complémentaires ont été établies, avec la pomme comme modèle d’étude, pour la double détermination de ces hydrocarbures aromatiques volatils et semi-volatils au niveau du µg/kg.Une micro-extraction en phase solide dans l’espace de tête (HS-SPME) a été utilisée pour l'extraction des hydrocarbures aromatiques les plus légers (MM entre 78 et 178 g/mol). La récupération des composés les plus lourds (MM entre 202 et 278 g/mol) a quant à elle été permise par une méthodologie basée sur l'extraction assistée par les ultrasons (UAE) suivie d'une extraction en phase solide (SPE). Une alternative plus sensible à cette stratégie combinant l’HS-SPME et l’UAE/SPE pour la détermination globale des hydrocarbures aromatiques a également été développée. Le remplacement de l’étape de SPE par une micro-extraction sur solide compacté (MEPS) a permis une récupération plus rapide, sensible et plus large des HAP (MM entre 152 et 278 g/mol).L’ensemble des stratégies d'extraction, associées à des analyses en chromatographie gazeuse couplée à la spectrométrie de masse, a ensuite pu être appliqué à la détermination des 20 composés aromatiques ciblés dans des pommes récoltées en Normandie ou des fruits provenant du commerce contaminés ou non par des gaz d’échappement ou des fumées de combustion de biomasse. Ces applications ont révélé la faible contamination des pommes normandes étudiées, l’adsorption prédominante des composés aromatiques au niveau de la peau des fruits et leur faible transfert vers la chair. / Monocyclic aromatic hydrocarbons (BTEX) and polycyclic aromatic hydrocarbons (PAHs) are considered and recognized as toxic compounds via ingestion. Their monitoring in food product is thus a significant concern. Sensitive and complementary experimental approaches were investigated with apple as model for the dual determination of volatile and semi-volatile aromatic hydrocarbons at the ppb level.A solid-phase micro-extraction in the headspace of samples (HS-SPME) was used for the most volatile aromatic hydrocarbons (MW between 78 and 178 g/mol). The recovery of the least volatile aromatic hydrocarbons (MW between 202 and 278 g/mol) was implemented with an ultrasound assisted extraction (UAE) followed by a purification step using a solid phase extraction (SPE). The methodology consisting in HS-SPME and UAE/SPE enabled the global determination of BTEX and PAHs. The SPE step was then substituted by a micro-extraction with a packed sorbent (MEPS) to elaborate a most sensitive and rapid methodology for the quantification of a wider range of PAHs (MW between 152 and 278 g/mol).Extraction procedures associated with analyses using gas chromatography and mass spectrometry were then applied to the determination of 20 targeted aromatic compounds in apples harvested in Normandy or in commercial fruits exposed or not exposed to exhaust gas or to wood smoke. These applications revealed low levels of contaminants in the studied apples from Normandy. A predominant adsorption of aromatic compounds occurs on the skin of fruits and the transfer to the pulp is almost inexistent.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Polychlorinated biphenyls (PCBs) are toxic, non-biodegradable, and lipophilic chemical compounds of industrial origin. In Brazil, PCBs are still used in old electrical transformers and capacitors which were installed up to 1981 when their use and commercialization were banned. However, the permission to use devices with PCBs installed up to the early 80s was maintained up to the moment that their dielectric fluids or the entire device be fully replaced for products free of PCBs. Currently, PCBs are classified as persistent organic pollutants (POPs) because they pollute and accumulate in the environment affecting all organisms in the food chain. The main form of human intake is the ingestion of contaminated food, especially food of animal origin. Fish are the main means of transmission of PCB congeners to the human body. The concern about PCBs is centered on their toxicological potential, especially as inducers of cancer. Thus, the aim of this study was to investigate the possible contamination by residues of 11 individual PCB congeners in fresh marine fish from the state of Rio Grande do Sul (RS), Brazil. The methodology for the analysis of PCBs in fish included sampling on the coast of RS, the extraction and purification of these compounds by the use of acidified silica gel and solvents, as well as their identification and quantification using gas chromatography coupled to a micro electron capture detector (GC - μECD). Results were confirmed by gas chromatography coupled to mass spectrometry (GC - MS). PCBs investigated were not found in all samples. PCBs 28 and 52 were not detected. PCBs 153, 138, 180, 118, 101, 77, and 81 were found in 98.33 %, 95%, 93.33%, 60%, 33.33 %, and 31.67 % of the samples, respectively. PCBs 126 and 169 were found at lower frequencies, 18.33% and 15%, respectively. The individual congeners found in higher mean concentrations were PCB 153 (184.36 ng/g fat), followed by PCB 138 (159.27 ng/g fat), PCB 118 (98.16 ng/g fat), PCB 180 (69.04 ng/g fat), PCB 101 (32.99 ng/g fat), and PCB 77 (12.95 ng/g fat). PCBs 81, 126 and 169 were found in lower concentrations (4.97 ng/g, 4.49 ng/g and 2.08 ng/g fat, respectively). Results confirm the contamination by PCBs of all marine species of consumption analyzed and can be used as indicators of environmental contamination by these compounds in the coast of RS. / Bifenilos policlorados (PCBs) são compostos químicos de origem industrial, tóxicos, não biodegradáveis, lipofílicos, e que foram produzidos a partir da década de 30 até os anos 70, quando foram proibidos. No Brasil, PCBs estão presentes, por exemplo, em transformadores e capacitores elétricos instalados até 1981, quando a comercialização e utilização desses compostos foram proibidas no país. Porém, manteve-se a permissão de utilização desses equipamentos contendo PCBs e instalados até o início dos anos 80, até que ocorra a substituição integral dos mesmos ou a troca de seus respectivos fluidos dielétricos por produtos isentos de PCBs. Atualmente, PCBs são classificados como poluentes orgânicos persistentes (POPs), pois poluem e se acumulam no meio ambiente, influenciando todos os organismos da cadeia alimentar. A principal forma de contaminação humana é a ingestão de alimentos contaminados, principalmente alimentos de origem animal. Dentre esses, os pescados constituem-se no principal meio de veiculação de congêneres de PCBs até o organismo humano. A preocupação acerca dos PCBs está centrada no seu potencial toxicológico, principalmente como indutores de câncer. Diante disso, o objetivo desse trabalho foi investigar a possível contaminação, por resíduos de 11 congêneres individuais de PCBs, em pescados marinhos in natura do estado do Rio Grande do Sul, Brasil. A metodologia empregada para análise de PCBs nos pescados incluiu a coleta de amostras do litoral do RS, a extração e purificação desses compostos com a utilização de sílica gel acidificada, solventes e, finalmente, a identificação e quantificação dos mesmos utilizando-se cromatografia gasosa acoplada a um micro detector de captura de elétrons (GC-μECD). Os resultados obtidos foram confirmados por cromatógrafo gasoso acoplado à espectrometria de massas (GC-MS). Os PCBs investigados não foram encontrados em todas as amostras analisadas. Os PCBs 28 e 52 não foram detectados. O PCB 153 foi encontrado em 98,33% das amostras analisadas, seguido pelos PCBs 138 e 180 encontrados em 95% das amostras, pelo PCB 118 encontrado em 93,33% das amostras, pelo PCB 101 encontrado em 60% das amostras, pelo PCB 77 e PCB 81, encontrados em 33,33% e 31, 67% das amostras, respectivamente. Os PCBs 126 e 169 foram encontrados com menores freqüências, 18,33% e 15%, respectivamente. Os congêneres individuais encontrados em maiores concentrações médias foram o PCB 153 (184,36 ng/g de gordura), seguido pelo PCB 138 (159,27 ng/g de gordura), PCB 118 (98,16 ng/g de gordura), PCB 180 (69,04 ng/g de gordura), PCB 101 (32,99 ng/g de gordura) e PCB 77 (12,95 ng/g de gordura). Os PCBs 81, 126 e 169 foram encontrados nas menores concentrações (4,97 ng/g, 4,49 ng/g e 2,08 ng/g de gordura, respectivamente). Os resultados obtidos confirmam a contaminação por PCBs de todas as espécies marinhas de consumo analisadas e podem ser utilizados como indicativos da contaminação ambiental, por esses compostos, no litoral do RS.

O consumo dos diferentes tipos de drogas está associado a problemas sociais, econômicos e de saúde pública, em todas as regiões no mundo. Dentre os problemas de saúde pública, podemos destacar a alta prevalência de comorbidade entre o uso de drogas e os transtornos mentais. A Cannabis, a cocaína e o crack são as drogas ilícitas mais consumidas no Brasil. A utilização do cabelo como matriz biológica para determinação destas drogas permite avaliar o uso crônicos pelos indivíduos, uma vez que o cabelo é uma matriz estável, de fácil manipulação e a janela de detecção depende apenas do comprimento do cabelo. Entretanto, a análise em cabelo ainda representa um desafio analítico. Foram desenvolvidos dois métodos para a detecção das diferentes drogas no cabelo, com diferentes enfoques analíticos. O primeiro método (desenvolvido durante o estágio de doutorado sanduíche na Itália) identificou e quantificou cocaína e metabólitos usando a técnica de column switching e detecção por LC-MS/MS. O segundo método foi desenvolvido para determinação de canabinóides nas amostras de cabelo utilizando GC-MS. O diferencial deste método foi a utilização de um novo dispositivo de extração em fase sólida (as ponteiras DPX) para concentração e purificação do extrato, utilizando menor quantidade de solventes. A determinação dos canabinóides e cocaínicos foi realizada nas amostras de cabelo da população atendida no CAPS - AD de Ribeirão Preto, São Paulo. Além da coleta da amostra de cabelo, o sujeito foi submetido a uma entrevista, onde os seguintes instrumentos de avaliação foram aplicadas: Questionário sobre a saúde do Paciente 9, Inventário de fobia social, Self Report Questionnaire, Questionário de Ansiedade de Beck, Inventário de Depressão de Beck e Questionário sobre o uso da Cannabis, bem como um questionário elaborado pelo pesquisador para coleta de dados sociodemográficos, consumo de substâncias e dados sobre a amostra de cabelo, como comprimento, cor, tintura ou coloração. As amostras de cabelo foram analisadas e a média das concentrações de cada droga encontrada no cabelo foi correlacionada com os indicadores clínicos de transtorno mentais, obtidos através dos instrumentos de avaliação psiquiátrica. A maior prevalência de indicadores clínicos positivos para transtornos psiquiátricos entre a população estudada foi de transtornos mentais comuns, entre eles a ansiedade e depressão. A comparação da média de concentração de Cannabis, cocaína e crack no cabelo com os indicadores clínicos positivos para os transtornos não apresentou resultados estatisticamente significantes. Entretanto, podemos inferir que os sujeitos que apresentaram maior concentração média de Cannabis e cocaína no cabelo possuíam mais indicadores clínicos positivos para sintomas mentais comuns e depressão maior enquanto que os sujeitos usuários de crack possuíam mais indicadores clínicos positivos para sintomas ansiedade. Apesar de algumas limitações, podemos concluir que o estudo possibilitou estimar a prevalência da morbidade entre abuso de drogas ilícitas e transtornos psiquiátricos na população atendida no Centro de Atenção Psicossocial Álcool e drogas de Ribeirão Preto. / Different kinds of drug use have been associated to social, economic and health public problems worldwide. Among the high prevalent public health problems is the comorbidity between drug abuse and psychiatric disorders. Cannabis, cocaine and crack are the most consumed illicit drugs in Brazil. Hair use as the biological matrix for the determination of these drugs allows to evaluate chronic use, once hair is a stable matrix, easy to manipulate and the window detection only depends on the size of the hair. However, hair analysis still represents an analytical challenge. It was developed two methods for the detection of the drugs in hair, with different analytical approach. The first method (developed during doctoral stage in Italy) had identified and quantified cocaine and metabolites using column switching technique and LCMS/ MS detection. The second method was developed for determination of cannabinoids in hair samples using GC-MS. Decontamination procedure was the same cited above. The differential of this method was the use of a new device in solid phase extraction (DPX tips) for the extracts concentration and purification, using less solvents volumes. Cannabinoids and cocaine analysis were performed in hair samples from people who were enrolled in the CAPS - AD of Ribeirão Preto, São Paulo. Besides hair collection, the individual was submitted to an interview, where it was applied the following evaluation scales: Patient health questionnaire - 9, Social phobia inventory, Self report questionnaire, Beck anxiety inventory, Beck depression inventory and Cannabis research questionnaire as well as a questionnaire made by the author to collect sociodemographic data, substance consume and hair data. Hair samples were analyzed and the concentrations were correlated with positive clinical factors of mental disorders obtained through evaluation scales. The highest prevalence of positive clinical indicators for psychiatric disorders among the population studied was of common mental disorders, including anxiety and depression. The comparison of the average concentration of Cannabis, cocaine and crack in the hair with the positive clinical indicators for the disorders did not show statistically significant results. However, we can infer that the subjects who had higher average concentration of Cannabis and cocaine in hair had showed positive indicators for common mental symptoms and major depression as well as the subjects who had higher average concentration of crack in hair had showed positive indicators for anxiety. Despite of some limitations, we can conclude that the study had allowed estimating the prevalence of morbidity among illicit drugs abuse and psychiatric disorders in the population enrolled in the psychosocial care center in Ribeirão Preto.

Os antidepressivos pertencem a uma importante classe de medicamentos investigados na toxicologia forense. Em casos de amostras provenientes de cadáveres, o intervalo entre o óbito e a obtenção da espécie biológica pode proporcionar a redistribuição postmortem destes fármacos. Com o objetivo de elucidar esse fenômeno, métodos analíticos foram desenvolvidos e aplicados utilizando sangue total (ST), humor vítreo (HV) e fígado. Para as amostras de ST e HV, o método de extração escolhido e validado foi a microextração em fase líquida (LPME) trifásica. Fibras ocas constituídas de polipropileno, com a extensão de 8 cm cada, foram tratadas com o solvente orgânico dodecano (fase orgânica), resultando em um membrana com permeabilidade seletiva. No lúmen destas fibras, adicionou-se ácido fórmico 0,1 mol/L (fase aceptora). Em frasco de fundo chato com 5 mL de capacidade, pipetou-se 3,5 mL de NaOH 0,1 mol/L (fase doadora) e 0,5 mL de ST ou HV. Ao término da extração, as amostras foram introduzidas no GC-MS, sem a necessidade de reações de derivatização. O estudo com ST contemplou os antidepressivos amitriptilina (AMI), nortriptilina (NTR), imipramina (IMI), desipramine (DES), clomipramina (CLO), desmetilclomipramina (DMC), fluoxetina (FLU) e norfluoxetina (NFL). Os limites de quantificação para estas substâncias ficaram inferiores aos níveis terapêuticos (20 ng/mL). As médias dos coeficientes de variação intradia e interdia foram, respectivamente, de 9,7 e 9,8%. As curvas de calibração apresentaram linearidade entre as concentrações de 20 até 1200 ng/mL. A validação do parâmetro integridade da diluição assegurou a mensuração de quantidades superiores ao limite apresentado na curva de calibração. O método foi aplicado em sete amostras reais postmortem e em apenas um caso foi observada uma diferença significativa (300%) entre os valores quantificados no ST periférico e central. Os antidepressivos tricíclicos AMI, NTR, IMI e DES foram avaliados no HV e o efeito matriz foi detectado para os dois últimos analitos. O método foi otimizado e validado utilizando solução salina adicionada de AMI e NTR. O limite de detecção igual a 5 ng/mL, foi obtido com a redução da voltagem da fonte de íons do espectrômetro de massa para 50 eV. Coeficientes de variação foram inferiores a 15%. Os procedimentos validados foram aplicados em seis amostras reais de HV. A relação encontrada entre os valores obtidos no ST periférico e HV foi de aproximadamente 0,1. A extração acelerada por solvente (ASE) e, posteriormente, a extração em fase sólida (SPE) foram as técnicas de separação dos analitos da matriz fígado. Ao término das citadas extrações, os antidepressivos foram analisados no GC-MS. Para esta matriz sólida, são necessários mais estudos, pois os valores encontrados nos ensaios analíticos estão em desacordo com as diretrizes utilizadas na validação dos métodos. / Antidepressants belong to an important class of drugs investigated in forensic toxicology. In cases of samples from corpses, the interval between death and obtaining the biological specimens can provide the postmortem redistribution of these drugs. Aiming to elucidate this phenomenon, analytical methods were developed and applied using whole blood (WB), vitreous humor (VH) and liver. For samples of WB and HV, the extraction method chosen and validated was the three-phase liquid phase microextraction (LPME). Hollow fibers consist of polypropylene, with a length of 8 cm each were treated with dodecane organic solvent (organic phase) resulting in a membrane with selective permeability. Into the lumen of these fibers was added formic acid 0.1 mol/ L (acceptor phase). In the vial containing 3.5 mL of NaOH 0.1 mol / L (donor phase) was spiked 0.5 ml of biological fluids (WB or VH). Subsequently, the samples were injected in GC-MS without derivatization reactions. The study of the ST included antidepressants amitriptyline (AMI), nortriptyline (NTR), imipramine (IMI), desipramine (DES), clomipramine (CLO), desmethylclomipramine (DMC), fluoxetine (FLU) and norfluoxetine (NFL). The quantification limits for these substances were below the therapeutic levels (20 ng / ml). The mean coefficients of variation and separate intradays were respectively 9.7 and 9.8%. The calibration curves showed linearity between concentrations of 20 to 1200 ng / mL. The validation of the integrity of the dilution parameter assured measurement higher than the limit shown in the calibration curve quantities. The method was applied to seven real postmortem samples and in one case a significant difference (300%) between the measured values in the peripheral and central ST was observed. The tricyclic antidepressants AMI, NTR, IMI and DES were evaluated in VH and the matrix effect was detected in the last two analytes. The method was optimized and validated using saline spiked AMI and NTR. The limit of detection (5 ng/ml) was obtained by reducing the voltage of the ion source of the mass spectrometer 50 eV. Coefficients of variation were below 15%. The procedures were validated in six real samples of HV. The relationship found between the values obtained in the peripheral ST and HV was approximately 0.1. Accelerated solvent extraction (ASE) and subsequently the solid phase extraction (SPE) were the techniques of separation of analytes liver matrix. At the end of the cited extractions, antidepressants were analyzed in GC-MS. To this solid tissue, further studies are needed, because the values found in the analytical tests were not in accordance with the guidelines used in the validation of the methods.

Grâce au développement des technologies de métagénomique au cours des dix dernières années, il a été constaté que les micro-organismes représentent la plus grande ressource de diversité métabolique et génétique sur Terre. En effet, un gramme de sol contient 109 cellules bactériennes et 103-104 différentes espèces bactériennes. Certaines sont en mesure de réaliser des réactions enzymatiques conduisant à la dégradation complète de certains polluants toxiques pour l’environnement comme les composés organiques tels que la quinoléine. Cependant, l'immense réservoir de molécules et enzymes microbiennes n'a pas encore été exploité, car plus de 99% d'entre elles ne sont, pour l’instant, pas cultivables in vitro. Mon travail s’inscrit dans le cadre d’une collaboration entre l’Université SJTU (Shanghai Jiao Tong Université en Chine) et le groupe de G. M.E (Génomique Microbienne Environmentale) du laboratoire Ampère à l’Ecole Centrale de Lyon. Nos partenaires à l’Université SJTU ont construit un réacteur de dénitrification à l'échelle du laboratoire capable de dégrader la quinoléine en retirant la demande chimique en oxygène. Un nouvel outil appelé "Genefish" a été developpé dans notre laboratoire comme une méthode alternative de la métagénomique pour aider à la découverte de nouveaux gènes d’intérêt industriel ou environnemental. A la suite des premiers travaux réalisés dans notre laboratoire, ma thèse présentée ici comporte deux parties.Dans la première partie de ce travail, nous avons étudié le potentiel de dégradation de la quinoléine présente dans les bactéries d’un sol de référence largement étudié au laboratoire. Pour cela nous avons mis en place des expériences de microcosme qui visent à révéler la diversité potentielle des bactéries responsables de la dégradation de la quinoléine. Des analyses comparatives des profils RISA (Ribosomal Intergenic Spacer analysis) nous ont permis de mettre en évidence des changements dans la structure de la communauté des bactéries du sol incubé en conditions aérobie et anaérobie en présence de quinoléine. La dégradation de la quinoléine a été confirmée par technique de GC/MS (Gas Chromatography-Mass Spectrometry). Les travaux futurs seront de vérifier la communauté de bactéries responsables de la dégradation de quinoléine en utilisant la technique de NGS (Next Generation Sequencing).Le deuxième objectif de ma thèse a été d'utiliser Genefish dont la finalité est de capturer des gènes ciblés (le gène bcr qui serait responsable de la degradation de quinoléine dans le réacteur de nos partenaires) dans l'ADN métagénomique extrait du sol. Genefish consiste à élaborer une souche d’E.coli incluant un plasmide de capture permettant de pêcher les gènes recherchés dans un échantillon d’ADN metagénomique par recombinaison homologue. Le plasmide de capture comprend une cassette de deux gènes toxiques pour la souche qui activés par induction chimique vont permettre la sélection positive directe des clones recombinants, et deux sites multiples de clonage dans lesquels sont insérées les zones de recombinaison qui vont jouer le rôle d’hameçons. Nous avons testé la capacité de Genefish à capturer des produits PCR du gène bcr, l'efficacité de recombinaison reste faible à cause de la persistance de plusieurs copies du plasmide suicide dans la cellule après l’ évenement de recombinaison. Par conséquent, trois stratégies ont été essayées pour améliorer l’efficacité: la co-électroporation, la ségrégation de plasmide et la construction de plasmide suicide en mono-copie. Finalement, la stratégie de la ségrégation plasmidique fonctionne mais l'efficacité de recombinaison est encore trop faible peut-être due à l’incertitude des modèles de recombinaison homologue. Les travaux futurs se concentreront sur l'amélioration des fréquences de recombinaison par transfert de fragments du plasmide de capture dans le chromosome de la souche Genefish. / As the development of metagenomic technologies in the past ten years, it is unquestionned that microorganisms encompass the largest resource of metabolic and genetic diversity in the world. Actually, one gramme of soil contains more than 109 bacteria and 103-104 species. Some of their members are able to carry out enzymatic reactions leading to the complete degradation of pollutants (such as quinoline). So, the biodegradation of some highly toxic or organic compounds by microorganisms will be a general trend for pollutant treatment. However, the huge reservoir of molecules and enzymes from microorganisms still need to be explored because more than 99% of microorganisms cannot be cultivated in vitro.My work was based on collaboration between the University SJTU and Ecole Centrale de Lyon. Our partners at the University SJTU have built a laboratory scale denitrification reactor which was capable of degrading quinoline by removing the chemical oxygen demand. A new tool called "Genefish" has been developed in our laboratory as an alternative method for metagenomics which aims to discover novel industrial or environmental genes of interest. Following the early work in our laboratory, my thesis is presented here in two parts.In the first part, we set up a quinoline microcosm experiment both under aerobic and anaerobic condition using reference soil extensively studied in the laboratory at Ecole Centrale de LYON. This work aimed to reveal the potential bacterial diversity and even genes responsible for quinoline degradation. We used RISA(Ribosomal intergenic Spacer analysis) to analyze the bacteria community structure changes and GC/MS (Gas Chromatography-Mass Spectrometry) was also used to detect the quinoline degradation and reveal potential quinoline metabolic pathways under aerobic and anaerobic condition. Results showed great bacteria community structure changes and high quinoline degradation activity after the quinoline addition under aerobic condition. The future work is to investigate the bacteria community which may be responsible for quinoline degradation using the technique of NGS (Next Generation Sequencing).The second object of my thesis was to use the Genefish tool to capture targeted genes (the bcr gene responsible for the quinoline degradation in the wastewater treatment bioreactor) from the soil metagenome. The aim was to construct an E.coli strain containing a capture plasmid and Red system for capturing targeted genes from metagenomic DNA by homologous recombination. The capture plasmid includes a toxic cassette consisting of two suicide genes which can be activated by chemical induction, finally support the positive recombinants selection. It also contains two multiply cloning sites in which highly conserved sequences were inserted and works as the bait during recombination. We have tested the capacity of Genefish to capture the PCR products of bcr gene; the efficiency was low because of the persistence of several copies of the capture plasmid into the Genefish strain after recombination events. So, three strategies were tried to improve the recombination efficiency: co-electroporation, plasmid segregation and mono-copy capture plasmid construction. Finally, the strategy of plasmid segregation works but the recombination efficiency was still low maybe caused by the uncertain model of homologous recombination. The further research will focus on the transfer of the toxic cassette and homologous arms into the host strain chromosome, this new strategy will exclude the bad effect of low copy number capture plasmid, uncertain model of λ Red induced homologous recombination and the homologous arms site in the capture plasmid which are the most important factors influencing the homologous recombination efficiency in Genefish.

Ces travaux ont pour but d’identifier des métabolites dans des teintures-mères (TMs) utilisées en homéopathie et en phytothérapie pour mieux contrôler la qualité des TMs et de mieux comprendre leur mode d’action sur la santé humaine. Nous avons étudié, par une approche de métabolomique globale, 19 TMs et un produit leader : le L52, fabriqués par les Laboratoires Lehning. Premièrement, nous avons utilisé des approches non-ciblées en construisant des banques de données GC-MS et UPLC-MS/MS, afin d’identifier un maximum de molécules dans chaque extrait. Puis, nous avons fractionné certaines TMs et purifié des métabolites inconnus pour une identification fine en HRMS. Nous avons identifié de nombreuses molécules dans chaque TM, montrant que ces dernières sont très riches en molécules pouvant être utilisées pour le contrôle-qualité des TMs et valorisées pour la santé humaine. / Tinctures defined as hydro-alcoholic extracts have been used from centuries in homeopathy and phytotherapy, but their chemical compositions remain still unknown. During my PhD, metabolomics analyses of nineteen tinctures and one leader product, L52, made by Laboratoires Lehning, were conducted using untargeted metabolomic approach. We build GC-MS and UPLC-MS/MS databases to identify a large amount of metabolites. Then, we used semi-preparative HPLC with both UV and mass detection to isolate some compounds from tinctures. We used UPLC-HRMS to obtain chemical formula, a prerequisite for metabolites identification. Finally, we identified a broad range of different metabolites in each tincture, highlighting the metabolic complexity of the TMs. These molecules can now be used for quality-control and valued for a better understanding of these products on human health.

Le méconium constitue les premières selles du nourrisson. Cette matrice complexe est analysée dans le cadre du projet « PENEW » (Pregnancy Environment and NEWborn malformations) afin de déterminer si le degré d'exposition fœtale aux xénobiotiques joue un rôle dans la survenue de malformations congénitales. Le but de cette étude était le développement analytique pour la détection et la quantification dans le méconium d’une quarantaine de composés de familles différentes (composés organiques volatils, pesticides, éthers de glycol et les métabolites associés). Trois techniques analytiques différentes et une préparation d’échantillon spécifiques ont été développées pour la détection et la quantification de ces composés dans le méconium. L’application des ces méthodes à 246 échantillons de méconium a montré une exposition fœtale à plusieurs des substances recherchées. / Meconium is the earliest stool of newborns. This complex matrix was analyzed through the "PENEW" project (Pregnancy and Newborn malformations Environment) to determine if the degree of fetal exposure to xenobiotics has an influence in the occurrence of birth defects. The objective of this thesis was developed analytical methods for the detection and quantification of several different families of compounds (volatile organic compounds, pesticides, glycol ethers and their metabolites) in meconium. Three different analytical methods and a specific sample preparation have been developed for the detection and quantification of these compounds in the meconium. The application of these methods to 246 meconium samples showed a fetal exposure to several target compounds.

La miniaturisation et la complexification croissante des composants microélectroniques induit une sensibilisation et une fragilisation accrue des composants vis-à-vis des contaminations présentes dans les zones de productions appelées “salles blanches”. Dans ces espaces, le contrôle actuel de la contamination organique n’est pas suffisant puisqu’il ne permet pas d’éviter la contamination de surface des plaquettes de silicium et des optiques des robots de production utilisés pour la photolithographie. Un contrôle accru des concentrations des contaminants organiques dans les atmosphères des salles blanches devient donc nécessaire et de nouvelles méthodes analytiques doivent être développées et validées. Dans le cadre de ce travail, des méthodes d’analyse ont été développées et validées afin de disposer d’une gamme d’outils permettant un suivi rigoureux des contaminations. Ces outils permettent d’identifier et de quantifier les contaminations surfaciques des plaquettes de silicium par des composés organiques semi-volatils (phtalates et organophosphorés) mais aussi de déterminer les concentrations de composés organiques volatils présents dans l’atmosphère des salles blanches. Ces méthodes font appel aux technologies du WOS/ATD-GC-MS « Wafer Outgassing System/Automated Thermal Desorber–Gas Chromatography–Mass Spectrometry » et de la DART-ToF-MS « Direct Analysis in Real Time-Time of Flight–Mass Spectrometry » pour les analyses de surfaces et au PTR-ToF-MS « Proton Transfer Reaction – Time of Flight - Mass Spectrometry » pour l’analyse de l’atmosphère. / The recent advances in the miniaturization and complexification of microelectronic components induce an increase in the sensitivity of these components regarding the organic contamination present in the production zone called “clean room”. Although, the control of organic contamination in the clean room is very rigorous it does not avoid the contamination of silicon wafer surfaces and robot lenses used in the photolithography process. The later implies that new analytical methodologies should be developed and validated. In this work, analytical methods were developed and validated in order to have a panel of tools which allows careful monitoring of organic contaminants. These tools allow the identification and quantitation of the contamination of silicon wafer surface by semi-volatiles organic compounds (phthalates and organophosphates) and the determination of volatile organic compounds concentrations in the clean room atmosphere. These methods uses the WOS/ATD-GC-MS « Wafer Outgassing System/Automated Thermal Desorber–Gas Chromatography–Mass Spectrometry » technology and the DART-ToF-MS « Direct Analysis in Real Time-Time of Flight–Mass Spectrometry » technology for wafer surface analysis and the PTR-ToF-MS « Proton Transfer Reaction – Time of Flight - Mass Spectrometry » technology for gas-phase analysis.

L'évaluation de l'inertie des matières plastiques au contact de denrées alimentaires est régie par le règlement européen N°10/2011. Au Liban, l'absence de législations relatives à l'usage des matières en plastiques a conduit à des emballages non contrôlés au niveau de la production et du stockage, ce qui peut induire un risque sanitaire pour le consommateur. L'objectif de ce travail a été d'apprécier les phénomènes de migration des contaminants du PET vers l'eau. Pour cela, l'analyse de certains migrants potentiels a été initiée tels que des COV et des SV afin de vérifier la conformité de leur teneur par rapport aux LMA dans les règlementations internationales. Le dosage des aldéhydes dans l'eau a été effectué par dérivation avec la 2,4-DNPH, suivie d'une étape de préconcentration sur une cartouche en C18, puis séparation et quantification par HPLC-UV. L'analyse des composés SV a été réalisée par SPME-GC-FID sur une fibre en PDMS/DVB. L'analyse des BTEX et du styrène a été réalisée par HS-Trap-GC-FID. Ces méthodes ont été validées conformément à la norme NF T 90-210 avec des LQ inférieures aux LMA. L'analyse des COV dans l'eau embouteillée et dans le PET a été réalisée par HSTrap- GC-MS. La présence de FA, d'AA, de DEP et des BTEX dans l'eau embouteillée en PET a été confirmée. Il a été mis en évidence que la température et l'exposition solaire influencent la migration des aldéhydes. Les concentrations d'AA trouvées après incubation à 40ºC ne sont pas négligeables et peuvent modifier les propriétés organoleptiques de l'eau. L'analyse du PET montre la présence des hydrocarbures, des aldéhydes, des alcools, et des cétones / The inertness’ value of plastics that are on contact with foodstuffs is governed by the European rule N°10/2011. In Lebanon, the lack of the Rules and Regulations of plastics’ usage led for a non-controlled packing in production as in storage, which will cause a sanitary risk to the consumer and to the population. The purpose of this research was to appreciate the migration’s phenomena of contaminants from the PET to the water. For this, there was an analysis of potential migrants such as VOC and SV to verify their tenor’s conformity relative to the MCL in international regulations. The aldehydes’ dosage in the water was performed by the derivation with the 2,4-DNPH, followed by a concentration on a C18 cartridge, then separation and quantification was done by HPLC-UV. The SV compounds’ analysis was realized by SPME-GC-FID method on a PDMS / DVB fiber. The BTEX and the styrene’s analysis was done by HS-trap-GC-FID method. These methods were validated in accordance with standard NF T 90-210 with a LQ less than the MCL. The volatile compounds in the bottled water and in the PET were analyzed by HS-trap-GC-MS method. The presence of FA, AA, DEP and BTEX in the bottled water with the PET was confirmed. It was highlighted that temperature and sun exposition influence the aldehydes’ migration. The acetaldehyde’s concentration after incubation at 40ºC wasn’t negligible and can modify the water’s organoleptic properties. The PET’s analyze reveals the presence of hydrocarbons, aldehydes, alcohols and ketones

Les huiles essentielles extraites de plantes aromatiques, médicinales et à parfum, sont utilisées depuis des millénaires pour leurs propriétés médicinales ou olfactives. Pour des raisons de rentabilité, les distributeurs sont contraints à réduire leur coût au maximum et à commercialiser des huiles essentielles au plus bas prix. Certains ont recours à la fraude et proposent, des essences de qualité inférieure sous la dénomination produits naturels de haute qualité. Il existe des normes (AFNOR, pharmacopées) qui doivent être respectées. Mais leur seule application est souvent limitée. Cette étude a donc permis une nette amélioration du contrôle de la naturalité des huiles essentielles de citrus, et lavande. La connaissance des compositions moléculaires des volatiles et des polymethoxyflavones (citron), l'étude énantiomérique de molécules cibles telles que l'alpha-pinène, le sabinène (citrus), le linalol et l’acétate de linalyle (bergamote et lavande), les analyses isotopiques du carbone 13, deutérium, des linalol, acétate de linalyle (bergamote et lavande), citral, acétate de géranyle (citron) ainsi que les analyses isotopiques du deutérium et azote 15 du N-méthyle anthranilate de méthyle (mandarine) sont les analyses permettant la mise en évidence de nombreux types de fraudes. Pour une méthodologie efficace, les analyses en composante principale et discriminante ont été indispensables. Elles ont permis l’identification d’échantillons fraudés via l’étude des volatiles. Les analyses chirales et isotopiques permettent l'identification d'échantillon plus “ finement ” adultérés ou de mieux comprendre les processus de fraude / Essential oils extract from aromatics, medicinal and perfume plants, are used since ancient times for their medicinal or olfactory properties. They are quite expensive, and owing to the actual growing demand for getting high quality materials at lowest prices, fraudulent essential oils are frequently found on the market. In order to fight this problem, norms exist (AFNOR, pharmacopeas) which have to be respected. However those techniques are often limited. This work allowed great improves in the quality control of citrus and lavender essential oils. The knowledge of the chemical composition of volatiles and polymethoxy-flavones (lemon), of the chirale ratios of alpha-pinene, le sabinene (citrus), linalool et linalyl acetate (bergamot et lavender), isotope ratio analyses of carbon 13, deuterium, of linalool, linalyl acetate (bergamot et lavender), citral, geranyl acetate (lemon) as wall as isotope ratio analyses of deuterium of nitrogen 15 du Nmethyl anthranilate de methyl (mandarin) permitted the detection of various type of frauds. In order to have an efficient methodology, principal component and discriminant analyses have been proved indispensables. It allowed the identification of fraudulent samples via volatiles molecules study. Chirale analysis and isotope ratio highlight frauds on more “discrete” adulterated samples or a better understanding of adulteration’s process