Au cours de ce travail de thèse intitulé " Identification chimique de métabolites secondaires de certains microorganismes, évaluation de leurs effets dans les domaines pharmaceutiques et agronomiques ", nous nous sommes intéressés à l'étude des effets des conditions de culture sur la production des composés organiques volatils microbiens à partir de deux souches bactériennes co-existantes dans le sol Français : Burkholderia sp. et Bacillus megaterium. A partir des différents extraits préparés, plus que cent composés ont été identifiés, comprenant les dicétopipérazines, les alcools, les composés soufrés, les esters et les acides carboxyliques, par le biais de plusieurs techniques chimiques, analytiques et spectroscopiques. Les résultats obtenus ont montré que les conditions de culture sont les pricipales responsables de la production des différentes familles chimiques des volatiles. Nous avons identifiés des composés qui sont rapportés pour la première fois à partir des bactéries tel que: la N-butylbenzènesulfonamide, triacontane, le proponaoate de 3- (3,5-di-tert-butyl-4-hydroxyphényl), (E) -5-chloro-3-(hydroxyimino) indoline-2-one et 1,3,5-triméthyl-2-octadecylcyclohexane. Sur le plan biologique, on a montré que les résultats obtenus sont fortement influencés par les conditions de culture utilisées pour cultiver les bactéries testées. En parallèle à cette investigation, nous avons montré que les extraits de Burkholderia sp. sont dotés d'un très important potentiel allélopathique. Enfin, une série des analogues de dicétopipérazines a été préparée et évaluée pour leurs activités anti-xanthine oxydase, anti a-amylase et anti 5-lipoxygénase ainsi que pour leurs activités cytotoxiques contre les lignées cellulaires suivantes ; OVCAR, MCF7 et HCT116. Un certain nombre de ces dérivés de dicétopiperazine ont montré des activités anti a-amylase et cytotoxique importantes. / In this thesis entitled " Chemical identification of secondary metabolites from microorganism, evaluation of their effects on pharmaceutical and agronomic fields ", we are interested in studying the effect of culture conditions on the production of microbial volatiles organic compounds by two bacteria that inhabit French soil which are: Burkholderia sp. and Bacillus megaterium. From different prepared extracts, more than one hundred compounds were identified, including diketopiperazine, alcohols, sulfur containing compounds, esters and carboxylic acids, by means of several chemical, analytical and spectroscopic techniques. Results showed that culture conditions of different bacteria are the mainly responsible of production of different blend of volatiles. Many identified compounds including N-butylbenzenesulfonamide, triacontane, octadecyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl)propanoate, (E)-5-chloro-3-(hydroxyimino)indolin-2-one and 1,3,5-trimethyl-2-octadecylcyclohexane are reported for the first time from bacteria.Biologically, we have shown that obtained results are greatly influenced by the cultures conditions used in cultivation of tested bacteria. In addition to that, we have shown that Burkholderia sp. extracts possessed a very good allelopathic potential. Finally, a series new protested deketopiperazine derivatives have been prepared and evaluated in vitro against xanthine oxidase, a-amylase and 5-lipoxygenase enzymes, OVCAR, MCF7 and HCT116 cancer cell lines. Some of these molecules have been shown to be potent inhibitors of a-amylase and different cancer cell lines.

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.

Chez les Vibrio spp., le QS est impliqué dans de nombreuses fonctions comme la colonisation de niche écologiques, les stratégies de survie ou encore la virulence. Cependant, pour la majorité des espèces de Vibrio, la diversité des AHLs produites reste largement sous-estimée et l'étude du QS est encore limitée à quelques espèces modèles ou pathogènes. Toutefois, dans les environnements aquatiques, ces espèces sont minoritaires et les espèces les plus abondantes ne sont que très peu étudiées. Nos résultats ont révélé une importante diversité d'AHLs mais aussi, de façon surprenante, une hétérogénéité dans les phénotypes de production d'AHL au sein d'une même espèce de Vibrio. Pour la première fois, nous avons mis en évidence qu'une même souche de Vibrio pouvait présenter des phénotypes de production d'AHLs différent au cours du temps et une approche statistique a révélé l'implication de certains déterminants biotiques et abiotiques dans ces variations temporelles. Par ailleurs, une approche à micro-échelle a révélé une structuration des populations de Vibrio en unités fonctionnelles constituées de souches phylogénétiquement proches qui partagent des niches écologiques spécifiques et des comportements sociaux. Nos résultats ont mis en évidence que les modalités de communication pouvaient être hétérogènes suggérant l'absence d'un langage commun au sein de ces unités fonctionnelles. En conclusion, ce travail de thèse a permis d'apporter de nouvelles connaissances sur le QS chez les Vibrio dans l'environnement marin, de la souche à la population, et propose une vision intégrée des mécanismes de régulation de la production d'AHLs dans l'environnement. / Quorum sensing is an important mechanism among Vibrio species and is involved in many vital functions such as niche colonization, survival strategies or virulence. However, AHL diversity still largely underestimated for the majority of Vibrio species and the current knowledge on AHL-mediated QS is limited to a few pathogenic or bioluminescent species. Nonetheless, these species are weakly abundant in seawater while dominant species in the environment are poorly studied. Our results revealed a unexpected diversity of AHL molecules but also a quite surprising intra-species diversity of AHL production phenotypes. For the first time, we showed that different isolates of a single genotype switched between different AHL production phenotypes among time and we revealed the potential involvement of abiotic and biotic parameters in these variations. However, it appears that when studied at a microscale, Vibrio populations are showing a functional structuration in ecological units consisting of phylogenetically close strains sharing habitat and social traits. In this context, it was necessary to determine if these different AHL production phenotypes were associated to different micro-habitats in the water column. We did not demonstrate that a common language was spoken within ecological populations. This thesis work provide new insights on AHL-mediated QS among a broader range of species and among Vibrio populations and depicts the potential impact of multiple aspects of marine environments on AHL production.

Wood chemical composition research is of significant importance for various fields like wood-manufacturing, food and pharmaceutical industries. Much research work has been motivated by the fundamental knowledge that wood of some tree species demonstrate significantly higher native durability against biological degradation and therefore is much convenient to use in more demanding applications, e.g. in contact with soil or water. This important wood property was assign mainly to presence of extractive chemical compounds or secondary metabolites, produced by wood when heartwood is formed. The presented doctoral thesis aims to find the possibilities of how to utilize extractive chemical compounds found in Black locust (Robinia pseudoacacia L.) as treatment solutions. These can be used to increase the durability of low durable wood species. The experimental part of the presented thesis was carried out using the laboraties and equipment of the Mendel University in Brno, and their external laboratories of research centre in Útěchov. Paper 01 aims to establish methodology for extraction apparatus fexIKA. Describes mainly possibilities for quantitative gains of extractives when using various organic solvents. Within this papers research scope heartwood, sapwood and bark of Black locust were used as the source material. Black locust was choosen mainly due to its extremely high native durability, together with oak considered highest in Central Europe, and for longer term experimental plans and reason. The established methodology for primal solvents (acetone, benzene, cyclohexane, ethanol and distilled water) was the result of this paper. The influence of extraction inputs (particles size, solvent type and temperature) on quantitative and qualitative result of extraction process was the topic for research design presented in Paper 02. In the paper comparable results with literature and established fexIKA methodology were proven. Also the effect of the above mentioned inputs are described and extracted chemical content was identified using HPLC-HRMS technique. The possibility of utilizing chemical compounds obtained, based on Paper 01 extractive methodology, aimed for higher content of phenolic compounds according to Paper 02 results, were the topic of Paper 03. This research paper proves antifungal activity of Black locust (Robinia pseudoacacia L.) and African padauk (Pterocarpus soyauxii Taub.) extracts after impregnation into European beech (Fagus sylvatica L.) sapwood. This wood species is assessed as class 5 (not durable), with an average mass loss result after the durability test of untreated samples 43.6 %. Native durability of European beech wood was significantly improved after impregnation with extractives, mass loss resulted in average 12.7 %. This revalued treated beech sapwood into durability class 3. Paper 04 continues with a study of extractives retention in wood matrix after impregnation whilst introducing adjusted leaching tests. Despite standard EN 84, this research employed miniaturized Bravery wood blocks for impregnation and leaching tests, which were at the end shortened to 144 hours. In order to increase the biologicaly active chemical compounds retention in the specimens, heat treatment was used. The results of the experiment proved the significance between heat treatment and retention.

Une nouvelle méthode, simple et rapide, a été développée pour isoler dans le miel différents antimicrobiens usuellement recherchés en contrôle sanitaire et appartenant à quatre classes différentes: les sulfamides, les tétracyclines, les macrolides et lincosamides associés et les aminoglycosides. Ces molécules antimicrobiennes sont analysées par chromatographie liquide couplée à la spectrométrie de masse en tandem (CL-SM/SM) après avoir été extraites de l’échantillon de miel par une méthode d’extraction unique. Afin de définir les conditions optimales de séparation et de détection, l’influence de la nature et de la concentration d’un agent d’appariement d’ions tel que le HFBA ou le PFPA, introduit dans la phase mobile, a pu être évaluée sur une colonne analytique en chromatographie de phase inversée de type C18. Plusieurs paramètres ont été pris en compte et étudiés lors de l’élaboration de la méthode d’extraction tels que la nature du solvant d’extraction, le pH, l’étape d’hydrolyse acide, l’efficacité de l’extraction par ultrasons et enfin la purification de l’extrait avant injection. La méthode développée a ensuite été validée suivant les recommandations de la Décision de la Commission Européenne (CE) No 2002/657 puis a subi une étape de validation supplémentaire en participant à une comparaison inter-laboratoire organisée sur des matériaux de miel contaminés et gérée par un organisme extérieur accrédité suivant la norme ISO17043. Par la suite, une démarche de transfert de la méthode analytique validée en CL-SM/SM a été mise en place pour son utilisation en chromatographie liquide couplée à la spectrométrie de masse à haute résolution (CL-SMHR). Une validation de cette démarche a été menée par l’application d’une étude statistique descriptive basée sur la notion de profil d’exactitude. Finalement, un programme expérimental de surveillance a été entrepris sur une série d’échantillons de divers miels collectés sur des marchés locaux pour tester la qualité des produits commercialisés au Liban. Contrôlés au regard de leur contamination en résidus d’antimicrobiens en CL-SM/SM parmi la trentaine de molécules prédéfinies dans l’étude, la positivité et/ou la non-conformité de certains échantillons ont pu être confirmées par l’utilisation de la CL-SMHR. / A new, simple and rapid method has been developed for the determination of multiclass antimicrobial residues in honey (sulfonamides, tetracyclines, macrolides, lincosamides and aminoglycosides). All the compounds were extracted from honey within single extraction method and analyzed by liquid chromatography - tandem mass spectrometry (LC-MS/MS) operating in positive electrospray ionization mode. In our study, we examined the behavior of volatile perfluorinated carboxylic acids (HFBA and PFPA) used as ion-pairing reagents for the separation of multiclass of antibiotic residues by reversed phase Zorbax SB C18 column. Furthermore, the extraction and clean-up steps were investigated and optimized by using ultrasonic-assisted extraction and dispersive solid phase extraction (d-SPE). Different parameters affecting the extraction efficiency including type of solvent, pH, breaking efficiencies of N-glycosidic linkage by hydrochloric acid, ultrasonic extraction and its duration compared to shaking technique, along with dispersive SPE clean-up were examined prior sample injection. The method was then validated according to European Commission Decision (EC) No 2002/657. Furthermore, the method was tested for its validity through participation in proficiency testing scheme organized by FAPAS for the analysis of tetracycline in honey. Afterwards, a transfer of the validated LC-MS/MS analytical method has been applied for the determination of antimicrobial residues in honey from low resolution to High Resolution Mass Spectrometry (HRMS). For that purpose, descriptive statistical approach was performed to assess the performance of the method based on simultaneous evaluation of the trueness and the intermediate precision. Finally, the method was applied for the determination of antimicrobial residues in honey collected from local markets at different regions in Lebanon. Positive samples were then analyzed by the LC-HRMS to confirm the presence of analytes detected by LC-MSMS.

The recent developments and improvements of instrumental methods for the analyses of the environmental contaminants, especially the persistent organic pollutants (POPs), have made it possible to detect and quantify these at very low concentrations in environmental and biotic matrices. The main objective of this thesis is to demonstrate the capability of the atmospheric pressure chemical ionization technique (APCI), using gas chromatography coupled to tandem mass spectrometry for the determination of a wide range of environmental contaminants, including the POPs regulated by Stockholm Convention, such as polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs), polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs), but also the derivates of PBDEs and novel brominated flame retardants (NBFRs). The APCI was operated in charge transfer condition, preferably producing molecular ions. Multiple reaction monitoring (MRM) experiments were optimized by adjusting cone voltage, collision energy and dwell time. Optimization of source parameters, such as gas flows and temperatures was also performed. Low concentration standards were analyzed, achieving a visible chromatographic peak for 2 fg 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) demonstrating the excellent sensitivity of the system. Adequate linearity and repeatability were observed for all the studied compounds. The performance of APCI methods was validated against the conventional methods using gas chromatography coupled to high resolution mass spectrometry for chlorinated compounds in a wide range of matrices including environmental, air, human and food matrices. The GC-APCI-MS/MS method was successfully applied to a set of 75 human serum samples to study the circulating levels of POPs in epidemiologic studies. Moreover the method was utilized to establish temporal trends of POPs in osprey eggs samples collected during the past five decades.

Biopharmaceuticals are becoming one of the most promising drugs on the market mainly due to their successful treatment of a vast array of serious diseases, such as cancers, immune disorders, and infections. Structurally, biopharmaceuticals are proteins and it is important to mention that more than 60 % of biopharmaceuticals are glycosylated. Glycosylation is one of the most common posttranslational modifications. It is also the most demanding and the most complex posttranslational modification. The research showed that glycosylation can significantly impact on the safety, efficiency, and quality of the therapeutic glycoproteins. In the first part of the introduction of the present thesis, the development of the therapeutic glycoproteins and their classification were reviewed. Glycosylation process and nomenclature were also discussed. The second part of the introduction revealed current issues in the field of the production and the characterization of the therapeutic glycoproteins. In the context of the doctoral thesis, we introduced new approach, namely hydrophilic interaction liquid chromatography coupled to a high-resolution mass spectrometer (HILIC-HR-MS) combined with Principal Component Analysis (PCA) and classification through Soft Independent Modelling by Class Analogy (SIMCA) data treatment. Accordingly, N-glycans were first enzymatically released using peptide-N-glycosidase F (PNGase F) and reduced using sodium borohydride. Then those N-glycans were separated by HILIC and detected by HR-MS. PCA and SIMCA simplified interpretation of the MS data collected in the huge tables. PCA was applied to test whether it is possible to visualize N-glycosylation differences between samples and to help identifying within which N-glycans changes occurred. SIMCA, which is a more complex data analysis technique, was applied to build and validate a classification models. SIMCA was also applied to verify whether it is possible to use built models to classify real samples. Described approach enabled us to detect small changes in N-glycosylation of the therapeutic glycoproteins (a change of only 1% in relative glycan abundance). It was applied to assess changes in N-glycosylation of therapeutic glycoproteins. Accordingly, we tested N-glycosylation consistency between batches of infliximab, trastuzumab, and bevacizumab and monitored the N-glycosylation of bevacizumab over storage time in plastic syringes.Furthermore, we worked on the faster sample preparation technique, where online-solid-phase extraction (SPE)-LC was combined to the previously mentioned HILIC-MS-PCA/SIMCA method. Online-SPE-LC allowed us to faster the sample preparation in terms of avoiding time-consuming cleaning steps. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

À l’heure où l’intégration des enjeux environnementaux dans le développement de procédés éco-efficients joue un rôle essentiel dans le moteur de l’innovation responsable, la chimie verte est devenue l’un des sujets de préoccupation majeure. Ainsi, le développement de nouveaux procédés éco-respectueux pour la production d’ingrédients naturels issus de matières premières végétales renouvelables est devenu une démarche incontournable dans le modèle de recherche. L’objectif de cette thèse a consisté au développement d’une stratégie d’éco-valorisation innovante employant les fluides sub/supercritiques pour l’extraction, la caractérisation, la production et l’imprégnation sur support cosmétique de produits naturels d’origine végétale. Pour cela, nous avons utilisé comme modèle végétal : les graines oléagineuses de la plante Kniphofia uvaria, sélectionnée pour des applications cosmétiques grâce à ses propriétés bioactives antioxydantes et anti-âge. Dans un premier temps, le développement de méthodes complémentaires en SFC ainsi que le développement du couplage SFC-MS a été réalisé à l’aide de la source APCI afin d’identifier les molécules responsables des activités bioactives des graines de Kniphofia uvaria. Ainsi, le développement d’un système hybride (U)HPLC/SFC-HRMS a été réalisé afin de mettre en place ce couplage. Des optimisations en termes de proportion et nature de solvant make-up ainsi qu’un travail au niveau des paramètres SFC et MS ont été faits afin de d’améliorer la sensibilité et la spécificité des analyses lipidiques. Dans un second temps, nous nous sommes attachés au développement d’une stratégie d’enrichissement en composés bioactifs à l’aide des méthodes : SFE et CPC. Ainsi, en SFE, des optimisations en termes de température, pression, nature/proportions de co-solvant dans le fluide ont été réalisées alors qu’en CPC, des optimisations au niveau de l’injection ont été faites. Des conditions optimales pour le fractionnement sélectif des anthraquinones et des triglycérides ont été déterminées en SFE et CPC. Dans un dernier temps, ce travail a consisté à développer un couplage en-ligne pour extraire et imprégner sélectivement sur silice cosmétique : les anthraquinones. Le développement et l’optimisation de ce procédé en-ligne ont été réalisés à l’échelle du laboratoire et ont démontré la faisabilité de ce couplage ainsi qu’un intérêt certain pour l’obtention de produits naturels sous une première forme galénique, destinée à une future incorporation dans la formulation de cosmétiques. / Nowadays, green chemistry is a great challenge. It seeks innovation in the development of eco-efficient processes. The production of natural products from renewable materials by these new environmentally friendly processes is more and more used. The aim of this Ph.D thesis is to develop an eco-valuation strategy to extract, characterize, produce and impregnate natural products onto a cosmetic support using sub/supercritical fluids. Consequently, we used oleaginous plant seeds from Kniphofia uvaria as a plant model, which was selected for its interesting cosmetic properties such as antioxidant or anti-ageing. Firstly, the SFC-MS hyphenation with the APCI as an ionization source was developed to screen bioactive molecules; responsible of cosmetic properties. This coupling was performed by the hybrid combination of (U)HPLC/SFC-HRMS. Various optimizations in terms of the solvent make-up (nature and proportion), modulation with SFC and MS parameters were carried out in order to improve sensitivity and selectivity of lipid analysis. Secondly, an enrichment strategy to concentrate bioactive compounds in the final extract was developed by SFE and CPC. Thus, in SFE, experimental parameters (temperature, pressure, nature/proportion of the modifier in the CO2 fluid) were optimized while in CPC, the injection optimization was realized. Methods for the selective fractionation of anthraquinones and triglycerides were obtained in CPC and SFE. Finally, an on-line sub/supercritical extraction-impregnation process was developed to extract and for simultaneously impregnating anthraquinones onto a cosmetic silica. Development and optimization of this process was realized on a laboratory scale. Consequently, this study demonstrated the feasibility of this concept and it presents a great interest to provide natural products as a galenic form, which could be used in the cosmetic formulation.

<p>  </p> <p>Atmospheric aerosols formed through primary emissions, secondary gas-particle formations, and multi-phase chemical processes are composed of solid, semi-solid, or liquid-like particles suspended in the air that have direct implications towards the global radiative balance and human health as air pollutants.  Direct emissions of primary organic aerosols (POA; e.g. soot, BrC) and multi-phase formation of secondary organic aerosols (SOA) from the oxidation of biogenic monoterpene isomers represent two important sources/classes of particulate matter in the atmosphere. Multi-phase chemical processes driving the atmospheric and environmental aging through the photochemistry of iron(III), FeIII in organic aerosol particles and aqueous media drives the multiphase chemistry leading to systematic aging of their chemical composition and modifications to resulting light-absorption properties. The molecular composition, organic structures, physical properties, and sources of emissions are complex requiring development of powerful multi-modal analytical metrology, such as high-resolution mass spectrometry (HRMS) hyphenated with liquid chromatography (LC), photodiode array optical detection, drift tube ion mobility (IM) spectrometry, and desorption and ambient ionization of multi-components mixtures in atmospheric particles using temperature programmed desorption Direct analysis in real time (TPD-DART). Disseminating the molecular-specific composition, chemical and physical properties of complex mixtures in atmospheric organic particles and mixed inorganic/organic systems will help improve our understanding of their formation mechanisms, transformative chemical ageing processes, as well as improved detection of individual components in complex mixtures. </p> <p>     </p> <p>Chapter 1 and 2 of dissertation introduces complexity of atmospheric organic, carbonaceous aerosols, and complex environmental mixtures and discusses analytical metrology, experiments, and data analysis procedures used for detailed molecular-level characterization of mixtures. Chapter 3 the development of a robust analytical method for untargeted screening and determination of the physical and chemical properties (e.g. vapor pressures, enthalpies of sublimation, and saturation mass concentrations) of single components out of complex SOA particles using temperature programmed desorption Direct analysis in real time ionization – high resolution mass spectrometry (TPD-DART-HRMS).  Chapter 4 introduces the use of ion mobility - mass spectrometry (IM-MS) separation and multidimensional characterization of structural isomers in complex SOA mixtures. The chapter discusses the advanced usage of IM-MS to investigate the molecular and structural properties of isomers of alpha-pinene and limonene derived SOA, use of advanced data analysis procedures to resolved complex conformational and structural isomers, and investigate single-molecule structural changes from atmospheric-like ageing in SOA particles using IM-MS.  Chapter 5 discusses the chemical characterization and analysis of individual brown carbon (BrC) chromophores out of mixture of colorless organic carbon constituents and insoluble soot particles generated from controlled flame combustion of ethane fuel, a surrogate system representing gasoline combustion of motor vehicles. The chapter focuses on the quantitative method development and use of state-of-the-art liquid chromatography coupled to photodiode array followed by dopant assisted atmospheric pressure photoionization and HRMS (LC-PDA-HRMS) analysis, followed by conversion to quantitative optical information for comparisons with retrieved literature reports. Chapter 6 examines the complex multiphase photochemical cycling of Fe(III)-citrate, a relevant proxy for [FeIII-carboxylate]2+ complexes in atmospheric water using complementary analytical metrology of optical spectroscopy, LC-PDA-HRMS, oil immersion flow microscopy. Multi-modal datasets from these complementary techniques provide a unique experimental description of various stages of FeIII-citrate photochemistry, elucidate individual components of this reacting system, determine mechanistic insights, and quantify environmental parameters affecting the photochemistry. </p>