L'écosystème aquatique est le résultat d'un équilibre entre l'environnement naturel et les organismes qui s'y développent. Cet équilibre peut être modifié par l'introduction de substances chimiques dues aux activités humaines. Aujourd'hui, l'impact de cette micropollution est encore mal connu, particulièrement pour les organismes constituant les premiers maillons des chaînes trophiques qui requièrent des efforts analytiques importants en raison de leur faible taille. L'objectif de ces travaux est centré sur le développement, la validation et l'application d'outils analytiques permettant d'étudier la bioaccumulation et la biotransformation de polluants émergents chez des invertébrés benthiques. Cette étude s'est focalisée sur 3 organismes aquatiques d'eau douce : C. riparius, G. fossarum et P. antipodarum. Une méthode analytique pour la quantification de 35 polluants émergents chez les 3 espèces sélectionnées a ainsi été développée. Elle permet d'accéder aux premières données de bioaccumulation des substances d'intérêts à l'échelle d'un individu grâce à la mise en oeuvre d'une stratégie analytique entièrement miniaturisée, incluant une extraction MicroQuEChERS suivie d'une analyse par nanochromatographie liquide couplée à la spectrométrie de masse en tandem. Afin de mieux comprendre l'impact d'une telle pollution sur les espèces sélectionnées et d'obtenir une vue globale des capacités de biotransformation de celles-ci, une approche métabolomique a été mise en place. Enfin, un nouveau mode de quantification par MRM3 a été utilisé pour dépasser la complexité de telles matrices, et fournir une évaluation fiable des cinétiques de bioaccumulation de potentiels traceurs de pollution anthropique / The aquatic ecosystem is the result of a balance between the natural environment and the organisms that inhabit it. This balance can be modified by the input of excessive amount of substances generated from human activities. Nowadays, the impact of this pollution is still little known, especially regarding the risk of bioaccumulation in the first trophic levels. This lack of data could be partially explained by the lack of suitable analytical method for small organisms. In this context, the aim of this study is to establish the development, the validation and the application of analytical tools for the assessment of bioaccumulation and biotransformation of emerging pollutants in freshwater invertebrates. Three benthic invertebrates are chosen for this project: C. riparius, G. fossarum and P. antipodarum. Analytical method has been developed for the quantification of 35 emerging pollutants in the selected species. This method provides the first bioaccumulation data of targeted substances in individual scale through the implementation of miniaturized analytical strategy, including an extraction step based on MicroQuEChERS followed by nanoliquid chromatography coupled to tandem mass spectrometry analysis. To better understand the impact of such pollution and to obtain a global view of the biotransformation capacities of the selected organisms, metabolomics approach has been put in place. Finally, a new quantification mode based on MRM3 was used to overcome biotic matrix complexity and assess the bioaccumulation kinetics of potential tracers of anthropic pollution

Le Cancer, l’une des pathologies les plus fréquentes au sein de la population, possède encore actuellement un taux de morbi-mortalité important tous sexes confondus, et ce malgré les importants progrès diagnostiques et thérapeutiques réalisés. D’un point de vue diagnostique, dans une approche dite de « Biologie de systèmes », en complément de l’étude anatomo- pathologique qui reste la référence, de nouvelles techniques ont été développées pour la caractérisation de profils métaboliques (Métabolomique) d’échantillons tissulaires pathologiques ou non, parmi lesquelles la Spectroscopie RMN HRMAS. Après un bref rappel théorique et avoir dressé le bilan des applications de cette technique en Cancérologie, nous avons exposé les différentes étapes du protocole à mettre en place afin d’envisager son implémentation dans un cadre hospitalier. L’ensemble des résultats présentés permettent d’envisager l’utilisation de cette technique en pratique clinique courante. Il faut néanmoins valider la robustesse des modèles statistiques élaborés et confirmer ces résultats sur de plus grandes cohortes d’échantillons. Des développements technologiques, analytiques et statistiques sont également nécessaires. / Cancer, one of the most frequent pathologies among the population, has still an important morbidity-mortality rate all sex confounded, despite the important diagnostical and therapeutical progresses achieved. From a diagnostical point of view, in a so called “Systems Biology approach”, as a complement of the gold standard histopathological study, some new techniques have been developed for the characterization of metabolic profiles (Metabolomics) of tissular samples pathological or not, among which HRMAS NMR Spectroscopy. After some brief theoretical considerations and after reporting the applications of this technique in Cancerology, we exposed the different steps of the protocol to design in order to consider its implementation in a hospital set up. All the results presented allow considering the use of this technique in a clinical routine. Nevertheless, it is necessary to validate the robustness of the statistical models built and to confirm these results on much larger cohorts of samples. Some technical, analytical and statistical developments are also needed.

Submitted by Viviane Lima da Cunha (viviane@biblioteca.ufpb.br) on 2016-03-29T14:36:11Z No. of bitstreams: 1 arquivototal.pdf: 5620328 bytes, checksum: cc12fb802b3be6b2f1e66e8519ace0cf (MD5) / Made available in DSpace on 2016-03-29T14:36:11Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 5620328 bytes, checksum: cc12fb802b3be6b2f1e66e8519ace0cf (MD5) Previous issue date: 2015-02-26 / The species Peperomia obtusifolia (L.) A. Dietr, which belongs to the Piperaceae family, is a well-known plant, distributed from Mexico to South America. Previous phytochemical studies demonstrated the presence of compounds from several classes, such as: lignans, flavonoids, amides, phenolic compounds and chromenes, which demonstrated interesting biological activities. This work describes the metabolomic analysis of Peperomia obtusifolia, which study in real time, the metabolism of this species in many kinds of scenarios. However, to accomplish this study, firstly it was developed a standard protocol, capable of preparing the samples in a reproducible way, enabling the following metabolomic studies to generate reliable results. Later, it was conducted the metabolomic study of Peperomia obtusifolia and its varieties, by fingerprinting analysis, in order to characterize the metabolomic profiles of each one of them. Furthermore, it was also evaluated the behavior of the species against the stress induced by the herbivory of Monoplatus sp. In general, the analyzes were conducted by NMR of 1H (which the data was interpreted by PCA), mass spectrometry, HPLC, GC and its hyphenated techniques. As a result, it was found that, despite the fact that the Peperomia obtusifolia varieties belong to the same species, they had different metabolomes. Regarding the study of the biotic stress against the plant, changes were detected on the metabolomic profile of the attacked plants, indicating a response against the herbivore attack. As a major result of this research, it can be highlighted the fact that the same changes were also detected on the metabolome of the intact plants closed to the attacked ones, indicating a sort of interplant communication by volatile compounds, capable to induce the neighborhood yet not attacked, to be prepared for an imminent attack, giving them a better chance of survival. / A espécie Peperomia obtusifolia (L.) A. Dietr, pertencente à família Piperaceae, é uma planta amplamente conhecida, cuja ocorrência vai desde o México até a América do Sul. Estudos fitoquímicos anteriores revelaram a presença de compostos de diversas classes, como: lignanas, flavonoides, amidas, compostos fenólicos e cromenos, os quais demonstraram atividade biológica bastante promissora. Este trabalho aborda a análise metabolômica de Peperomia obtusifolia, que visa estudar em tempo real o metabolismo desta espécie em diversos tipos de cenários. Entretanto, para que este estudo fosse realizado, primeiramente foi desenvolvido um protocolo padrão, capaz de preparar amostras de maneira reprodutível, possibilitando assim uma análise metabolômica que gerasse resultados fidedignos. Posteriormente foi realizado o estudo metabolômico de Peperomia obtusifolia e suas variedades, por análise de fingerprinting, que visa caracterizar os perfis metabólicos de cada organismo. Além disso, também foi avaliado o metaboloma da espécie frente ao estresse biótico provocado pela herbivoria do besouro Monoplatus sp. As análises das amostras foram realizadas por meio de RMN de 1H (cujos dados foram interpretados por PCA), espectrometria de massas, CLAE, CG e técnicas hifenadas. Como resultado, descobriu-se que apesar das variedades de Peperomia obtusifolia pertencerem à mesma espécie, elas possuíam metabolomas diferentes. Já em relação ao estudo de estresse biótico aplicado à planta, foram detectadas nas plantas predadas, alterações nos perfis metabolômicos, configurando-se uma resposta contra o ataque provocado. Considera-se um resultado de maior importância, o fato de que plantas intactas que estavam próximas às predadas, responderam ao ataque de maneira semelhante, indicando assim, uma comunicação inter-plantas por meio de compostos voláteis, capaz de induzir a vizinhança ainda não atacada a se preparar para reagir ao ataque, possibilitando assim, maior chance de sobrevivência.

Les dilatations des voies urinaires de diagnostic anténatal (DVU) peuvent être transitoires, ou correspondre à une anomalie significative de l'écoulement des urines pouvant entrainer une dégradation de la fonction rénale. L'identification de biomarqueurs urinaires pourrait contribuer à différencier précocement les uropathies des dilatations transitoires. La métabolomique permet l'identification de toutes les molécules de bas poids moléculaire présentes dans un échantillon biologique et la mise en évidence d'une signature métabolique associée à un événement physiopathologique. L'objectif principal de cette thèse est l'identification de marqueurs urinaires pronostiques chez les nouveaux nés présentant une DVU de diagnostic anténatal, par spectroscopie RMN et analyse métabolomique.Soixante-dix nouveau-nés ayant une DVU unilatérale et 90 témoins ont été inclus dans cette étude prospective. Tout d'abord, nous comparons la précision de différentes classifications échographiques pour l'évaluation du risque d'intervention chirurgicale.Ensuite, nous montrons l'absence de différence significative entre les profils métaboliques urinaires des nouveau-nés ayant une DVU et ceux des témoins. Dans une troisième partie, nous démontrons l'influence significative de l'âge, du poids et de la taille sur le profil métabolique urinaire des nouveau-nés. Enfin, nous montrons l'évolution du profil métabolique urinaire avec la croissance chez les nourrissons ayant une DVU.Ce travail permet une meilleure compréhension de la physiopathologie des DVU et de la maturation métabolique des nouveau-nés. Il contribue à identifier des biais potentiels dans les études métaboliques en néonatologie / The prenatal finding of unilateral Urinary Tract Dilatation (UTD) can be transient or represent a significant urinary flow impairment that would lead to progressive deterioration of renal function. Identifying urinary biomarkers could help to differentiate uropathy requiring surgical management from transient dilatation at an early stage.Metabolic phenotyping studies provide untargeted quantification of all detectable low molecular-weight molecules by profiling without any a priori the metabolic signatures of biological samples in connection to physiopathological events.The main objective of this study is to identify diagnosis and prognosis urinary markers for uropathy in newborns with prenatally diagnosed unilateral UTD using 1H-NMR spectroscopy combined with multivariate statistical analyses.A total of 70 newborns with unilateral UTD and 90 controls were included in this prospective study. First, the usefulness of different ultrasound grading systems in predicting the need for surgical intervention is evaluated. Then, we report the absence of significant difference between the urinary metabolic profiles of newborns with UTD and controls. In thethird part, the influence of age, weight and height on the urinary metabolic profiles of healthy newborns is highlighted for the first time, and key-metabolites responsible for this evolution are identified. Finally, we demonstrate the influence of age on the urinary metabolic profiles of children with UTD. This work allows a deeper understanding of the metabolic maturation of healthy newborns. It contributes to identify potential confounding factors for metabolomics investigations in neonatology. It represents a step toward a better comprehension of thephysiopathology of UTD

Submitted by JÚLIO HEBER SILVA (julioheber@yahoo.com.br) on 2017-04-06T17:53:33Z No. of bitstreams: 2 Dissertação - Jorge Candido Rodrigues Neto - 2017.pdf: 4621226 bytes, checksum: 93409e3a28aced62d8c3f5dab02c58a7 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-04-07T11:07:20Z (GMT) No. of bitstreams: 2 Dissertação - Jorge Candido Rodrigues Neto - 2017.pdf: 4621226 bytes, checksum: 93409e3a28aced62d8c3f5dab02c58a7 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-04-07T11:07:20Z (GMT). No. of bitstreams: 2 Dissertação - Jorge Candido Rodrigues Neto - 2017.pdf: 4621226 bytes, checksum: 93409e3a28aced62d8c3f5dab02c58a7 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-03-03 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Oil palm (Elaeis guineensis) is the world’s major source of vegetable oil and it has been able to adapt to the Brazilian subtropical area. Its growth, largely done in Para, is affected by Fatal Yellowing (FY), a condition that can cause plant’s death and has unknown cause. The aim for this master’s project was to prospect and identify, using a metabolomics approach, possible biomarkers of FY. To accomplish this study, for the first time different transport and extraction methods were evaluated in order to verify the metabolic profile stability in this species. Significant differences were noted and described using a metabolic fingerprinting approach, with ultra high precision liquid chromatography – mass spectrometry (UHPLC-MS) as the chemical analysis and ANOVA simultaneous component analysis (ASCA) as the multivariate statistic tool. After this study, leaves with and without FY symptoms were transported using the previously optimizes protocol, analyzed and five potential biomarkers were identified and related to their metabolic pathways, using PCA as the chemometric tool. Finally, for the first time, the use of advanced analytical techniques as MALDI imaging, which has been highlighted as a powerful tool for identification of chemical compounds present on tissue surface with spatial localization, has been applied to oil palm leaves in order to perform a metabolic screening and search for the previously identified biomarkers. An analytical protocol was established for analysis with and without matrix application, for a wide range mass coverage and one of the possible biomarkers was detected using the matrix-free approach. / O dendê, fruto do dendezeiro (Elaeis guineensis) é a maior fonte de óleo vegetal do mundo e teve boa adaptação ao clima subtropical brasileiro. Seu cultivo no Brasil, que tem maior extensão no Pará, é afetado pelo Amarelecimento Fatal (AF), uma condição que pode provocar a morte da planta e tem causa desconhecida. O objetivo deste projeto de mestrado foi prospectar e identificar marcadores químicos relacionados ao AF utilizando uma abordagem metabolômica. Para que o estudo dessa causa fosse realizado, pela primeira vez foram avaliados diferentes métodos de transporte e extração de folhas para verificar a estabilidade do perfil metabólico desta espécie. Diferenças significativas foram observadas utilizando a estratégia de metabolic fingerprinting, com análise dos metabólitos por cromatografia de ultra alta eficiência – espectrometria de massas (UHPLC-MS) e análise quimiométrica dos dados por análise de componente simultâneo e análise de variância (ASCA). Após este estudo, folhas com e sem sintomas de AF foram transportadas utilizando protocolo otimizado anteriormente, analisadas e cinco potenciais biomarcadores foram identificados e relacionados as suas respectivas vias metabólicas, utilizando a análise de componentes principais (PCA) como ferramenta quimiométrica. Por fim, fez-se uso da recente e avançada técnica de imagem química em espectrometria de massas, com ionização por dessorção a laser assistida por matriz (MALDI-MS Imaging), que vem se destacando como uma poderosa ferramenta para a identificação de compostos químicos presentes na superfície de um tecido com localização espacial e foi utilizada pela primeira vez em um screening de metabólitos em folhas de dendê na busca dos marcadores previamente identificados. Um protocolo analítico foi desenvolvido para análise com aplicação de matriz e sem aplicação de matriz, com o intuito de abranger uma maior faixa de massas e um dos possíveis marcadores foi detectado utilizando a técnica de imagem sem matriz.

Muitas substâncias das espécies de Lychnophorinae (Asteraceae) são relatadas como inibidoras da síntese de mediadores da cascata do processo inflamatório. Nesse processo, duas enzimas são essenciais e atuam no metabolismo do ácido araquidônico, formado em processos inflamatórios: ciclooxigenase (COX) e lipoxigenase (LOX). A análise de impressões digitais metabólicas é um método capaz de fornecer informações sobre o objeto de estudo através da utilização de ferramentas estatísticas, além de possibilitar a correlação desses dados com outros utilizando métodos in silico. O objetivo deste estudo foi analisar 26 espécies da subtribo Lychnophorinae quanto à atividade inibitória in vitro das vias COX- e LOX-dependentes e identificar as substâncias responsáveis por essa atividade através de análises in silico de correlação entre bioatividade e impressão digital metabólica. As impressões digitais metabólicas dos extratos hidrometanólicos das espécies de Lychnophorinae foram obtidas utilizando UHPLC-UV-(DAD)-MS (OrbitrapTM). Os ensaios de triagem de inibição das vias COX-1 e 5-LOX-dependentes revelaram que 20 espécies possuem atividade inibitória dupla para ambas as vias com valores de IC50 menores que 100 ?g.mL-1. Dentre essas, 11 espécies apresentaram valores de IC50 menores que 40 ?g.mL-1, e cinco apresentaram valores de IC50 menores que 10 ?g.mL-1. Utilizando-se as impressões digitais metabólicas e os resultados de inibição enzimática foram realizadas análises estatísticas multivariadas supervisionadas e não supervisionadas (PCA, PLS e OPLS) visando à identificação de substâncias discriminantes (ativas). Através das análises de correlação, obtiveram-se as prováveis substâncias que detém o potencial inibitório. As cinco substâncias com maior potencial discriminante foram identificadas por técnicas de desreplicação e são: a lactona sesquiterpênica (4,5-diidro-15-desoxigoyazensolido), dois flavonóides glicosilados (3-O-(acetil-hexosídeo)-quercetina e 7-O-(cumaroil-hexosídeo)-apigenina), e um hidroxinerolidol. Assim, este estudo revelou o ótimo potencial inibidor das enzimas COX-1 e 5-LOX dos extratos hidrometanólicos das espécies de Lychnophorinae. Além disso, indicou as substâncias mais discriminantes responsáveis por essa atividade, sendo as substâncias acima mencionadas as propostas como as principais responsáveis pela atividade inibidora das enzimas COX e LOX / Several compounds from Lychnophorinae species (Asteraceae) are reported as inhibitors of cascade mediators that elicits the inflammatory process. During this process, two enzymes are essential in the metabolism of the arachidonic acid: cyclooxygenase (COX) and lipoxygenase (LOX). The analysis of metabolic fingerprinting is a method that provides information about the object of study by using statistical tools, and enables the correlation of these data with others using in silico methods. This work therefore aimed to analyze 26 species of the Lychnophorinae subtribe for in vitro inhibition of COX-1 and 5-LOX and (to) identify the bioactive compounds responsible for this activity by in silico correlation analysis between bioactivity and metabolic fingerprint. The metabolomic analysis was carried out using UHPLC-DAD-ESI-Orbitrap and a metabolic fingerprint for each extract was obtained. The in vitro inhibition screening assays of COX-1 and 5-LOX revealed that 20 extracts presented dual inhibitory activity on both enzymes with IC50 values lower than 100 ?g.mL-1. Among them, 11 species showed IC50 values lower than 40 ?g.mL-1 and five lower than 10 ?g.mL-1. In order to identify discriminant substances (active), supervised and non-supervised multivariate statistical analysis (PCA, PLS e OPLS) were performed using the metabolic fingerprints and the results of enzyme inhibition. Through correlation analysis, it was possible to locate the substances most likely to be responsible for the pharmacological activity in both enzymes simultaneously; among them, five were chosen as the most likely. The substances were identified by dereplication as: a sesquiterpene lactone (4,5-dihydro-15-desoxygoyazensolide), two flavonoids (3-O-(acetil-hexoside)-quercetin and 7-O-(cumaroil-hexoside)-apigenine), and a hidroxynerolidol. In summary, in this work it was possible to reveal crude extracts with outstanding inhibitory potential of both, COX-1 and 5-LOX, enzymes as well as to propose the most probable compounds responsible for this action, and the compounds mentioned above were proposed as the main responsible for the inhibitory activity.

O gênero Eucalyptus é uma das principais espécies arbóreas comercialmente plantadas em todo o mundo. Apresenta inúmeras características favoráveis para a produção e comercialização de sua madeira, tais como o rápido crescimento, rotação de ciclo curto e produção de biomassa renovável, com potencial para geração de biocombustível. Apesar de sua grande importância econômica, pouco é conhecido sobre os processos moleculares que envolvem a formação da madeira e casca, principalmente no que diz respeito ao metabolismo primário. Ademais, não se têm muitas informações sobre mudanças moleculares que ocorrem durante a formação desses tecidos em resposta a variações sazonais. Sabe-se que a região cambial das árvores apresenta maior atividade metabólica durante o verão, quando comparada ao inverno. Deste modo, visando compreender mudanças dinâmicas ao nível transcricional, protéico e metabólico, principalmente em relação ao metabolismo primário, o presente trabalho teve por objetivo comparar os tecidos da região cambial e casca, coletados em diferentes períodos climáticos (verão e inverno). A expressão do padrão transcricional foi analisada por PCR em tempo real, seguido de normalização e análise estatística, usando os programas NormFinder e Rest, respectivamente. O perfil protéico foi obtido por eletroforese bidimensional (2D-PAGE), seguido de análise por espectrometria de massas associada a cromatografia líquida (LC-MS/MS) e posterior identificação das proteínas pelo programa Mascot Daemon. Já o perfil metabólico foi obtido por espectrometria de massas associada à cromatografia gasosa (GC/MS), com posterior análise dos picos identificados pelo programa MatLab. Em seguida, as análises estatísticas foram geradas pelo programa SIMCA P+ e \"R\". Para os transcritos, foi possível observar tanto para a região cambial quanto para a casca padrão diferencial na expressão dos genes analisados, principalmente aqueles atuantes na glicólise, durante os dois períodos sazonais. Quanto ao perfil protéico, foram identificadas um total de 77 e 75 proteínas estatisticamente significativas na região cambial e casca, respectivamente. Proteínas pertencentes ao metabolismo primário foram identificadas em ambos os tecidos. Metabólitos relacionados ao metabolismo de açúcares também foram encontrados. / Eucalyptus genus is the most widely planted hardwood crop in the world because of its superior growth, broad adaptability and multipurpose wood properties. In today\'s \"new carbon economy\", eucalypts are receiving attention as fast-growing, short-rotation, renewable biomass crop for energy production. In spite of its economical importance, little information is available about the molecular changes that occur in primary metabolism in the wood and bark forming tissues. Furthermore, there is less information about molecular changes that occur during wood and bark formation in response to seasonal variation. It\'s known that Eucalyptus cambial region presents higher metabolic activity in summer than in winter. Thus, in order to observe the dynamic changes in transcript, protein and metabolite levels, mainly related to primary metabolism, we compared cambial tissue and bark collected in two different seasons (summer high temp + high rainfall) and winter (lower temp and little rainfall). Transcript expression patterns were analyzed by Real-Time PCR, normalization chosen by NormFinder and statistical analysis carried out using REST. The protein profile was obtained by bidimensional electrophoresis (2D-PAGE) followed by liquid chromatography associated with mass spectrometry (LC-MS/MS) and analyzed by Mascot Daemon. Metabolite profile was obtained by GC-TOF/MS, peaks were analyzed in MatLab and statistical analyses were done using SIMCA and \"R\". The results obtained with transcripts indicate differential gene expression in the cambial region and bark during summer and winter. A total of 77 and 75 proteins in cambium and bark, respectively, presented statistically significant alterations and were identified and classified into functional categories. We identified many proteins from primary metabolism. Metabolites from carbohydrate metabolism were also identified.

Orientadores: José Andrés Yunes, Ana Carolina de Mattos Zeri / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-19T13:46:58Z (GMT). No. of bitstreams: 1 Canevarolo_RafaelRenatino_M.pdf: 3376497 bytes, checksum: f3e8edf217e0d57089ffcffbe4a8097a (MD5) Previous issue date: 2012 / Resumo: O uso intensivo e combinado de diferentes quimioterápicos tem permitido a cura de 70-80% das leucemias linfóides agudas (LLA) da infância, sendo que a recaída da doença decorre em grande parte da resistência intrínseca das células leucêmicas à quimioterapia. Alguns dos quimioterápicos utilizados na LLA são inibidores metabólicos, como o metotrexato (MTX), antagonista do ácido fólico, que impede a divisão celular ao inibir a síntese de nucleotídeos. Em uma abordagem metabolômica, foi investigada a associação entre linhagens leucêmicas resistentes ou sensíveis ao MTX e metabólitos biondicadores de cada um destes fenótipos. Seis linhagens celulares B-derivadas e oito T-derivadas foram classificadas como sendo resistentes ou sensíveis ao MTX pelo método do 3-(4,5-dimetiltiazol-2il)-2,5-difenil brometo de tetrazolina (MTT) após 48h de co-cultura com diferentes concentrações da droga. Cinco linhagens foram classificadas como resistentes e nove como sensíveis ao MTX. Após 24h de cultura na presença ou ausência de MTX (ambos em triplicata), os metabólitos intracelulares das linhagens foram acessados por ressonância magnética nuclear (RMN) numa abordagem metabolômica. Ao total, oitenta e quatro metabólitos foram quantificados, dos quais 72 foram também identificados. A análise de componentes principais (PCA) não conseguiu segregar as amostras de acordo com sua resistência, ao passo que a análise discriminante por mínimos quadrados parciais (PLS-DA) foi efetiva nesta separação. Os metabólitos mais relevantes para a construção dos modelos de classificação quanto à resistência ao MTX, tanto para amostras tratadas quanto controles foram: ATP, dimetilglicina, fosfocolina e sarcosina (associados à resistência); carnitina, CB-09 (composto não identificado), colato, fumarato, glicocolato, lactato, malato e succinato (associados à sensibilidade ao MTX). A capacidade de classificar corretamente as amostras em sensíveis ou resistentes foi obtida com a construção de curvas da característica operativa do receptor (ROC) para os metabólitos individualmente. Os metabólitos com desempenho bom ou excelente na análise ROC (AUC>0,8) foram selecionados para comporem "testes diagnósticos" de classificação de amostras. De todas as combinações possíveis dentre os metabólitos selecionados, o teste que considerou a combinação de carnitina, sarcosina e succinato em amostras não tratadas com MTX apresentou sensibilidade de 100% (identificou todas as 15 amostras resistentes) e especificidade de 92,3% ao classificar corretamente 24 de 26 amostras sensíveis. O melhor teste diagnóstico para amostras tratadas com MTX considerou as concentrações de CB-MTX, glicocolato, sarcosina e succinato; apresentou sensibilidade de 100% (identificou as 15 amostras resistentes) e especificidade de 85,2%, equivocando-se na classificação de 4 dentre 27 amostras sensíveis. As concentrações metabólicas diferenciais apontaram para uma superativação dos metabolismos energético e de lipídeos em linhagens sensíveis ao MTX, ao passo que linhagens resistentes teriam superativado o metabolismo da glicina. As análises metabolômicas e de integração bioquímica dos metabólitos revelaram interações gênicas, enzimáticas e metabólicas que podem estar alteradas em linhagens sensíveis ou resistentes ao MTX, bem como permitiram a especulação sobre possíveis alvos moleculares que poderiam tornar sensíveis células resistentes ao quimioterápico / Abstract: The intensive use of different and combined chemotherapics has allowed curing 70-80% of pediatric acute lymphoblastic leukemia (ALL), and the relapse of the disease stems largely from the intrinsic resistance of leukemic cells to chemotherapy. Some of the chemotherapics used in ALL are metabolic inhibitors such as methotrexate (MTX), a folic acid antagonist, which prevents cell division by inhibiting the synthesis of nucleotides. The association between leukemic strains resistant or sensitive to MTX and the metabolites associated with each of these phenotypes were investigated. Six B- and eight T-derived cell lines were classified as resistant or sensitive to MTX by the 3-(4,5-Dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) assay, after 48h in co-culture with different concentrations of the drug. Five lineages were classified as resistant, and nine as sensitive to MTX. After 24 hours of culture in the presence or absence of MTX (both in triplicates), the intracellular metabolites of the lineages were assessed by nuclear magnetic resonance (NMR), in a metabolomic approach. In total, 84 metabolites were quantified, 72 of which were also identified. The principal component analysis (PCA) did not segregate the samples according to their resistance, whereas the supervised partial least square discriminate analysis (PLS-DA) was effective in this separation. ATP, dimethylglycine, sarcosine and phosphocholine were associated with MTX resistance in both models constructed for treated and untreated samples, whereas carnitine, CB-MTX (unidentified compound), cholate, fumarate, glycocholate, lactate, malate and succinate were associated with sensitivity to MTX. The ability to correctly classify the samples into sensitive or resistant groups was checked with the construction of the receiver operating characteristic (ROC) curves for metabolites individually. Metabolites with good or excellent performance in ROC analysis (AUC> 0.8) were selected to compose "diagnostic tests" for classifying samples. Of all the possible combinations among the selected metabolites, the test composed by the comination of carnitine, sarcosine and succinate in untreated samples exhibited sensitivity of 100% (identified all 15 resistant samples) and specificity of 92.3% in classifying correctly 24 of 26 sensitive samples. The best diagnostic test for samples treated with MTX took into consideration concentrations of CB-MTX, glycocholate, sarcosina, succinato. It had a sensitivity of 100% (identified 15 resistant samples) and specificity of 85.2%, classifying incorrectly 4 out of 27 sensitive samples. Differential metabolic concentrations pointed to an over activation of energy and lipids metabolism in MTX-sensitive strains, whereas resistant strains seemed to have overactive the glycine metabolism. Metabolomic and biochemical integration analysis revealed genetic, enzymatic and metabolic interactions that might be altered in strains sensitive or resistant to MTX, as well as allowed speculations about possible molecular targets on which intervention could make resistant cells susceptible to chemotherapy / Mestrado / Ciencias Biomedicas / Mestre em Ciências Médicas

Propionibacterium freudenreichii est une bactérie très utilisée par l’industrie laitière. Elle appartient aux Actinomycètes connus pour leur survie pendant de longues périodes, dans des conditions environnementales défavorables. Pour mieux comprendre ce phénomène, la caractérisation phénotypique de 8 souches de P. freudenreichii a été réalisée sur 11 jours dans un milieu en carence nutritionnelle. Le taux de survie bactérienne a été mesuré par densité optique, par énumération et évaluation de la viabilité cellulaire. En outre, l’absence de lyse cellulaire a été évaluée par PCR quantitative. La croissance de P. freudenreichii a été décrite en phases exponentielle, stationnaire, stationnaire tardive et survie à long terme.Dans nos conditions expérimentales pendant la période de survie à long terme, les bactéries sont restées viables. La caractérisation phénotypique a montré que P. freudenreichii CIRM-BIA138 était la plus résistante à la carence nutritionnelle et entrait dans un état viable mais non-cultivable. Cette souche a été utilisée pour une étude fonctionnelle par RNA-Seq ainsi que pour des analyses biochimiques sur les surnageants de culture, en phases exponentielle et stationnaire. L’association de ces données transcriptomiques et métabolomiques a permis de déduire les stratégies impliquées dans la survie de cette bactérie. La préparation à l’état de dormance, la diminution du métabolisme et l’utilisation de sources alternatives d’énergie semblent impliquées dans l’adaptation et la persistence de P. freudenreichii CIRM-BIA138 en carence nutritionnelle durant de long / Propionibacterium freudenreichii is a dairy bacterium belonging to the Actinobacteria group, which is known to survive for long periods in harsh environmental conditions. In order to investigate the long-term survival phenomenon in P. freudenreichii, 8 strains were phenotypically characterized for a period of 11 days in nutrient shortage condition. Bacterial survival rate was assessed by optical density, CFU counting and live-dead cellular viability. In addition, the absence of cell lysis was evaluated by quantitative PCR. P. freudenreichii growth phases were classified as exponential, stationary, late stationary and long-term survival. Moreover, it was observed that bacterial viability was maintained during long-term survival.Phenotypical characterization indicated that P. freudenreichii CIRM-BIA138 was more resistant to nutrient shortage being able to enter into a viable but nonculturable dormant state. In addition, functional studies of this strain were conducted by RNA-Seq on cultures sampled in exponential and stationary growth phases. Concomitantly, several biochemical analyses were carried out on the culture supernatant. An integrative approach of metabolomic and transcriptomic data allowed us to infer strategies associated with the survival of this bacterium, such as preparation for the dormant state, slow down of metabolic activity and utilization of alternative sources of energy, which altogether might allow P. freudenreichii CIRM-BIA 138 to adapt and persist through nutrient shortage for long periods.

La spectroscopie RMN Haute Résolution en Rotation à l’Angle Magique (HRMAS) permet la caractérisation métabolomique tissulaire. Nous avons caractérisé par RMN HRMAS le profil métabolomique des glandes parathyroïdiennes hypersécrétantes. Celui de l’hyperparathyroïdie primaire (HPT1) a été comparé à celui des HPT rénales. Au sein des HPT1, la distinction a pu être faite entre pathologie uni- et multi-glandulaire. Le profil métabolomique du tissu pancréatique sain a été comparé à celui du tissu tumoral. Aussi, les patients longs/courts-survivants ont pu être distingués. La relation entre le phénotype métabolique et la survie des patients a été étudiée. Le profil métabolomique des TIPMP a été caractérisé. Les TIPMP non dégénérées et dégénérées ont été comparées. Le risque de dégénérescence a été corrélé au profil métabolomique. Nos résultats montrent que la spectroscopie RMN HRMAS est une technique prometteuse pour l’étude du profil métabolomique des HPT et des pathologies pancréatiques. / High Resolution Magic Angle Spinning (HRMAS) NMR spectroscopy allows metabolomics of intact tissues. Metabolomics profiling of hyperfunctioning parathyroid glands were characterized were characterized by using HRMAS NMR spectroscopy. Primary hyperparathy- roidism (PHPT) was compared to renal HPT. Among PHPT, we distinguished single gland disease from multiple gland disease. Pancreatic parenchyma and adenocarcinoma were compared. Thus, long-term and short-term survival patients were distinguished. The relationship between the survival of patients and their metabolic phenotype was studied. Metabolomics profiling of IPMN was also examined. IPMN with no degeneration and de- generated IPMN were compared. Finally, the risk of degeneration was correlated with the metabolomics profile. Our results show that HRMAS NMR spectroscopy is a promising technique in view of studying metabolomic profiling of HPT and pancreatic diseases.