Les microalgues présentes à la fois dans les eaux douces et salées compteraient plus de 200 000 espèces. Cette diversité en fait une source potentielle de métabolites spécialisés originaux. Parmi les principales familles de substances naturelles valorisées actuellement, on peut citer les pigments, lipides, protéines, polysaccharides, caroténoïdes. Une vision plus globale du métabolome de chacune des espèces apparaît aujourd’hui nécessaire pour mieux mettre en valeur le potentiel commercial que représente cette « microbiodiversité ». Pour cela, nous avons tout d’abord choisi d’approcher le métabolome de différentes souches de microalgues cultivées au sein de la Société Greensea en s’appuyant sur les techniques d’HPTLC, de RMN et d’UHPLC-QTOF pour une visualisation large. Cette étude nous a permis de regrouper les espèces par analogie métabolique après traitement statistique des données. Une seconde partie a consisté en une étude phytochimique approfondie de certaines souches et a conduit à l’isolement et la caractérisation de plusieurs molécules. Ainsi, en plus de métabolites connus, un peptide original portant un motif isoprényl, le cumbriamide a été caractérisé au sein de Lyngbya sp. et une première évaluation de son potentiel thérapeutique a été entreprise. Une large diversité en glycolipides s’est montrée prépondérante dans de nombreuses souches et une méthode de caractérisation a pu être mise au point pour leur identification par UHPLC-QTOF. Enfin, différentes applications des approches métabolomiques ont été envisagées. Ainsi, des études chimiotaxonomiques ont été menées sur les différentes souches de microalgues et l’influence de changements de conditions de culture sur la production de métabolites chez Nannochloropsis oculata a été observée. / Microalgae are present both in Oceans and freshwaters and could include more than 200 000 species. This diversity is a source of original specialized metabolites that can find a large array of applications. Pigments, lipids, proteins, polysaccharides and carotenoids are usual compounds produced by microalgae that have found commercial applications. A global vision of the metabolome of each species has showed promises to highlight the commercial value of this “microdiversity”. We then decided to assess the metabolome of several microalgae species grown at the Greensea company by using HPTLC, NMR and UHPLC-QTOF techniques for a rapid and global overview. A classification of the species according to their metabolomics similarities was obtained after statistics treatment of the data. A second part was dedicated to a phytochemical study of the extracts of selected strains and led to the isolation and characterization of several metabolites. Thus, in addition to known molecules, an original peptide substituted by an isoprenyl moiety and named cumbriamide has been characterized in Lyngbya sp and a first assessment of its therapeutical potential has been undertaken. Glycolipids have been identified as the major metabolites in the extracts of numerous strains and a UHPLC-QTOF method was developed for their identification. Finally, several applications of the metabolomics approaches were considered. Chemotaxonomic studies were first carried out and the influence of growth conditions on the metabolome of Nannochloropsis oculata was observed.

Orientador: Anita Jocelyne Marsaioli / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Quimica / Made available in DSpace on 2018-08-13T13:31:18Z (GMT). No. of bitstreams: 1 Martins_LucasGelain_M.pdf: 1200827 bytes, checksum: 8708dd4e868d8749ef0f4a5d27b03248 (MD5) Previous issue date: 2009 / Resumo: Técnicas de RMN de H foram aplicadas no estudo de metabolômica de Theobroma cacao e interações proteínas ¿ ligantes. Esses dois tópicos são reportados em dois capítulos. O primeiro descreve a análise quantitativa por RMN de H de 8 metabolitos de Theobroma cacao endógenos ou produzidos durante a fermentação de 10 diferentes variedades resistentes ao fungo Moniliophthora perniciosa. Os espectros foram obtidos de extratos aquosos saturando o sinal da água com pressaturação. Os metabólitos monitorados foram: glicose, sacarose, frutose, etanol, cafeínas e os ácidos acético, lático e succínico. O teor dos metabólitos presentes em 7 dessas variedades são similares àqueles de T. cacao comum e análise sensorial selecionou aquelas com maior teor de cafeína como as melhores. O segundo capítulo descreve as interações acetilcolinesterase/alcalóides de Amaryllidaceae e proteína de membrana bacteriana/fosfomicina, com os mapas de epitopo de interação dos ligantes com as macromoléculas determinados por RMN de H ¿ STD (Saturation Transfer Difference). O mapa de epitopo para acetilcolinesterase/fisostigmina foi confirmado por cálculos de ¿doking¿ molecular. A constante de dissociação aparente da fisostigmina com acetilcolinesterase foi obtida através das medidas de T1 seletivo. Interação entre células integras de Escherichia coli CCT 5050, Serratia liquefaciens CCT 7262 e fosfomicina indicaram que microrganismo resistente ao antibiótico (Serratia liquefaciens) não apresentaram sinais no experimento de RMN de H ¿ STD enquanto que o microrganismo não resistente (Escherichia coli) apresenta sinais no mesmo experimento. Esses resultados certamente contribuirão para a elucidação do mecanismo de ação da fosfomicina / Abstract: H NMR tecniques were applied to study Theobroma cacao metabolomics and protein-ligand supramolecular interactions. These two topics are reported in two chapters. First chapter describes the H NMR quantitative analysis of 8 Theobroma cacao secondary metabolites which are endogenous or produced during the fermentation process of 10 different varieties resistent to the Moniliophthora perniciosa fungus. The spectra were obtained from the aqueous extracts saturating the water signal with the PRESAT tecnique. The monitored metabolites were: glucose, sacharose, fructose, ethanol, cafeine and the acids acetic, latic and succinic. The abundance of the metabolites present in 7 of these varieties were similar to those in comon T. Cacao and sensory analysis selected those with high cafeine content as the best. The second chapter describes the supramolecular interactions of acetylcholisterase/Amaryllidaceae alkaloids and bacterial membrane protein /fosfomicine, with the epitope mapping of the interactions of the ligands to the macromolecules obtained by H NMR STD (saturation transfer difference). The epitope mapping of acetylcholisterase/Amaryllidaceae alkaloids was confirmed by molecular docking calculations. The apparent dissociation constant of the fisostigmine which is an acetylcholinesterase inhibitor was obtained by applying selective T1. Interactions of Escherichia coli CCT 5050, Serratia liquefaciens CCT 7262 whole cells and fosfomycin indicated that microorganisms resistent to this antibiotic (Serratia liquefaciens) did not show signal in the H NMR STD experiment while non resistent microorganism (Escherichia coli) showed signals in the same experiment. This data will certainly contribute to the elucidation of fosfomycin action mechanism / Mestrado / Quimica Organica / Mestre em Química

Orientador: Anita Jocelyne Marsaioli / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-21T04:07:12Z (GMT). No. of bitstreams: 1 Lopes_ThiagoInacioBarros_M.pdf: 3472847 bytes, checksum: 6ec44b5e5d2ad869887d1d1a884a0682 (MD5) Previous issue date: 2012 / Resumo: A organização mundial de saúde (OMS) estima que existam atualmente mais de 1,5 bilhões de adultos com sobrepeso, número que é esperado dobrar até 2015. Obesidade e sobrepeso são enfermidades caracterizadas pelo acúmulo excessivo de gordura corporal e têm sido associadas a vários problemas de saúde. Vários fármacos têm sido utilizados no tratamento desta enfermidade, entre os mais utilizados se encontra o Orlistate, um inibidor de lipases gástricas utilizado na redução da absorção de gordura da dieta, auxiliando na perda e manutenção do peso. Esta dissertação teve como objetivo principal avaliar as alterações metabólicas sofridas por pacientes com sobrepeso tratados com Orlistate por 120 dias. Para tornar mais didático, o trabalho foi divido em duas Partes: Parte I, implementação da metodologia analítica para análise de ácidos graxos por CG-EM e avaliação do perfil de ácidos graxos em indivíduos com sobrepeso tratados com Orlistate; e Parte II, avaliação do perfil metabólico de plasma por RMN de ¹H de indivíduos com sobrepeso tratados com Orlistate. Na primeira etapa do trabalho, análise de componentes principais foi aplicada na seleção íons (m/z) para quantificação de ácidos graxos, após preparação de ésteres metílicos correspondentes, por CG-EM usando monitoramento de íons selecionados. Quatro íons foram então selecionados de forma a aumentar a detectividade sem perda completa de informação qualitativa. Os íons de m/z 74, 79, 81 e 87 foram selecionados e permitiram a quantificação de vários ácidos graxos, além da determinação do número de insaturações dos mesmos relacionando a abundância relativa dos íons à presença de insaturações. A metodologia analítica implementada permitiu quantificar ácidos graxos esterificados em vários lipídios presentes no sangue, após transesterificação para produção dos ésteres metílicos de ácidos graxos, com adequada precisão, repetitividade e baixos limites de detecção e quantificação. Na segunda etapa, a metodologia analítica foi aplicada no estudo do perfil de ácidos graxos de 20 mulheres com sobrepeso tratadas com Orlistate durante 120 dias. O tratamento não reduziu o índice de massa corpórea contribuiu para diminuição significativa dos níveis de colesterol-HDL no plasma e do conteúdo de colesterol na membrana de eritrócitos, além de alterar as proporções relativas de vários ácidos graxos essenciais e exógenos em vários lipídios estudados. Adicionalmente foi observado um perfil de ácido graxo significativamente diferente para os indivíduos magros (controles) em comparação com indivíduos com sobrepeso. Na última etapa, o perfil metabólico de plasma por RMN de ¹H foi estudado por uma abordagem metabolômica. A análise discriminatória por quadrados mínimos parciais (PLS-DA) revelou que alterações nos níveis de lactato e magnésio são importantes na diferenciação entre indivíduos com sobrepeso tratados e não-tratados com Orlistate, sinalizando diminuição destes metabólitos relacionada ao tratamento. Não há relatos anteriores da alteração dos níveis de lactato devido ao tratamento. Adicionalmente, os níveis de triacilglicerídios, alanina e lactato contribuíram significativamente na distinção entre indivíduos magros e com sobrepeso / Abstract: The World Health Organization (WHO) estimates that there are currently more than 1.5 billion overweight adults, a number that is expected to double until 2015. Obesity and overweight are diseases characterized by excessive accumulation of body fat and linked to various health problems. Several drugs have been used to treat such diseases. Orlistat is a gastric lipase inhibitor largely used to reduce the absorption of dietary fat, helping weight loss. Thus this thesis aimed at the metabolic variation of overweight subjects treated with Orlistat for 120 days. This work is divided into two parts: Part I, implementation of the analytical methodology for analysis of fatty acids by GC-MS and evaluation of the fatty acid profile in overweight subjects treated with Orlistat; and Part II, evaluation of the metabolic profile of plasma of overweight subjects treated with Orlistat using ¹H NMR. In the Part I of this work, principal component analysis was applied to selected ions (m/z) for determination of fatty acids, after preparation of the corresponding methyl esters, by GC-MS using selected ion monitoring. Four ions were selected. The ions of m/z 74, 79, 81 and 87 allowed the quantification of various fatty acids and determination of the number of double bounds in theo fatty acids through the relative abundance of these selected ions. The analytical methodology implemented permitted quantify esterifing fatty acids in various lipids in blood, after transesterification for production of methyl esters of fatty acids, with adequate accuracy, repeatability and low limits of detection and quantification. In the second step, the analytical methodology was applied to study the fatty acid profile of 20 overweight women treated with Orlistat for 120 days. Although there was no significant reduction in body mass index, the treatment contributed to significant reduction of HDL-cholesterol levels in plasma and the cholesterol content in erythrocyte membranes. The Orlistat also alters the relative proportions of various fatty acids in the several lipids studied. Additionally, a significantly different fatty acid profile was observed for lean subjects (controls) compared to overweight subjects. In Part II, the metabolic profile of plasma obtained by ¹H NMR was studied by a metabolomics approach. The partial least squares discriminant analysis (PLS-DA) revealed changes in lactate and magnesium level, these metabolites were important to differentiating between overweight subjects treated and not-treated with Orlistat, suggesting that the level of these metabolites decreased with treatment. There are no previous reports of changes in lactate levels. Additionally, levels of triglycerides, alanine and lactate were highlighted by PLS-DA into distinguishing between lean and overweight individuals / Mestrado / Quimica Organica / Mestre em Química

Le paludisme est une des maladies tropicales les plus dévastatrices au monde causée par des parasites protozoaires intracellulaires du genre Plasmodium. Cinq espèces de plasmodies sont responsables du paludisme chez l'homme et causent 600 000 décès par an principalement chez les enfants de moins de 5 ans et les femmes enceintes vivant dans les régions les plus pauvres du globe. Les parasites ont généré une résistance contre les chimiothérapies existantes et aucun vaccin efficace n'est encore disponible. Il est donc impératif d'identifier et de valider de nouvelles cibles qui peuvent être exploitées pour la découverte de nouveaux médicaments.Cette étude a porté sur la caractérisation d'un enzyme, la phosphoénolpyruvate carboxylase (PEPC), produit d'un gène spécifique au parasite et absent chez l'hôte humain, ce qui constitue l'un des pré-requis d'une cible potentielle pour la découverte de médicaments. Le gène avait été montré comme essentiel pour des parasites seulement en absence de malate ou de fumarate, suggérant un rôle de la protéine dans le métabolisme du carbone intermédiaire des parasites.Mes études de thèse avaient pour but de caractériser le rôle de la PEPC en utilisant la métabolomique. J'ai d'abord établi et normalisé une méthodologie d'analyses métabolomiques des globules rouges infectés par Plasmodium et optimisé l'analyse des métabolites hydrophiles présents dans le parasite intracellulaire et sa cellule hôte. Nous nous sommes concentrés sur les métabolites du métabolisme du carbone intermédiaire, où la PEPC pouvait jouer un rôle déterminant par analogie avec les plantes et les bactéries. Des analyses ciblées utilisant un marquage isotopique du métabolome à partir de 13C-U-glucose, 13C-bicarbonate et 13C, 15N-glutamine ont aussi été réalisées permettant de mieux appréhender les conséquences d'un KO de l'enzyme PEPC sur le métabolisme du parasite.Les données montrent que l'enzyme PEPC permet une fixation du bicarbonate et catalyse une réaction anaplérotique conduisant à du malate qui est introduit dans le cycle de l'acide tricarboxylique mitochondrial, transférant ainsi des équivalents réducteurs du cytoplasme à la mitochondrie et fournissant aussi un point d'entrée du squelette carboné dans le cycle. Les résultats montrent surtout que les parasites possèdent un cycle complet et de type oxydatif de l'acide tricarboxylique mitochondrial. Il parait y avoir trois points d'entrée: 1. l'acétyl CoA résultant du pyruvate généré par la glycolyse et décarboxylé dans la mitochondrie; 2. l'acide alpha-cétoglutarique provenant du glutamate, qui lui-même résulte de la désamination de la glutamine essentiellement fournie par l'environnement externe; 3. le malate, produit en aval de la malate déshydrogénase qui réduit l'oxaloacétate produit par la PEPC. En aval de la PEPC, la biosynthèse des pyrimidines opère grâce à l'activité de l'aspartate aminotransférase agissant sur oxaloacétate.En dehors du malate, le fumarate est le seul autre métabolite qui permet de s'opposer au défaut de croissance des parasites déficients en PEPC, ce qui a conduit à évaluer le rôle de la fumarase. À cette fin, l'étiquetage du gène endogène fumarase avec une étiquette HA, a permis de montrer que la protéine est exprimée dans les stades intra-érythrocytaires de P. falciparum et de montrer que la protéine se trouve à la fois dans la mitochondrie et le cytoplasme. La protéine recombinante a été exprimée avec succès et partiellement caractérisée biochimiquement. De nombreuses tentatives visant à générer des mutants de délétion génétique de P. falciparum n'ont pas abouti, laissant en suspens la question du caractère essentiel du gène pour les parasites. Cependant, il est possible de cibler le locus du gène via un marquage C-terminal. Ceci suggère que l'enzyme peut être essentielle pour la survie du parasite et donc une cible exploitable pour la découverte d'un type nouveau de médicament antipaludique. / Malaria is one of the world's most devastating tropical diseases caused by obligate intracellular protozoan parasites of the genus Plasmodium. Five species of these parasites cause malaria in humans and infection results in ~600,000 deaths annually primarily in children under the age of 5 and pregnant women living in the poorest areas of the globe. The parasites have an outstanding ability to generate resistance against existing chemotherapies and an efficacious vaccine is not available yet. Therefore it is imperative that attempts are being made to identify and validate new targets that can be exploited for future drug discovery.This study focused on the validation and elucidation of a parasite-specific gene product namely phosphoenolpyruvate carboxylase (PEPC), which is not present in the human host and thus has one of the pre-requisites of a potential drug target. The gene had been previously genetically validated and it was demonstrated that mutant parasites lacking pepc were only viable in the presence of malate or fumarate, suggesting a role of the protein in intermediary carbon metabolism of the parasites.My studies had the goal to assess the role of PEPC using a metabolomics approach. Initially the methodologies to perform metabolomics analyses of Plasmodium-infected RBCs were established and standardised and it was assessed how to best analyse the hydrophilic metabolites present in the intracellular parasites and its host cell. We focused on metabolites of intermediary carbon metabolism, as it is likely that PEPC is important for metabolic functions linked to this in the parasites, in analogy to plants and bacteria. While global metabolomics analyses were appealing, it was decided to apply a targeted metabolomics and comparative approach using stable isotope labelling of the parasite metabolomes with 13C-U-glucose, 13C-bicarbonate and 13C-,15N-glutamine to assess the consequences of the pepc knockout on parasite metabolism.The data demonstrated that PEPC has an anaplerotic function fixing bicarbonate and leading to generation of malate that is fed into the mitochondrial tricarboxylic acid cycle and so transfers reducing equivalents from cytoplasm to mitochondrion as well as providing an entry point of carbon skeleton into the cycle. The most important findings with respect to parasite mitochondrial metabolism were that the parasites possess a complete and oxidative tricarboxylic acid cycle, which appears to have three entry points: 1. Acetyl CoA resulting from glycolytically generated pyruvate that is decarboxylated in the mitochondrion; 2. α-ketoglutarate from the reaction of glutamate dehydrogenase and 3. malate, which is a downstream product of malate dehydrogenase that reduces oxaloacetate the reaction product of PEPC. Other downstream reactions supported by PEPC activity are pyrimidine biosynthesis through the activity of aspartate aminotransferase also acting on the PEPC-derived oxaloacetate.Apart from malate, fumarate was the only other metabolite that reversed the growth defect of pepc mutant parasites. Hence the role of fumarase in the parasites was also assessed. To this end the endogenous fumarase gene of P. falciparum was tagged with an HA-tag, which showed that the protein is expressed in the intra-erythrocytic stages of P. falciparum and demonstrated that the protein is located in both mitochondrion and cytoplasm. In addition, the recombinant protein was produced and partially biochemically characterised. Numerous independent attempts to generate genetic deletion mutants of P. falciparum were unsuccessful, leaving the question whether the gene is essential for the parasites unanswered. However, it was possible to manipulate the locus by C-terminal tagging of the fumarase gene suggesting that fumarase might be indeed essential for parasite survival and therefore possibly suitable for future drug design and discovery.

Obesity is an independent risk factor for ischaemic stroke and may worsen stroke outcome. Therefore, the objective of this thesis was to identify potential interactions between obesity and stroke, with particular focus on inflammatory mechanisms. Ischaemic stroke was surgically induced in obese (ob/ob) and control (ob/-) mice by middle cerebral artery occlusion (MCAO). Outcome was worse in ob/ob mice, which showed rapid and severe ischaemic and vascular damage, characterised by blood vessel haemorrhage as early as 30 minutes post-stroke. The early vascular damage is likely an important driver of subsequent ischaemic damage by exacerbating inflammation in the brain. Assessment of systemic inflammation in ob/ob mice found that stroke had triggered a deregulated inflammatory response in the plasma, spleen, liver and bone marrow, and increased adipose tissue inflammation. Increased vascular damage was hypothesised to mediate the worse outcome found in obese rodents. The structural and inflammatory state of the vasculature was therefore assessed in ob/ob mice prior to and after stroke. Prior to stroke, increased vascular inflammation was found in ob/ob mice, though no differences in tight junction expression or structural alterations were noted. Stroke resulted in vascular damage that was similar on structural and molecular levels between genotypes, though ob/ob mice showed an increase in transcytosis in endothelial cells which may mediate their enhanced blood-brain barrier breakdown. Some studies have found obese patients have better outcome after stroke, perhaps because they are protected from detrimental post-stroke weight loss. The metabolic response to MCAO was therefore assessed, with ob/ob mice showing altered lipid metabolism post-stroke, such as increased fatty acid release from adipose tissue into the plasma. The potential role of fatty acid release in triggering adipose inflammation was also assessed in adipose tissue explants, though limited evidence for an inflammatory response to lipolysis was found. Overall, these findings suggest rapid and enhanced vascular damage may drive worse stroke outcome in ob/ob mice, as may their altered immunological and metabolic states.

La résonance magnétique nucléaire (RMN) est devenue une des techniques spectroscopiques les plus puissantes et polyvalentes de la chimie analytique avec des applications multiples dans des différents domaines de la recherche. Cependant, un des inconvénients majeurs de la RMN est le processus fastidieux d'analyse de donnée qui nécessite fréquemment des interventions humaines. Ces dernières font diminuer non seulement l'efficacité et l'objectivité des études mais également renferment les champs d'applications potentielles de la RMN pour les non-initiés. Par conséquent, le développement des méthodes computationnelles non supervisées se trouve nécessaire. Les travaux réalisés ici représentent des nouvelles approches dans le domaine de la métabolomique et de la biologie structurelle. Le défi ultime de la RMN métabolomique est l'identification complète de l'ensemble des molécules constituant les échantillons biologiques complexes. Cette étape est vitale pour toute interprétation biologique. Dans la première partie de cette thèse, une nouvelle méthode numérique a été développée pour analyser des spectres à deux dimensions HSQC et TOCSY afin d'identifier les métabolites. La performance de cette nouvelle méthode a été démontrée avec succès sur les données synthétiques et expérimentales. La RMN est une des principales techniques analytiques de la biologie structurale. Le processus conventionnel de détermination structurelle est bien établie avec souvent une attribution explicite des signaux. Dans la seconde partie de cette thèse, une nouvelle approche computationnelle est présentée. Cette nouvelle méthode permet de déterminer les structures RMN sans attributions explicites des signaux. Ces derniers proviennent de données spectrales tridimensionnelles TOCSY et NOESY. Les structures ont été résolues en appliquant cette nouvelle méthode aux données spectrales d'une protéine de 12kDa. / Nuclear Magnetic Resonance (NMR) has become one of the most powerful and versatile spectroscopic techniques in analytical chemistry with applications in many disciplines of scientific research. A downside of NMR is however the laborious data analysis workflow that involves many manual interventions. Interactive data analysis impedes not only on efficiency and objectivity, but also keeps many NMR application fields closed for non-experts. Thus, there is a high demand for the development of unsupervised computational methods. This thesis introduces such unattended approaches in the fields of metabonomics and structural biology. A foremost challenge to NMR metabolomics is the identification of all molecules present in complex metabolite mixtures that is vital for the subsequent biological interpretation. In this first part of the thesis, a novel numerical method is proposed for the analysis of two-dimensional HSQC and TOCSY spectra that yields automated metabolite identification. Proof-of principle was successfully obtained by evaluating performance characteristics on synthetic data, and on real-world applications of human urine samples, exhibiting high data complexity. NMR is one of the leading experimental techniques in structural biology. However the conventional process of structure elucidation is quite elaborated. In this second part of the thesis, a novel computational approach is presented to solve the problem of NMR structure determination without explicit resonance assignment based on three-dimensional TOCSY and NOESY spectra. Proof-of principle was successfully obtained by applying the method to an experimental data set of a 12-kilodalton medium- sized protein.

Les expositions environnementales aux pesticides chez la femme enceinte sont une préoccupation de santé publique. L’association d’analyses métabolomiques par RMN chez l’Homme et l’animal a permis de tester l’utilité de cet outil dans l’étude des mélanges complexes. Les empreintes métaboliques proviennent des urines et du sang de cordon de plus de 300 femmes de la cohorte PELAGIE (Bretagne) et des urines, sang, foie et cerveaux de rates et de leurs fœtus exposés par voie orale durant la gestation à 8 pesticides représentatifs des expositions humaines. Une discrimination des profils métaboliques a été observée chez l’Homme en fonction de l’exposition estimée par la part de cultures de céréales dans la commune. Les modifications métaboliques suggèrent un mécanisme osmo-protecteur lié à un stress oxydant. Les données animales ont confirmé ces observations avec des modifications évoquant un dysfonctionnement mitochondrial. Les conséquences sanitaires associées restent à étudier. / Environmental exposure to pesticides is of growing interest in public health, especially in pregnant women. The combination of metabolomics analyses both in human and animal allows testing the utility of this tool in the study of complex mixtures and low-dose exposures. 1H NMR metabolic profiling was carried out on urinary samples collected at the first trimester of pregnancy and cord blood samples at birth for more than 300 women included in the PELAGIE cohort (Britany, France) and on urine, blood, liver and brain from rats and their fetuses orally exposed during the gestation to 8 pesticides representative of human exposures. Based on PLS-DA analyses, a good separation of the metabolic profiles was observed in human according to the percentage of area devoted to cereal crops in the town of residence. The metabolic modifications suggest an osmo-protective mechanism associated with an oxidative stress. Animal data confirmed this hypothesis with plasmatic, liver and brain metabolic changes suggesting a mitochondrial dysfunction. The associated health impacts are yet to be studied.

In recent years, the role of metabolomics has become increasingly more important in the advancement of many research fields including medical studies. Due to lack of metabolomics research in the area of infectious disease and the rise in antibiotic resistance, there is a need for further studies on the modes of antibiotic action and the mechanisms of resistance of pathogenic microorganisms at the metabolome level. This study aimed to investigate effects of DNA synthesis inhibitors on the metabolome of E. coli and to develop a workflow for discrimination between E. coli isolates down to the sub-species level using a variety of methods, which can inform the choice of analytical techniques in metabolomics research. A metabolomics-based approach was used to elucidate metabolic alterations in E. coli K-12 upon challenge with trimethoprim at two pH levels (5 and 7) which mimic human urine acidity. FT-IR spectroscopy was used as a preliminary experiment to produce bacterial fingerprints and GC-MS was applied to generate global metabolic profiles in each condition. At pH 7, as the drug molecules exhibited higher permeability, stronger direct effects of the antibiotic were observed, i.e. decreased levels of nucleotides. Trehalose, an osmoprotectant, was up-regulated in these stress conditions and this up-regulation was mirrored by a decrease in glucose levels. This also correlated with up-regulation of pyruvate-related products (e.g. alanine, citrate and malate). Other off-target related effects were observed such as alterations in the levels of various amino acids upon trimethoprim challenge. This study offered a wider view of drug action at pH levels similar to healthy human urine. A high throughput FT-IR spectroscopy method was developed to discriminate between pathogenic E. coli isolates based on sequence type. This method employed a Bioscreen as a micro-culture incubator instead of traditional sample preparation (shaking flasks), which can be labour intensive and time consuming. Excluding the washing step in the protocol enabled discrimination between isolates of different sequence types. Moreover, a reproducible workflow of lipid analysis based on LC-MS was developed and applied on four pathogenic isolates with different sequence type and susceptibility to ciprofloxacin. This workflow enabled detection of a wide range of lipid classes and determination of significant alterations in lipid levels related to susceptibility to ciprofloxacin. Stressed and control isolates were also analysed using the developed Bioscreen FT-IR approach to assess phenotypic fingerprint differences, which were in line with the LC-MS-ve class distribution. Further investigation by means of four analytical platforms (FT-IR, GC-MS, LC-MS-ve and LC-MS+ve) was applied on E. coli ST131 isolates characterised using classical microbiological tests (virulence factors and metabolic tests). Procrustes transformation was used to compare between the analytical methods and the microbiological tests in terms of their capacity to discriminate between the different isolates. As indicated above, the results from FT-IR and LC-MS-ve were comparable and in line with virulence tests, while GC-MS and metabolic tests were in agreement. Complementary information generated by different analytical techniques and microbiological tests may indicate the requirement for careful selection of the method of investigation and may suggest the need to continue using a combination of methods which are applied to study different features of bacterial physiology.

Introduction: Hypoxia is found in solid cancerous tumours. The presence of hypoxia within tumours inhibits anti-cancer treatment strategies such as chemotherapy from being completely effective and it is suspected that multiple mechanisms contribute to the resistance. Methods: In this project a systems biology approach was applied to determine how the toxicity of doxorubicin is affected by hypoxia at the metabolome level. A multitude of analytical techniques were applied to analyse the intracellular metabolism of a monolayer of cancer cells (MDA-MB-231). Metabolic profiling was used to determine metabolite markers related to hypoxia-induced chemoresistance. For this gas chromatography mass spectrometry (GC-MS) and ultra high performance liquid chromatography mass spectrometry (UHPLC-MS) were used. Furthermore, network-based correlation analysis was developed as a novel tool to bridge the gap between metabolomics dataset and systems biology modelling. This methodology was applied to elucidate novel metabolic pathways as potential therapeutic targets to overcome hypoxia-induced chemoresistance. This algorithm determines significant correlation differences between different physiological states, and through applying graph-theory on large genome scale models; it is possible to construct a metabolic network of the pathways connecting the pair-wise correlation. Finally, imaging mass spectrometry using time-of-flight secondary ion mass spectrometry (ToF-SIMS) was developed as a tool for in situ metabolite analysis to investigate the metabolic response to chemotherapy in multi-tumour spheroids (MTSs). Results: Metabolic fingerprinting analysis characterised a snapshot of cells exposed to various environmental perturbations. Metabolite markers associated with hypoxia-induced chemoresistance were related to metabolic pathways including gluconeogenesis, DNA synthesis and fatty acid synthesis. Furthermore, network-based correlation analysis revealed specific metabolites in the fatty acid synthesis pathways were contributing to drug resistance, which included malonyl-CoA, 3-oxoeicosanoyl-CoA, stearoyl-CoA and octadecanoic acid. To facilitate the detection of metabolites in ToF SIMS datasets, a series of metabolites standard spectra were acquired. Hypoxic metabolite markers detected in ToF-SIMS data of cell lysates included glycine, lactic acid and succinic acid, which were also shown to be metabolite markers in GC-MS metabolic data. Furthermore, MTS sections were imaged using ToF-SIMS to profile the chemical response to chemotherapy treatment within the oxygen gradient. Loadings from image PCA were explored to determine the metabolic response in the highly oxygenated outer region and hypoxic inner region of the MTS. Conclusion: A multitude of analytical techniques were able to contribute to elucidating the metabolic mechanisms associated with hypoxia-induced chemoresistance. Metabolic profiling combined with a systems biology approach was further able to identify potential underlying metabolic regulation of resistance. Finally ToF-SIMS was developed as a tool for metabolite analysis in complex biological systems in situ.

A seleção de animais para consumo alimentar residual (RFI) está intrinsecamente associada com a diminuição do consumo matéria seca e é independente do ganho de peso corporal, selecionando animais de eficiência produtiva e econômica, além também de diminuir a emissão de gases de efeito estufa provinda do gado. Neste estudo, amostras de soro de 16 animais selecionados divergentemente para eficiência de alimentação foram coletadas antes do confinamento (dia -21) e avaliadas em uma abordagem metabolômica global, com o objetivo de usar análise diferencial, análise de co-expressão e enriquecimento funcional, identificando marcadores para eficiência de alimentação antes do confinamento. Um analito foi diferencialmente presente entre os animais de baixo e alto RFI. A análise WGCNA identificou 22 e 25 módulos no modo positivo e negativo, respectivamente e, 1 módulo de cada modo foi fortemente associado a RFI (r = 0,53, p-valor <0,05 e r = 0,52, p-valor <0,1 nos modos negativo e positivo, respectivamente). A análise de enriquecimento funcional predize 13 processos biológicos associados à eficiência alimentar, incluindo alterações no metabolismo de vitaminas lipossolúveis, inflamação, estresse oxidativo, metabolismo de aminoácidos e metabolismo de ácidos graxos. Esse trabalho evidencia a possibilidade de se identificar um biomarcador para eficiência alimentar e também sugerem que as diferenças nas respostas ao estresse oxidativo e nos processos inflamatórios já influenciam na variação da eficiência alimentar previamente ao confinamento. / Animal selection for residual feed intake (RFI) is intrinsically associated with decreased consumption of dry matter independent of body weight gain, selecting yielding increased production and economic efficiency but also decreasing the greenhouse gas emission of livestock. In this study, serum samples of 16 animals selected for divergent feed efficiency were collected prior to feedlot (day -21) and evaluated in an untargeted metabolomics approach, with the goal of using differential analysis, co-expression analysis and functional enrichment to identifier markers for feed efficiency prior to the feedlot. One feature was differentially accumulated between low and high RFI. WGCNA analysis identified 22 and 25 modules in positive and negative mode, respectively, of 1 module of each mode was strongly associated with RFI (r= 0.53, p-value <0.05 and r=0.52, p-value < 0.1 to negative and positive mode, respectively). Pathway enrichment analysis yielded 13 biological processes associated with feed efficiency including alterations in vitamins liposoluble metabolism, inflammation, oxidative stress, amino acid metabolism and fatty acid metabolism. Our findings suggest the possibility to identify a biomarker for feed efficiency and also discuss that differences in oxidative stress responses and inflammatory processes could explain the feed efficiency variation prior to feedlot.