Yes / Hair fibres are extraordinary materials, not least because they are exquisitely formed by each of the 5 million or so hair follicles on our bodies and have functions that cross from physiology to psychology, but also because they have well known resistance to degradation as seen in hair surviving from archaeological and historical samples [1]. Hair fibres on the head grow at around 1cm each month, together totalling approximately 12km of growth per person per year. Each fibre is incredibly strong for its small diameter; with one fibre typically holding 100g and together a well-formed ponytail [allegedly] has the collective strength to support the weight of a small elephant! Hair – and from here I mean scalp hair – is under constant scrutiny by each of us; whether it be style, split ends, the first few grey hairs or the collection of hairs in the shower that should be firmly attached - leading to the fear that is hair loss.

Οι αναλύσεις συστατικών του αίματος αποτελούν σημαντική παράμετρο στην κλινική πράξη, επηρεάζοντας τη διάγνωση ασθενειών και το σχεδιασμό κατάλληλων θεραπευτικών αγωγών. Η απομόνωση πλάσματος με χρήση φιαλιδίων συλλογής (vacutainers), που περιέχουν κάποιο αντιπηκτικό, είναι ένα καθημερινό φαινόμενο σε νοσοκομεία και κλινικές. Η μη σωστή επιλογή του κατάλληλου αντιπηκτικού και πρωτοκόλλου επεξεργασίας των δειγμάτων μπορεί να οδηγήσει σε λανθασμένα αποτελέσματα με κόστος χρημάτων και κυρίως κόστος ανθρώπινων ζωών. Επίσης, οι καθιερωμένες σήμερα βiοχημικές εξετάσεις παρέχουν συνήθως μεμονωμένες και όχι ολιστικές μετρήσεις του μεταβολικού/βιοχημικού προτύπου του δότη. Αυτό σημαίνει πως πρέπει συνήθως να ληφθούν πολλά δείγματα ή και μεγάλη σχετικά ποσότητα δείγματος, με καταπόνηση του δότη, και να εφαρμοστούν πολλαπλά πρωτόκολλα διαφορετικής ακρίβειας για τις επιμέρους αναλύσεις. Υπάρχει επομένως αναγκαιότητα για την προτυποποίηση των πρωτοκόλλων συλλογής και χειρισμού των δειγμάτων αίματος στο πλαίσιο ολιστικών μελετών του προτύπου συγκέντρωσης μεταβολιτών του. Με την παρούσα εργασία διερευνάται η τυχόν επίπτωση του αντιπηκτικού και συγκεκριμένα των δύο ευρύτερα χρησιμοποιούμενων στις βιοχημικές αναλύσεις πλάσματος, δηλαδή EDTA και ηπαρίνης, στην μέτρηση του προτύπου συγκέντρωσης των μεταβολιτών μικρού μοριακού βάρους στο πλάσμα αίματος με τη χρήση χρωματογραφίας αερίων – φασματομετρίας μάζας. Για την επίτευξη του στόχου αυτού, αναλύονται και συγκρίνονται τα πρότυπα συγκέντωσης μεταβολιτών στο πλάσμα τριών εθελοντών, που απομονώθηκε (α) σε φιαλίδιο συλλογής αίματος (vacutainer) εσμυρισμένο με ηπαρίνη λιθίου (lithiumheparin) της εταιρείας BD και (β) σε φιαλίδιο falcon που περιέχει 400μL διαλύματος 0,5M EDTA pH 8,0. Τα αποτελέσματα αυτής της ανάλυσης έδειξαν πως το υγρό EDTA επηρέασε την παραγώγιση του εκχυλίσματος των μεταβολιτών, ένα στάδιο που είναι απαραίτητο στη μεταβολομική με χρωματογραφία αερίων – φασματομετρία μάζας και δεν είναι δυνατή η ποσοτική σύγκριση μεταξύ των δύο προτύπων. Όμως, ποιοτική σύγκριση μεταξύ των προτύπων των δειγμάτων στα δύο αντιπηκτικά, έδειξε ότι μετρώνται οι ίδιοι μεταβολίτες, με εξαίρεση την ιδιαίτερα αυξημένη συγκέντρωση της κλειστής μορφής της γλυκόζης στα δείγματα που απομονώθηκαν στο EDTA. Αυτό συνάδει με προηγούμενες μελέτες που ανέφεραν την επίδραση της ηπαρίνης στη μέτρηση της γλυκόζης στο αίμα. Περαιτέρω συγκριτική ανάλυση με τη χρήση εσμυρισμένων φιαλιδίων συλλογής με EDTA και ηπαρίνη, σε διαφορετικούς χρόνους παραμονής του αίματος στο αντιπηκτικό και περισσότερα δείγματα είναι απαραίτητες για την αριστοποίηση των πρωτόκολλων συλλογής δειγμάτων πλάσματος και ορού για μεταβολομικές και πρωτεωμικές αναλύσεις. / Blood tests constitute an important part of clinical practice, contributing significantly to disease diagnosis and the design of appropriate therapeutic treatments. Plasmaisolationusingcollection tubescontaining a specific anticoagulant, aka vacutainers, isa regular procedure taking place multiple times per day in hospitals and medical clinics. Erroneous selection of the appropriate anticoagulant, as well as the sample collection and handling protocol may lead to inaccurate measurementswith negative financial consequences, but mainly to the medical care of the patients. Moreover, current standard biochemical tests usually measure individual biochemical parametersand not the holistic metabolic and biochemical profile of the patient. This may mean multiple collections of relatively large volumes of blood samples from the subjects,while various analytical protocols of different accuracy have to be followed. Therefore, there is a need for the standardization of the protocols for the collection and handling of blood samples in the context of holistic analyses of its metabolite concentration profile. The present study aims at investigating the effect of anticoagulants, and more specifically of the two more widely used in biochemical analysis of blood plasma, EDTA and heparin, on the measurement of the concentration profile of low molecular weight metabolites in the blood plasma, using gas chromatography - mass spectrometry (GC-MS). To achieve this objective, the plasma metabolite concentration profilesof three donors were measured after plasma isolation in (a) BD lithium heparin glass vacutainers and (b) falcon tubes containing 400mL of n 0.5 M EDTA pH 8.0 solution. The results of this analysis showed that the liquid EDTA influenced the derivatization of metabolite extract, derivatization being a necessary step in GC-MS metabolomic analysis. Thus, the obtained metabolic profiles of plasma from isolation in EDTA and heparin cannot be quantitatively compared. However, a qualitative comparison between indicated that the same metabolites can be quantified in the two profiles. The only major difference was the observed high concentration of glucose in the form of glucopyranose in the plasma samples from the EDTA isolation compared to those isolated in heparin. This observation is consistent with previous studies that have reported the negative effect of heparin isolation on the measurement of plasma glucose. Further comparative analysis between the two isolation protocols using glass vacutainersfor both coagulants investigating also the effect of the duration of the isolation protocol in a larger number of samplesis necessary for optimizing the plasma and serumsamples collection protocols for metabolomics and proteomic analyses.

No description available.

Ce travail est composé de trois projets. Le premier projet a pour objectif de découvrir de nouveaux métabolites de la vitamine A. Il a consisté en la synthèse d’un analogue du rétinaldéhyde, portant une fonction azoture et permettant de suivre son devenir in vivo. Le second projet a consisté en l’élaboration de la sonde ATPP permettant l’analyse de l’ensemble des métabolites aldéhydiques d’un échantillon. La sonde permet un gain de sensibilité en LS-MS². Une analyse de sa biodistribution a été faite, et montre que la sonde ATPP, après injection intrapéritonéale, est distribuée in vivo. Concernant le troisième projet, un réactif de couplage homobifonctionnel « thiol-thiol » a été élaboré. Les produits du couplage ont montré une excellente stabilité plasmatique. Le réactif a d’abord été appliqué avec succès au couplage de petites molécules, puis au couplage d’un oligonucléotide modifié et d’un peptide. / This work consists of three projects. The first project aims to discover new metabolites of vitamin A. An analog of retinaldehyde, carrying an azide function was synthesized. It would allow to follow its fate in vivo. The second project consisted in the development of a probe allowing the analysis of all the aldehyde metabolites in a sample. The probe provides sensitivity gain in LS-MS². An analysis of its biodistribution has been done, and showed the ATPP probe is distributed after an intraperitoneal injection. Concerning the third project, a homobifunctional coupling reagent "thiol-to-thiol" has been developed. The coupling products showed excellent plasma stability. The reagent was first successfully applied to the coupling of small molecules and then to the coupling of a modified oligonucleotide and a peptide.

Le profilage métabolomique global de matrices biologiques dans de larges séries d'échantillon est un enjeu majeur. Dans ce contexte, notre travail vise à développer des approches LC-HRMS et outils bioinformatiques pour les analyses métabolomique et lipidomique de larges cohortes. Dans un premier temps, nous avons développé puis évalué la pertinence de 4 méthodes LC-HRMS dans l'annotation du métabolome/lipidome sérique humain. Ainsi, une base de données spectrales a été implémentée à l'aide de spectres MS, MS/MS et les temps de rétention de composés de référence afin d'assurer l'annotation de jeux de données. La combinaison de méthodes RP, HILIC et PFPP-HRMS a permis l'identification de 266 métabolites et 706 espèces lipidiques sériques répartis sur 20 et 24 classes chimiques respectivement dont 27% d'espèces isomères. Ces outils ont été appliqués, dans un second temps, à la stratification de 78 patients diabétiques. Outre le syndrome métabolique marqué (perturbation du métabolisme énergétique), nos analyses ont montré l'impact délétère de facteurs physiologiques confondants -âge et IMC-. Nous en avons évalué l'influence sur une cohorte de 227 salariés du CEA. Les empreintes lipidomiques sont robustes, néanmoins l'impact de l'IMC est marqué pour les lipides neutres. L'effet du genre démontre un catabolisme masculin important. L'effet de l'âge se manifeste par des activités enzymatiques altérées. Ces études combinent une analyse globale métabolomique et lipidomique des mêmes échantillons humains. Elles visent à construire une base de données relationnelle incluant données spectrales et biologiques servant à la caractérisation de biomarqueurs dans le cas d'études cliniques. / Global metabolomic profiling of biological media in large sample sets is a major challenge. In this context, our work aims to develop LC-HRMS approaches and data mining tools for metabolomics and lipidomics analysis of large cohorts. We have first developed and evaluated the reliability of four LC-HRMS methods in the annotation of human serum metabolome and lipidome. Thus, spectral database was implemented using MS spectra, MS/MS and retention times of reference compounds to further ensure datasets annotation. The combination of RP, PFPP and HILIC-HRMS methods allowed identification of 266 metabolites and 706 lipid species in human serum over 20 to 24 chemical classes respectively including 27% of isomeric species. These analytical tools were then applied for the stratification of 78 diabetic patients. Unsurprisingly, we highlighted a metabolic syndrome (energy metabolism disruption), moreover our analyses have shown the deleterious impact of confounding physiological factors on diabetes biomarker discovery –age and BMI-. We finally evaluated their influence on a cohort of 227 CEA employees. Lipidomic fingerprints are robust, however BMI impact is marked for neutral lipids. Gender effect shows significant male catabolism and age altered enzyme activities. These studies combine an overall metabolomics and lipidomics analyses of the same human samples. They aim to build up a relational database including spectral and biological data for biomarker characterization in clinical studies.

No description available.

In the first part of this work, LC-MS data were used to calculate the in-vitro intrinsic clearances (CLint) for the metabolism of p-methoxyrnethamphetamine (PMMA) and fluoxetine by the CYP2D6 enzyme using a steady-state (SS) approach and a new general enzyme (GE) screening method. For PMMA, the SS experiment resulted in a CLint of 2.7 ± 0.2 µL pmol 2D6-1min-1 and the GE experiment resulted in a CLint of 3.0 ± 0.6 µL pmol 2D6-1min-1. For fluoxetine, the SS experiment resulted in a CLint of 0.33 ± 0.17 µL pmol 2D6-1min-1 and the GE experiment resulted in a CLint of 0.188 ± 0.013 µL pmol 2D6-1min-1. The inhibition of PMMA metabolism by fluoxetine was also demonstrated.In the second part of the work, target factor analysis was used as part of a library search algorithm for the identification of drugs in LC-DAD chromatograms. The ability to resolve highly overlapped peaks using the spectral data afforded by the DAD is what distinguished this method from conventional library searching methods. A validation data set of 70 chromatograms was used to calculate the sensitivity (correct identification of positives) and specificity (correct identification of negatives) of the method, which were 92% and 94% respectively.Finally, the last part of the work shows the development of data analysis methods for four-way data generated by two-dimensional liquid chromatography separations with DAD. Maize seedlings were analyzed, specifically focusing on indole-3-acetic acid (IAA) and related compounds. Window target testing factor analysis was used to identify the spectral groups represented by the standards in the mutant and wild-type chromatograms. Two curve resolution algorithms were applied to resolve overlapped components in the data and to demonstrate the quantitative potential of these methods. A total of 95 peaks were resolved. Of those peaks, 45 were found in both the mutant and wild-type maize, 16 peaks were unique to the mutants, 13 peaks were unique to the wild-types, and the remaining peaks were standards. Several IAA conjugates were quantified in the maize samples at levels of 0.3 - 2 µg/g plant material.

La métabolomique permet une étude à large échelle du profil métabolique d'un individu, représentatif de son état physiologique. La comparaison de ces profils conduit à l'identification de métabolites caractéristiques d'une condition donnée. La métabolomique présente un potentiel considérable pour le diagnostic, mais également pour la compréhension des mécanismes associés aux maladies et l'identification de cibles thérapeutiques. Cependant, ces dernières applications nécessitent d'inclure ces métabolites caractéristiques dans un contexte plus large, décrivant l'ensemble des connaissances relatives au métabolisme, afin de formuler des hypothèses sur les mécanismes impliqués. Cette mise en contexte peut être réalisée à l'aide des réseaux métaboliques, qui modélisent l'ensemble des transformations biochimiques opérables par un organisme. L'une des limites de cette approche est que la métabolomique ne permet pas à ce jour de mesurer l'ensemble des métabolites, et ainsi d'offrir une vue complète du métabolome. De plus, dans le contexte plus spécifique de la santé humaine, la métabolomique est usuellement appliquée à des échantillons provenant de biofluides plutôt que des tissus, ce qui n'offre pas une observation directe des mécanismes physiologiques eux-mêmes, mais plutôt de leur résultante. Les travaux présentés dans cette thèse proposent une méthode pour pallier ces limitations, en suggérant des métabolites pertinents pouvant aider à la reconstruction de scénarios mécanistiques. Cette méthode est inspirée des systèmes de recommandations utilisés dans le cadre d'activités en ligne, notamment la suggestion d'individus d'intérêt sur les réseaux sociaux numériques. La méthode a été appliquée à la signature métabolique de patients atteints d'encéphalopathie hépatique. Elle a permis de mettre en avant des métabolites pertinents dont le lien avec la maladie est appuyé par la littérature scientifique, et a conduit à une meilleure compréhension des mécanismes sous-jacents et à la proposition de scénarios alternatifs. Elle a également orienté l'analyse approfondie des données brutes de métabolomique et enrichie par ce biais la signature de la maladie initialement obtenue. La caractérisation des modèles et des données ainsi que les développements techniques nécessaires à la création de la méthode ont également conduit à la définition d'un cadre méthodologique générique pour l'analyse topologique des réseaux métaboliques. / Metabolomics allows large-scale studies of the metabolic profile of an individual, which is representative of its physiological state. Metabolic markers characterising a given condition can be obtained through the comparison of those profiles. Therefore, metabolomics reveals a great potential for the diagnosis as well as the comprehension of mechanisms behind metabolic dysregulations, and to a certain extent the identification of therapeutic targets. However, in order to raise new hypotheses, those applications need to put metabolomics results in the light of global metabolism knowledge. This contextualisation of the results can rely on metabolic networks, which gather all biochemical transformations that can be performed by an organism. The major bottleneck preventing this interpretation stems from the fact that, currently, no single metabolomic approach allows monitoring all metabolites, thus leading to a partial representation of the metabolome. Furthermore, in the context of human health related experiments, metabolomics is usually performed on bio-fluid samples. Consequently, those approaches focus on the footprints left by impacted mechanisms rather than the mechanisms themselves. This thesis proposes a new approach to overcome those limitations, through the suggestion of relevant metabolites, which could fill the gaps in a metabolomics signature. This method is inspired by recommender systems used for several on-line activities, and more specifically the recommendation of users to follow on social networks. This approach has been used for the interpretation of the metabolic signature of the hepatic encephalopathy. It allows highlighting some relevant metabolites, closely related to the disease according to the literature, and led to a better comprehension of the impaired mechanisms and as a result the proposition of new hypothetical scenario. It also improved and enriched the original signature by guiding deeper investigation of the raw data, leading to the addition of missed compounds. Models and data characterisation, alongside technical developments presented in this thesis, can also offer generic frameworks and guidelines for metabolic networks topological analysis.

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

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.