Le premier acide aminé incorporé dans une chaîne polypeptidique naissante est toujours la méthionine. On identifie donc toujours ce premier résidu à la méthionine N-terminale. Cependant, les deux tiers des protéines accumulées à l’état stationnaire ne présentent plus leur méthionine initiatrice. Cet enlèvement résulte essentiellement d’une maturation protéolytique affectant chaque protéine. Ainsi, l’Excision de la Méthionine N-terminale (NME) concerne la majorité des protéines et ce dès que les premiers résidus émergent du ribosome. Ce mécanisme est retrouvé dans tous les compartiments cellulaires où une synthèse protéique a lieu : le cytoplasme, les plastes et les mitochondries. Les enzymes responsables du clivage de la méthionine initiatrice sont les METhionine AminoPeptidases (METAPs) ; les METAPs sont conservées dans le Règne vivant. Des études fonctionnelles de délétions géniques ont montré le caractère létal du maintien de la première méthionine dans tous les organismes. Il y a plus de dix ans, les METAPs ont été identifiées comme étant la cible de composés naturels ayant des effets anticellulaires. Aujourd’hui un nombre croissant d’études rapportent que la NME est une cible prometteuse pour le traitement de nombreuses pathologies. Néanmoins, les bases moléculaires qui expliquent le caractère essentiel de la NME restent très peu comprises, en particulier dans le cytoplasme des eucaryotes supérieurs. Grâce à un système inductible permettant de moduler finement la NME cytoplasmique dans la plante modèle Arabidopsis thaliana et différentes approches incluant des analyses protéomiques et métabolomiques, j’ai pu étudier les événements moléculaires précoces associés à l’inhibition de la NME cytoplasmique. J’ai également caractérisé la contribution relative des deux types de METAP cytoplasmiques au processus. Dans ce contexte, j’ai pu démontrer chez A. thaliana que la NME cytoplasmique agit sur deux voies de signalisation fréquemment dérégulées lors de conditions pathologiques : le statut des composés thiolés et la protéolyse. La diminution de la NME cytoplasmique induit une protéolyse accrue principalement via une augmentation du nombre de protéines destinées à une dégradation rapide. Ainsi, l’activité de la NME, en modulant la sensibilité de nombreuses protéines à subir la protéolyse, est un élément fondamental de la régulation de la demi-vie protéique. Finalement, mes résultats simialires obtenus également chez les Archées, levures et les lignées de cellules humaines suggèrent l’existence d’un mécanisme ubiquitaire associé à la NME. / The first amino acid incorporated in nascent polypeptide chain is always methionine so called N-terminale methionine. However, in a given proteome, more than fifty percent of proteins have not this first methionine. Indeed, the early proteolytic event affecting a majority of proteins is N-terminal Methionine Excision (NME) as soon as few residues exit from the ribosome. Enzymes ensuring NME process are conserved along species. This mechanism takes place in all compartments where protein synthesis occurs including cytoplasm, plastids and mitochondria and the enzymes responsible of N-methionine excision are METhionine AminoPeptidases (METAP). Early functional studies of gene deletion has quickly showed that NME is an essential process. Ten years ago, METAPs have been identified as the molecular target of natural compounds with anticancer activities. Now, a growing number of studies suggest that NME is a promising target for treatment of various deseases. Nevertheless, molecular mechanisms making NME an essential process is poorly understood in particular in higher eukaryote cytoplasms.Using a dedicated inducible system in the model organism Arabidopsis thaliana and multiple approaches, including proteomics and metabolomics, I examined the earliest molecular events associated with the inhibition of this process and the contribution of both METAP to NME process. In this context, I demonstrated that cytoplasmic NME in A. thaliana orchestrates a cross-talk between two fundamental signaling pathways frequently deregulated in pathological conditions: thiol status and proteolysis. In these studies, we demonstrated that developmental defects induced by cytoplasmic NME inhibition are associated with an increase of the proteolytic activity due to an increase of the proteins available for rapid degradation. Thus, NME activity that modifies the availability of several proteins for degradation is an integral and fundamental element protein turnover regulation. Finally my preliminary results obtained in Archea, Fungi and human cells seem to suggest the existence of a ubiquitous mechanism associated with NME process.

Considérées comme des menaces pour les écosystèmes marins tropicaux et subtropicaux, les proliférations de macroalgues sont susceptibles de modifier le fonctionnement et la structure des récifs coralliens. Le genre Asparagopsis (Rhodophyta) est connu pour être largement distribué, introduit et parfois invasif dans certaines régions comme en Méditerranée occidentale. Le premier objectif était de corréler le métabolisme spécialisé et la bioactivité de l’algue à son génotype, et éventuellement à son caractère proliférant. Aucune corrélation génétique/métabolomique n’a été démontrée, ce qui laisse entendre que le métabolisme macroalgal serait principalement influencé par l'environnement et/ou sa flore microbienne associée. En milieu tempéré, A. taxiformis et A. armata présentent des signatures métabolomiques globalement similaires et associées à une bioactivité significativement plus importante qu'en milieu tropical. Cependant, même lorsqu’elle a été introduite, une même lignée génétique et un même phénotype chimique peuvent présenter des caractères proliférants opposés. En milieu tropical, les extraits de macroalgues testés in situ sur 4 espèces de coraux n'ont provoqué que de faibles blanchissements. En milieu tempéré, aucun effet biologique de l’algue n’a été enregistré sur le corail Astroides calycularis. Par contre des expériences en aquarium ont permis de montrer qu’A. taxiformis pouvait exprimer un métabolisme spécifique avec une bioactivité augmentée après 10 jours de contact avec ce corail. En conclusion, qu’elle soit indigène ou introduite, A. taxiformis exerce peu d’effets sur la santé des coraux, et le caractère invasif de cette algue reste une source de débat. / Considered as a major threat for sub–tropical and tropical ecosystems, macroalgal proliferations are susceptible to modify the structure and the functioning of coral reefs. The genus Asparagopsis (Rhodophyta) is known to be widespread, introduced and sometimes invasive in certain regions such as the Western Mediterranean Sea. The first objective of this thesis was to correlate the algal specialized metabolism and its bioactivity with its genotype, and eventually with its proliferation trait. No correlation between genetics and metabolomics has been demonstrated, which would suggest the main influence of environmental factors and/or the associated microbial diversity on the algal metabolism. In temperate regions, A. taxiformis and A. armata showed similar metabolomic fingerprints with bioactivities significantly higher than in tropical regions. However, even when it is introduced, a given genetic lineage and a given chemical phenotype can exhibit opposite proliferative traits. In tropical areas, algal extracts tested in situ on 4 coral species did not lead to any coral bleaching. In temperate areas, no biological effect of the alga was recorded on Astroides calycularis. However, some aquarium experiments allowed to show that A. taxiformis can express a specific metabolism, with an increased bioactivity after 10–days of contact with this coral. To conclude, either indigenous or introduced, A. taxiformis poorly affects corals’ health, and thus the invasiveness of this alga remains a matter of debate.

No description available.

This thesis aimed to investigate several fundamental and perplexing questions relating to the phloem loading and transport mechanisms of <i>Cucurbita maxima</i>, by combining metabolomic analysis with cell biological techniques. This putative symplastic loading species has long been used for experiments on phloem anatomy, phloem biochemistry, phloem transport physiology and phloem signalling. Symplastic loading species have been proposed to use a polymer trapping mechanism to accumulate RFO (raffinose family oligosaccharides) sugars to build up high osmotic pressure in minor veins which sustains a concentration gradient that drives mass flow. However, extensive evidence indicating a low sugar concentration in their phloem exudates is a long-known problem that conflicts with this hypothesis. Previous metabolomic analysis shows the concentration of many small molecules in phloem exudates is higher than that of leaf tissues, which indicates an active apoplastic loading step. Therefore, in the view of the phloem metabolome, a symplastic loading mechanism cannot explain how small molecules other than RFO sugars are loaded into phloem. <br><br> Most studies of phloem physiology using cucurbits have neglected the possible functions of vascular architecture in phloem transport. It is well known that there are two phloem systems in cucurbits with distinctly different anatomical features: central phloem and extrafascicular phloem. However, mistaken conclusions on sources of cucurbit phloem exudation from previous reports have hindered consideration of the idea that there may be important differences between these two phloem systems. <br><br> The major results are summarized as below:<br> 1) O-linked glycans in <i>C.maxima</i> were structurally identified as beta-1,3 linked glucose polymers, and the composition of glycans in cucurbits was found to be species-specific. Inter-species grafting experiments proved that these glycans are phloem mobile and transported uni-directionally from scion to stock.<br> 2) As indicated by stable isotopic labelling experiments, a considerable amount of carbon is incorporated into small metabolites in phloem exudates. However, the incorporation of carbon into RFO sugars is much faster than for other metabolites.<br> 3) Both CO2 labelling experiments and comparative metabolomic analysis of phloem exudates and leaf tissues indicated that metabolic processes other than RFO sugar metabolism play an important role in cucurbit phloem physiology.<br> 4) The underlying assumption that the central phloem of cucurbits continuously releases exudates after physical incision was proved wrong by rigorous experiments including direct observation by normal microscopy and combined multiple-microscopic methods. Errors in previous experimental confirmation of phloem exudation in cucurbits are critically discussed.<br> 5) Extrafascicular phloem was proved to be functional, as indicated by phloem-mobile carboxyfluorescein tracer studies. Commissural sieve tubes interconnect phloem bundles into a complete super-symplastic network.<br> 6) Extrafascicular phloem represents the main source of exudates following physical incision. The major transported metabolites by these extrafacicular phloem are non-sugar compounds including amino acids, O-glycans, amines.<br> 7) Central phloem contains almost exclusively RFO sugars, the estimated amount of which is up to 1 to 2 molar. The major RFO sugar present in central phloem is stachyose. <br> 8) Cucurbits utilize two structurally different phloem systems for transporting different group of metabolites (RFO sugars and non-RFO sugar compounds). This implies that cucurbits may use spatially separated loading mechanisms (apoplastic loading for extrafascicular phloem and symplastic loading for central phloem) for supply of nutrients to sinks. <br> 9) Along the transport systems, RFO sugars were mainly distributed within central phloem tissues. There were only small amounts of RFO sugars present in xylem tissues (millimolar range) and trace amounts of RFO sugars in cortex and pith. The composition of small molecules in external central phloem is very different from that in internal central phloem.<br> 10) Aggregated P-proteins were manually dissected from central phloem and analysed by both SDS-PAGE and mass spectrometry. Partial sequences of peptides were obtained by QTOF <i>de novo</i> sequencing from trypsin digests of three SDS-PAGE bands. None of these partial sequences shows significant homology to known cucurbit phloem proteins or other plant proteins. This proves that these central phloem proteins are a completely new group of proteins different from those in extrafascicular phloem. The extensively analysed P-proteins reported in literature to date are therefore now shown to arise from extrafascicular phloem and not central phloem, and therefore do not appear to be involved in the occlusion processes in central phloem. / Phloem transportiert ein ausgedehntes Spektrum an Molekülen zwischen Pflanzenorganen, um Wachstum und Entwicklung zu koordinieren. Folglich ist eine umfassende und unvoreingenommene Metabolom-Analyse notwendig, um unser Verständnis über den Transport von Stoffwechselprodukten sowie über Phloemtransport zu vertiefen. Phloemexsudate von Kürbispflanzen werden unter Verwendung der Metabolom-Analyse analysiert. Bei diesen Pflanzen wird angenommen, dass sie symplastische Beladungswege verwenden, um Photoassmilate als Ausgangsschritt des Phloemtransportes zu konzentrieren. Zwei neue Familien Callose-verwandter Substanzen, 1,3-Overknüpfte Glycane, sowie eine Reihe anderer kleinerer Metabolite werden in den Phloemexsudaten detektiert. Metabolom-Daten und physiologische Experimente widersprechen früher berichtetem Verständnis des Phloemexsudationsprozesses in Kürbispflanzen. Folglich bestätigt sich der Phloemexsudationsprozeß durch Kombination unterschiedlicher mikroskopischer Techniken. Kürbispflanzen besitzen zwei Phloemsysteme mit eindeutigen anatomischen Eigenschaften. Es zeigt sich, daß Phloemexsudate in Kürbissen hauptsächlich vom extrafaszikulären Phloem, nicht vom zentralen Phloem, stammen. In den letzten Jahrzehnten wurde gewöhnlich mißverstanden, daß Phloemexsudate vom zentralen Phloem stammen. Die eindeutigen metabolischen Profile der unterschiedlichen Phloemsysteme, die durch Metabolom-Analysen in der räumlichen Auflösung beobachtet werden, bestätigen die unterschiedlichen physiologischen Funktionen der zwei unterschiedlichen Phloemsysteme: das zentrale Phloem transportiert hauptsächlich Zucker, während das extrafaszikuläre Phloem ein ausgedehntes Spektrum von Metaboliten transportiert. Es kann auch ein unterschiedliches metabolisches Profil kleiner Moleküle zwischen internem und externem zentralem Phloem beobachtet werden. Von Strukturproteinen des zentralen Phloems wurden auch Proben genommen und mittels Massenspektrometrie analysiert. Diese Proteine erweisen sich als neuartige Proteine, die sich zu denen im extrafaszikulären Phloem unterscheiden. Dies bestätigt ferner den Funktionsunterschied der unterschiedlichen Phloemsysteme in Kürbispflanzen. Basierend auf diesen neuartigen Entdeckungen des Phloem-Metaboloms und dem vorhergehenden Wissen über den Phloemtransport in Kürbispflanzen, wird ein neues Modell vorgeschlagen, um den Mechanismus des Phloemtransports in der symplastischen Beladung zu verstehen.<br>

Introduction Inflammatory bowel disease (IBD) is a chronic gastrointestinal disorder that encompasses two major subtypes; Crohn’s Disease (CD) and Ulcerative Colitis (UC). Our knowledge regarding disease pathogesis is rapidly increasing. However, these disease entities provide challenges in diagnosis, monitoring of disease activity and assessing individual response to treatment, because there is a lack of validated clinical biomarkers. Metabolomics involves the study of numerous analytes that have very diverse physical and chemical properties and occur in a wide concentration range. Early evidence suggests there is potential for metabolomic profiling to be used in the differentiation of CD and UC. However, knowledge is limited regarding the metabolic changes seen in relation to disease activity or to medical or surgical treatments. Aims A metabolomics approach was taken to determine whether metabolomic profiles could distinguish between patients with CD or UC and healthy controls. We also aimed to define the relationship between metabolomic profile and disease activity, and to determine the effect of medical (anti-TNFa agents) and surgical treatment on the metabolome. Methods A metabolomics approach was undertaken. Serum and urine sample sets were collected from a total of 41 patients with ulcerative colitis, 43 patients with Crohn’s disease, and 62 healthy controls (HC). In order to allow a comparison of metablomic profile and disease activity, 4 sample sets were taken from the same patient at 3 monthly intervals over the period of one year. Those patients undergoing either surgical or biological treatment had sample sets taken pre and post intervention. Metabolomic analysis using gas chromatography time of flight mass spectrometry (GC-ToF-MS) and ultra-high performance liquid chromatography Fourier Transform mass spectrometry (UHPLC-FTMS) was carried out on both serum and urine. Results Serum and urine GC-ToF-MS and UHPLC-FTMS metabolomic analyses show differentiation between UC, CD and healthy controls, most significantly in urine analyses. No significant differentiation was seen in pre- and post-surgical patients, or pre- and post-biological therapy patients. It was possible to differentiate surgical patients from healthy controls, especially in the urine analyses. Metabolite identification revealed consistently more dietary variation in the healthy controls than in the IBD patients. Significant differences (p < 0.05) were seen between healthy controls and IBD patients in classes of metabolites relating to the citric acid cycle and the uronic acid pathway, as well as amino acids, fatty acids and cholesterols. The behaviour or location of disease, or the disease activity score did not appear to influence the metabolome in either serum or urine analyses using GC-ToF-MS and UHPLC-FTMS. Conclusion Metabolomic profiling of urine and serum in IBD may provide a novel methodology aiding both clinical diagnosis through biomarker development, and advancing knowledge of disease pathogenesis.

Orientador: Rodrigo Ramos Catharino / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-18T04:45:53Z (GMT). No. of bitstreams: 1 Riccio_MariaFrancesca_M.pdf: 1073402 bytes, checksum: ad216531a7e88603fc492ee5c702fd1f (MD5) Previous issue date: 2011 / Resumo: Este trabalho direcionou-se ao controle de qualidade de óleos vegetais e gorduras animais pelo emprego de análise de baixa massa molecular pela técnica EASI-MS utilizando a ferramenta metabolômica. As matérias-primas graxas (óleos e gorduras vegetais e animais) e os produtos sintetizados a partir destes insumos são misturas complexas, com aplicações e valores agregados variados como por exemplo para a nutrição humana, aplicações industriais na produção de lubrificantes, biodiesel, plasticidas, surfactantes, entre outros. As técnicas disponíveis como Cromatografia gasosa (GC) com detector de ionização de chama (FID) ou acoplada a um espectrômetro de massas (GC-MS) tem sido as técnicas mais utilizadas para caracterizar óleos e gorduras, através da determinação da composição de ácidos graxos. Como a caracterização da composição graxa destas matrizes são muitas vezes restritas, exigindo a necessidade de procedimentos laboriosos como extrações, purificações que requerem muito tempo utilizamos neste trabalho uma recente técnica de ionização ambiente de espectrometria de massas: EASI-MS (easy ambient sonic-spray ionization mass spectrometry) na caracterização de óleos vegetais e gorduras animais que dispensa o emprego de processos de derivatização química e a separação cromatográfica. Neste trabalho aplicamos a metabolômica para elucidação de um conjugado de marcadores taxonômicos de óleos nunca observados ou analisados em conjunto antes, principalmente ácidos graxos e bifenóis os quais podem ser extraídos de maneira simples e eficaz. Para a caracterização dos componentes existentes no metaboloma de óleos de origem vegetal e animal e para a caracterização de azeites de oliva de diferentes procedências, uma simples extração com uma solução hidroalcoólica foi utilizada. O extrato foi adicionados à uma superfície de vidro para dessa forma, serem injetados para dentro do equipamento de massas e então analisados. O equipamento utilizado foi um Q-TrapTM utilizando uma fonte de EASI construída por pesquisadores do Laboratório ThoMSon de espectrometria de massas (IQ/UNICAMP). Os espectros de massas obtidos demonstraram a presença de ácidos graxos livres além de bifenóis característicos de cada tipo de óleo ou gordura analisado. A presença na mesma análise (espectro) demonstrou que mesmo para amostras complexas, a técnica se mostra aplicável para a identificação, caracterização e controle de qualidade de maneira inequívoca para óleos e gorduras / Abstract: This work is directed to quality control of vegetable oils and animal fats by the use of low molecular analysis of the EASI-MS technique using the metabolomics tool. Raw materials greases (oils and vegetable and animal fats) and the products synthesized from these inputs are complex mixtures, with varying applications and value added such as for human nutrition, industrial applications in the lubrificants production, biodiesel, plasticity, surfactants, among others. The available techniques such as chromatography (GC) with flame ionization detector (FID) and coupled to a mass spectrometer (GC-MS) has been the most widely used techniques to characterize oils and fats by determining the fatty acid composition. To characterize the grease composition of these matrices are often restricted, requiring the need of laborious procedures such as extractions, purifications that require much time, in this work we used a recent technique ambient ionization mass spectrometry: EASI-MS (Easy ambient sonic-spray ionization mass spectrometry) in the characterization of vegetable oils and animal fats does not require the use of chemical derivatization procedures and chromatographic separation. In this work we have applied metabolomics to elucidate a combination of taxonomic markers oils never observed or analyzed together before, mainly fatty acids and biphenols which can be extracted in a simple and effective process. To characterize the metabolome of existing components in vegetable oils and animal and for the characterization of olive oils from different sources, a simple extraction with a water-methanol solution was used. The extract was added to a glass surface to thereby be injected into the spectrometer and then analyzed. The equipment used was a Q-TrapTM using a source of EASI built by researchers at the Thomson Laboratory of mass spectrometry (Institute of Chemistry / UNICAMP). The mass spectra obtained showed the presence of free fatty acids besides biphenols characteristic of each type of oil or fat analyzed. The presence in the same analysis (spectrum) showed that even for complex samples, the technique proves to be applicable to the identification, characterization and quality control unequivocally for oils and fats / Mestrado / Ciencias Biomedicas / Mestre em Ciências Médicas

As doenças cardiovasculares ainda são as principais causas de mortalidade e morbidade em todo o mundo. E a aterosclerose é uma das principais precursoras de vários desfechos clínicos como isquemias e infarto do miocárdio. As placas ateroscleróticas se desenvolvem preferencialmente em regiões de bifurcação ou curvatura dos vasos, onde o shear stress (SS) encontra-se diminuído ou perturbado. A expressão de proteínas pró-aterogênicas em regiões de baixo SS e ateroprotetoras em regiões de SS alto foram relatadas na literatura, porém o mecanismo completo carece de elucidação. Este trabalho teve por objetivo integrar proteômica e metabolômica para um melhor entendimento das alterações moleculares que acontecem nas células endoteliais em situações de alto e baixo SS, que podem resultar no desenvolvimento de lesões e placas ateroscleróticas. Para esta finalidade, células endoteliais foram submetidas a alto e baixo SS em sistema cone plate, seguido de análise proteômica e metabolômica por espectrometria de massas. Nossos dados demonstraram que o metabolismo de lipídio e metabolismo de modificações pós-traducionais de proteínas (N-glicosilações) estavam diminuídos em baixo SS. Em relação ao metabolismo de lipídio, foi identificada diminuição na concentração de ácidos graxos e na expressão de enzimas e proteínas transportadoras de lipídios em células sob baixo SS. O receptor de LDL, proteína importante para a homeostase do colesterol, foi identificado em menor concentração na membrana, bem como com alteração no seu perfil de glicosilação em células após baixo SS. As células submetidas a baixo SS e, portanto, aquelas com perfil pró-aterogênico, quando tratadas com estatina para o aumento da expressão de LDLR, aproximaram seu fenótipo ao de células submetidas a alto SS, adquirindo parte de um fenótipo ateroprotetor, com recuperação dos níveis de aminoácidos, lipídios, açúcares e ácidos carboxílicos. Os dados deste trabalho sugerem que o metabolismo de lipídios é um processo importante na manutenção do perfil ateroprotetor de células submetidas a alto SS. Além disso, as evidências demonstraram que estatinas apresentam uma atividade protetora, não apenas sistêmica, com diminuição do LDL circulante, mas também no microambiente vascular, contribuindo para o bom funcionamento das células endoteliais / Cardiovascular diseases are the main cause of the mortality and morbidity worldwide. Atherosclerotic plaque development is closely associated to the hemodynamic forces applied to endothelial cells (EC). Among these, shear stress (SS) plays a key role in disease development since changes in flow intensity and direction could stimulate an atheroprone or atheroprotective phenotype. EC under low and/or oscillatory SS (LSS) have upregulation of inflammatory proteins, adhesion and cellular permeability molecules. On the contrary, cells under high/laminar SS (HSS) increase their expression of protective and anti-inflammatory factors. The mechanism behind the SS regulating an atheroprotective phenotype is not completely elucidated. Here we used proteomics and metabolomics to better understand the changes suffered by endothelial cells under LSS and HSS that promote the atheroprone and atheroprotective profile and how these modifications can be connected to atherosclerosis development. Our data showed that lipid metabolism and post translational modification protein metabolism were downregulated in cells under LSS. About lipid metabolism, we found the LDLR, one important protein in cholesterol homeostasis, showed significant alterations both at the quantitative expression level, as well as regarding post-translational modifications. Under LSS, LDLR was seem at lower concentrations and with a different glycosylation profile. Finally, modulating LDLR with atorvastatin led to the recapitulation of an HSS metabolic phenotype in EC under LSS. The phenotype was recovery based on increasing of amino acids, lipids, sugars and carboxylic acids. Altogether, our data suggest lipid metabolism is important in atheroprotective phenotype of endothelial cells under HSS. Statins showed benefits not only systemic, decreasing cholesterol level in blood, but also in vascular environment, contributing for protector phenotype of endothelial cells

Introduction: Inflammatory bowel disease is a common, complex relapsing disorder characterised by immune dysregulation, altered intestinal permeability and microbial insult. Limited knowledge is available regarding the metabolic changes observed during progression of the disease, and limited biomarkers of disease available that have been validated and shown to be of sound clinical value. Aim of Study: A two stage metabolomics approach was adopted to determine if metabolic signature profiles, could distinguish inflammatory bowel disease Crohn’s disease (CD) patients from ulcerative colitis (UC) patients and from healthy controls. Methods: A serum metabolomics approach was undertaken to define metabolic changes associated with UC and CD. Serum samples from a discovery study of 30 UC, 30 CD and 29 ethnically, age and gender matched controls were analysed by ultra-performance liquid chromatography mass spectrometry. A subsequent validation study was preformed using 28UC, 31CD, and 29 gender matched controls were also analysed using UPLC-MS.ResultsClasses of metabolites, identified as biologically interesting and at significantly different levels (p<0.05) in comparisons of control and CD and UC cohorts included: steroids and steroid derivatives, phosphocholine, Vitamin D metabolites, fatty acids and conjugates, glycerolipids, isoprenoids, amino acids, and phosphosphingolipids. There were fewer discriminatory metabolites differentiating the CD and UC cohorts. Conclusion: Serum Metabolomic profiling may represent a novel technology which could be used to distinguish individuals with CD from those with UC and healthy controls.

La pollution des produits agroalimentaires et des eaux environnementales par les substances phytosanitaires et les médicaments est une réelle préoccupation de santé publique. Il existe aujourd’hui de nombreuses méthodes d’analyses quantitatives développées sur des appareils de type triple quadripôle pour leur détection mais elles ne se préoccupent peu ou pas des résidus médicamenteux et des produits de dégradation abiotique ou biologique des polluants et sont limitées à un nombre restreint et figé de molécules.Dans ce contexte, nous avons développé une méthode d’analyse basée sur la métabolomique par chromatographie liquide haute performance couplée à la spectrométrie de masse à ultra-haute résolution (LC-HRMS). Deux matrices (miel et eau souterraine) ont ainsi été étudiées comme preuve de concept. Nous avons montré que la LC-HRMS associée à des outils de fouille de données permet (i) une recherche ciblée de polluants prioritaires, (ii) la détection sans a priori de contaminants inattendus ou inconnus et (iii) la recherche de métabolites nécessaire à la classification des échantillons en fonction de leur métadonnée (adultération, origine géographique, etc.). Ensuite, une approche semi-quantitative ciblée large spectre a été développée et validée pour la détection de pesticides, médicaments et métabolites dans l’eau par couplage de la SPE en ligne avec l’UHPLC-ESI-HRMS. Cette méthode permet la détection de 539 contaminants organiques en 36 minutes dans 5 millilitres d’eau au seuil minimum réglementaire de 0,1 µg/L. L’étude de 26 eaux du robinet de la région parisienne a permis de mettre en évidence la présence à l’état de trace de 37 polluants dont 6 médicaments. / Pollution of agrifood and environmental water by pesticides and drugs is a real public health concern. There are many quantitative methods developed on triple quadrupole mass spectrometer for their detection, but drug residues and abiotic or biological degradation of pollutants are generally not considered and are limited to a predetermined and restricted list of molecules.In this context, we have developed an analytical method based on metabolomic analyses by high performance liquid chromatography coupled with ultra-high resolution mass spectrometry (LC-HRMS). Two matrices (honey and groundwater) were investigated as proof of concept studies. We showed that LC-HRMS associated to data mining tools enables (i) targeted analyses of pollutants, (ii) detection of untargeted and unknown xenobiotics, and (iii) detection of metabolites useful for the characterization of matrices. Then, a large targeted and semi-quantitative approach has been developed and validated for the detection of pesticides, drugs and metabolites in water samples by using on line SPE and UHPLC-ESI-HRMS. This method allowed the detection of 539 organic contaminants at the MLR of 0.1 µg/L in 36 minutes with only 5 milliliter of water. A study of 26 tap water samples from the Paris region showed the presence of 37 pollutants including 6 drugs.

Mansoa hirsute (Bignoniaceae), popularly known as Cipo d'álho, is chemically little known, although it is popularly used in the treatment of many disesases. The present studiy aimed to study the biological potential of the extracts and fractions obtained from leaves of M. hirsuta, to isolate and purify the active principles and to study the metabolomics of the plant. Plant materials were collected in five different regions from Semi-Arid in northeastern Brazil and were submitted to classic phytochemistry and analysis of the metabolomics. For that, the brute extract of the plants of Gentio do Ouro was obtained by extraction with ethanol 96% and was submitted to partitioning with different solvents, while the volatile were extracted by Clavenger apparatus. The isolated compounds were identified by GC-MS and NMR. Extract and subfractions were submitted to biological tests to clarify their possible biological activities. The brute extracts of the plants provenientes of the five regions was submitted to NMR analysis RMN to adquire 1D 1H and 2D COSY and J-resolved spectra. NMR data were submitted to chemiometry (HCA, PC and PSL-of the) to classify the species according to their metabolome. Pentacyclic triterpenes ursólico and oleanolic acids, and the steroids stigmasterol, ß-stitosterol and lupeol were isolated, together with dialyl sulfide and trialyl sulfide. The ethyl acetate fraction was able to inhibit the production of NO (57.92%) and proliferation of linfocites (99.92%), while the acid ursólico and oleanolic presented inhibition values greater than 99%. Metabolic analysis allowed to establish that plants of Gentio do Ouro are rich in pentacyclic triterpens, while plants from Boninal contains mostly flavonoids in its chemical composition. The results also suggest that protoantocianidinas are the main constituents of the plants from Caetité, while saponinas are found in the plants from Campo Alegre de Lourdes. Plants of Morro do Chapéu were distinguished form the other regions for being rich in oligosaccharides and aminoacids. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A espécie Mansoa hirsuta (Bignoniaceae), popularmente conhecida como Cipó d'alho, é quimicamente pouco conhecida, embora seja popularmente utilizada no tratamento de muitas infecções. O presente trabalho objetivou estudar o potencial biológico dos extratos e frações obtidos das folhas de M. hirsuta, isolar e purificar os possíveis princípios ativos e estudar o perfil metabolômico da planta. O material vegetal foi coletado em cinco regiões diferentes do Semi-Árido nordestino e foi submetido à fitoquímica clássica e análise metabolômica. Para tanto, o extrato bruto das plantas de Gentio de Ouro foi obtido pela extração com etanol 96% e submetido a fracionamento com diferentes solventes, enquanto os voláteis foram extraídos por Clavenger. Os compostos isolados foram identificados por GC-MS e dados de RMN. O extrato e as subfrações foram submetidos a testes biológicos para esclarecer suas possíveis atividades biológicas. Os extratos brutos das plantas provenientes das cinco regiões foi submetido a análise por RMN para obtenção de espectros 1D 1H e 2D COSY e J-resolvido. Os dados provenientes do RMN foram então submetidos à métodos quimiométricos (HCA, PCA e PSL-DA) com vistas a classificar as espécies de acordo com seu metaboloma. Os triterpenos pentacíclicos ácidos ursólico e oleanólico e os esteróides estigmasterol, β-stitosterol e lupeol foram isolados, juntamente com o dissulfeto de dialila e trissulfeto de dialila. A fração acetato de etila inbiu a produção de NO (57,92%) e a linfoproliferação (99.92%), enquanto os ácidos ursólico e oleanólico apresentaram valores de inibição maiores que 99%. A análise metabólica permitiu estabelecer que as plantas oriundas de Gentio de Ouro são ricas em triterpernos pentacíclicos, enquanto as plantas de Boninal contêm principalmente flavonóides em sua composição química. Os resultados também sugerem que as protoantocianidinas são os principais constituintes das plantas oriundas de Caetité, enquanto saponinas são encontradas na plantas de Campo Alegre de Lourdes. As plantas de Morro do Chapéu diferem daquelas das demais regiões por serem ricas em oligossacarídeos e aminoácidos.