Tout au long du procédé d’élaboration d’un vin, des réactions d’oxydation peuvent se produire y compris au cours du vieillissement en bouteilles. Depuis quelques années, la profession viti-vinicole est confrontée au problème de l’oxydation prématurée des vins blancs.. A ce jour, peu d’études ont pu apporter des explications d’ordre chimique à ce phénomène, et les mécanismes réactionnels intervenant restent peu connus.L’objectif de ce travail de thèse est d’apporter, au travers d’une analyse moléculaire non ciblée en spectrométrie de masse à transformée de Fourier et à résonance cyclotronique des ions (FT-ICR-MS) couplée à une étude chimiométrique, des réponses originales aux questions posées par la problématique actuelle d’oxydation prématurée des vins blancs. Nous avons montré suite à l’étude d’une série de vins oxydés prématurément non seulement la grande diversité chimique des vins, mais aussi la présence d’un ensemble de masses typiques associées à ce phénomène. Pour une meilleure compréhension de l’origine de cette problématique, nous avons considéré deux autres types d’oxydation : une oxydation relative exclusivement à un apport contrôlé en oxygène et une autre relative à l'évolution naturelle de vins en bouteilles. Cette dernière consiste en le suivi de l'évolution des espaces chimiques de séries verticales de vins blancs et rouges en fonction du temps. Sur la série verticale des vins blancs allant de 1979 à 2006, une charnière à l’année 1990 a été observée avec des groupes de masses typiques de chacun des vins jeunes (1979-1990) et des vieux vins (1991-2006).La comparaison entre les espaces chimiques discriminants chacun de ces trois types d’oxydation ne révèle la présence que de trois masses en commun, ce qui appuie l'hypothèse de causes multiparamétriques à l’oxydation prématurée des vins blancs, qui ne serait donc pas un phénomène du exclusivement à une exposition non contrôlée à l'oxygène.Des essais d’élucidation structurale en FT-ICR-MS/MS des masses discriminantes des vins oxydés et de la série verticale ont été effectués et des schémas de fragmentation pour certaines masses sont proposés / During winemaking processes, many oxidation reactions may occur especially during the aging period. Recently, white wines are characterized by a problem of premature oxidation for which few studies have provided chemical explanation. To date, the involved mechanisms in this phenomenon remain poorly understood.The aim of this thesis project is to provide, through an untargeted molecular analysis using Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) coupled to chemometric analysis, original clue to understand the premature oxidation of white wines. Based on the study of a series of premature oxidized white wines, we were able to elucidate the high complexity and the chemical diversity of wine, and got out typical masses characterizing the oxidation state. In order to better understand the origin of this phenomenon, we considered two alternative possibilities of oxidation: the first one induced by oxygen, and the second through a natural evolution of wines in bottles. The latter included the monitoring of the chemical evolution of white and red wines as a function of time. A very interesting result was obtained on the vertical series of white wines from 1979 to 2006, where two groups were separated at the 1990 vintage to provide a group of old wines (1979-1990) and a group of new wines (1991-2006). Typical discriminant masses were found for each group.A comparison between the chemical spaces discriminating each of the three types of oxidation (premature oxidation, oxidation with oxygen and natural evolution of wine in bottle) revealed very few common masses that may indicate that the phenomenon of premature oxidation is indeed influenced by multiple factors.Finally, a structural elucidation of the typical masses of the groups of oxidized and aged wines were established using FT-ICR-MS/MS. Possible fragmentations schemes of some of these masses were proposed

Humic substances (HS), the complex mixture of organic molecules produced by microbial and abiotic degradation of plant and animals residues, are the major components of organic matter in soil and natural waters. As such, they are vital to the integrity of soil, play important roles in nutrient biogeochemical cycling and determine the mobility and fate of both nutrients and contaminants. How HS enact their varied roles, however, is largely unknown due to the lack of comprehension of their molecular make-up. What is known is that they contain aromatic (e.g. phenols, condensed aromatics) and aliphatic (e.g. carbohydrates, lipids) molecules with prevailing carboxyl and hydroxyl functionalities. As for other complex chromatographically inseparable mixtures, high-resolution analytical techniques such as Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICR MS) as well as Nuclear Magnetic Resonance (NMR) spectroscopy have been applied to study HS composition at the molecular level. While FT-ICR MS can provide molecular formulae, it is only NMR that can unveil structures. Until now, however, very few unambiguous structures of individual molecules have emerged from standard NMR experiments. This is because purposely designed multidimensional NMR experiments are required to achieve ‘spectroscopic separation’ where chromatography fails. In this work, ‘spectroscopic separation’ was accomplished with the aid of chemical modification in the form of 13C methylation of COOH and OH groups. 13C containing tags allow the observation of signals only from molecules carrying these tags. In combination with purposelydesigned isotope-filtered NMR experiments, these tags act as spies reporting on their surrounding structure. This is achieved by utilising scalar and dipolar couplings to transfer the polarisation between protons and carbons of the 13CH3O groups and nuclei of the parent molecules. The necessary spectral resolution is attained using 3D/4D NMR experiments. This approach provides access to an array of correlated chemical shifts of HS molecules and represents a paradigm shift in the use of tags in investigations of complex mixtures: instead of focusing only on the tags, they are used to obtain structural information from the molecules they are attached to. The compounds at the centre of this investigation are the phenols of HS. These molecules are thought to be important in many of the key processes in organic matter rich soils, particularly peat. For example, their accumulation is thought to impair the activity of extracellular enzymes essential for the degradation of peat HS. Understanding the nature and source of phenols would provide a more fundamental framework for rationalising their role in peat, as well as other carbon rich soils. The developed methodology was initially tested on model mixtures containing 3 or 9 phenolic compounds before applying it to a operational fraction of HS, fulvic acid, extracted from a Scottish raised peat bog. For this fulvic acid, over 30 major phenolic molecules/moieties were identified, many of which can be directly traced to the flora prevalent in the vicinity of the sampling site. For the first time in 150 years of HS research, a novel analytical methodology has yielded unambiguous structural information, providing the first steps towards understanding the various roles of HS in natural systems.

Dissolved organic matter (DOM), one of the most complex naturally occurring mixtures, plays a central role in the biogeochemistry and the photochemistry of natural waters. A complete understanding of the environmental role of DOM will come only from the elucidation of the relationship between its structure and function. This thesis presents new work on the separation, characterization, and reactivity of DOM in rainwater, freshwater, and seawater. A new separation technique based on counterbalance capillary electrophoresis was developed for the separation of Suwannee River NOM. A comparative study of the organic content of rainwater was accomplished using nuclear magnetic resonance (NMR) with spectral database matching ,Fourier transform ion cyclotron mass spectrometry (FT-ICR-MS), and comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry (GCxGC-TOFMS). Three complementary, non-overlapping datasets identified of over 400 compounds. Analysis of the FT-ICR-MS data using van Krevelen diagrams and the carbon oxidation state showed variation in the elemental composition and molecular size. Over 50% of the compounds identified in this study were known components of secondary organic aerosol (SOA) and volatile organic carbon (VOCs). Dissolved organic matter (DOM) plays a central role in the photochemistry of natural waters through the production of reactive oxygen species and the triplet excited state of DOM (3DOM*). These reactive species are central to the reactivity, transport, and fate of both natural and anthropogenic chemicals in the environment. Laser flash photolysis (LFP) was used to demonstrate that particulate organic matter (POM) generates a triplet excited state species (3POM*). LFP of seawater from the Pacific Ocean and the Bermuda Atlantic Time Series Station detected similar excited state species from surface to 4535m. Metal speciation has been implicated in the photochemistry of natural waters. Copper immobilized metal affinity chromatography (IMAC) of seawater and freshwater isolated a low and a high affinity fraction that generated excited state transients. Excitation-emission matrix spectroscopy showed that while the seawater fractions were autochthonous, freshwater fractions enriched in chromophoric DOM (CDOM), were allochthonous. The discovery of these different classes of compounds in freshwater and seawater has important implications both for the mineralization of DOM and the removal of xenobiotics in the aquatic environment.

Dissolved organic matter (DOM), one of the most complex naturally occurring mixtures, plays a central role in the biogeochemistry and the photochemistry of natural waters. A complete understanding of the environmental role of DOM will come only from the elucidation of the relationship between its structure and function. This thesis presents new work on the separation, characterization, and reactivity of DOM in rainwater, freshwater, and seawater. A new separation technique based on counterbalance capillary electrophoresis was developed for the separation of Suwannee River NOM. A comparative study of the organic content of rainwater was accomplished using nuclear magnetic resonance (NMR) with spectral database matching ,Fourier transform ion cyclotron mass spectrometry (FT-ICR-MS), and comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry (GCxGC-TOFMS). Three complementary, non-overlapping datasets identified of over 400 compounds. Analysis of the FT-ICR-MS data using van Krevelen diagrams and the carbon oxidation state showed variation in the elemental composition and molecular size. Over 50% of the compounds identified in this study were known components of secondary organic aerosol (SOA) and volatile organic carbon (VOCs). Dissolved organic matter (DOM) plays a central role in the photochemistry of natural waters through the production of reactive oxygen species and the triplet excited state of DOM (3DOM*). These reactive species are central to the reactivity, transport, and fate of both natural and anthropogenic chemicals in the environment. Laser flash photolysis (LFP) was used to demonstrate that particulate organic matter (POM) generates a triplet excited state species (3POM*). LFP of seawater from the Pacific Ocean and the Bermuda Atlantic Time Series Station detected similar excited state species from surface to 4535m. Metal speciation has been implicated in the photochemistry of natural waters. Copper immobilized metal affinity chromatography (IMAC) of seawater and freshwater isolated a low and a high affinity fraction that generated excited state transients. Excitation-emission matrix spectroscopy showed that while the seawater fractions were autochthonous, freshwater fractions enriched in chromophoric DOM (CDOM), were allochthonous. The discovery of these different classes of compounds in freshwater and seawater has important implications both for the mineralization of DOM and the removal of xenobiotics in the aquatic environment.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第24325号 / 農博第2530号 / 新制||農||1096(附属図書館) / 学位論文||R5||N5427(農学部図書室) / 京都大学大学院農学研究科森林科学専攻 / (主査)教授 德地 直子, 教授 柴田 昌三, 教授 舘野 隆之輔 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

La glycosylation est la forme la plus courante de modification post-traductionnelle (PTM) des protéines humaines, puisque plus de 70% d’entre elles sont glycosylées. Celle-ci régule de nombreuses propriétés biologiques comme leur stabilité, leur demi-vie et leur activité. Néanmoins, les protéines peuvent également présenter d'autres types de PTM, ce qui peut conduire pour une protéine donnée à un très grand nombre d'isoformes variant par leur masse, leurs propriétés biologiques et physico-chimiques et leur concentration dans les échantillons biologiques. Ainsi, caractériser une glycoprotéine comporte de nombreux défis et nécessite la mise en œuvre de méthodes séparatives très performantes et de détection très sensibles et informatives.La gonadotrophine chorionique humaine (hCG) est l’hormone spécifique de la grossesse humaine. Elle est essentielle au développement du placenta et du fœtus. Elle est composée de deux sous-unités hCGα et hCGβ qui sont fortement glycosylées (4 sites de N-glycosylation et 4 sites d’O-glycosylation). Récemment, des travaux ont montré une corrélation entre sa glycosylation et une bonne implantation du fœtus. Une caractérisation des ces glycoformes s’avère donc nécessaire.Par conséquent, de nouvelles méthodes en LC/CE-MS ont été développées pour la caractérisation de la hCG à l’échelle intacte en utilisant deux médicaments à base de hCG ayant des glycosylations différentes. Alors que la méthode en CZE-MS (TQ) a permis de différencier les profils des glycoformes de la sous-unité hCGα des deux médicaments, la complémentarité des méthodes RP- et HILIC-MS (qTOF) a conduit à leur identification.Pour limiter les erreurs potentielles d’identification dues au chevauchement des profils isotopiques, le profil de chaque isoforme a été résolu par FT-ICR MS. Dans ce but, une séparation au format nanoLC en mode RP a été développée, améliorant ainsi la sensibilité de la méthode d’un facteur 500 par rapport au format conventionnel. Cette méthode a permis de confirmer l’identification des glycoformes de la sous-unité hCGα. D’autre part, il a été possible d’obtenir des profils différents de glycosylation de la sous-unité hCGβ en favorisant leur ionisation par réduction de la hCG. Enfin, un traitement à la PNGase a conduit à l’élimination des N-glycanes pour l’obtention des profils d’O-glycosylation de la sous-unité hCGβ. / Glycosylation is the most common form of post-translational modifications (PTMs) of human proteins, since more than 70% are glycosylated. It regulates numerous biological properties including their stability, half-life, and activity. Nevertheless, proteins can also exhibit other types of PTMs that lead to a very large number of isoforms, varying in mass, properties and concentration in the biological samples. Therefore, the characterization of a glycoprotein is highly challenging and requires the use of powerful separation techniques and sensitive and informative detection modes.The human chorionic gonadotropin (hCG) is the hormone specific to human pregnancy. It is essential for the development of placenta and fetus. It is based on two heavily glycosylated subunits, hCGα and hCGβ, having 8 glycosylation sites (4 N- and 4 O-glycosylation sites). Some recent studies demonstrated that here is a correlation between the hCG glycosylation state and the fetus implantation. This is why the characterization of the hCG glycoformes is needed.Therefore, new LC/CE-MS methods were developed for the characterisation of hCG at the intact level using two hCG-based drugs having different glycosylation profiles. While the CZE-MS (TQ) method showed its potential for glycosylation fingerprinting, the complementarity of LC-(qTOF) MS methods in RP and HILIC modes allowed the identification of the glycoforms of the hCGα subunit.To limit the identification errors due to the overlapping of isotopic distribution patterns, the profile of each isoform was resolved by FT-ICR MS. For this purpose, a nanoLC separation in RP mode was developed, thus improving the sensitivity of the method by a factor 500 compared to the conventional format. This method allowed the confirmation of the identification of hCGα glycoforms. Then, it was possible to obtain different glycosylation patterns of the hCGβ by promoting its ionization after hCG reduction. Then, a PNGase treatment was carried out to remove the N-glycans in order to obtain the O-glycoprofiles of hCGβ isoforms.

Le vin en tant qu’écosystème complexe est un modèle particulièrement intéressant pour l’étudie des interactions entre les microorganismes. L’interaction sans contact celluaire (interaction indirecte) entre la levure Saccharomyces cerevisae et la bactérie lactique Oenococcus oeni a un effect direct sur l’induction et l'achèvement de la fermentation malolactique (FML), une fermentation très importante pour la qualité du vin. Une souche levurienne peut être classée FML+ si elle stimule la croissance bactérienne et FML- si elle a un effet inhibiteur. Les métabolites connus qui inhibent ou stimulent la FML ne permettent pas toujours d’expliquer cette distinction phénotypique. Dans ce travail de thèse, nous avon développé un workflow multidisciplinaire qui combine l’approche métabolomique non ciblée, l’analyse classique ciblée, les statistiques et les réseaux. L’objectif premier était de dévoiler des métabolites levuriens impliqués dans l’interaction entre levures et bactéries par une comparaison directe des exométabolome des deux phénotypes.À cet effet et pour la première fois dans l’éude d’interactions inter-espèces, la Spectrométrie de Masse à Résonance Cyclotronique des Ions et à Transformée de Fourier (FT-ICR-MS) et la Chromatographie Liquide couplée à la Spectrométrie de Masses (UPLC-Q-TOF-MS) ont été combinées. Pour mieux visualiser les données à haut débit générées par les deux plate-formes, une méthode statistique non supervisée MetICA a été developpée et validée. Par rapport à l’analyse en composantes principales (ACP), cette nouvelle méthode peut réduire la dimension des données d'une façon plus robuste et fiable. Afin d’extraire des métabolites impliquées dans la distinction phénotypique, nous avons comparé différentes methodes de classification et choisi la meilleure pour chaque jeu de données. Les structures putatives de ces biomarqueurs ont été validés par la spectrométrie de masse MS/MS et leurs rôles physiologiques sur la croissance bactérienne ont été confirmées in vitro. La découverte de biomarqueurs a été complétée par l’analyse ciblée réalisées par Chromatographie en Phase Liquide à Haute Performance (HPLC). La complémentarité entre les différentes techniques métabolomiques a conduit à l’identification de nouveaux biomarqueurs de familles distinctes, comme des composés phénoliques, des sucres, des nucléotides, des acides aminés et des peptides. En outre , l'analyse des réseaux métaboliques a révélé des liens entre les biomarqueurs de levure et a suggéré des voies bactériennes influencés par l’exo-métabolome de levure.Notre workflow multidisciplinaire a révélé une réelle capacité à identifier des signatures moléculaires nouvelles et inattendues de l’interaction levure-bactérie. / As a complex microbial ecosystem, wine is a particularly interesting model for studying interactions between microorganisms. Contact-independent interactions (indirect interactions) between the yeast Saccharomyces cerevisae and the lactic acid bacterium Oenococcus oeni have a direct effect on malolactic fermentation (MLF), induction and completion, which is an important factor in wine quality. Yeast strains could be classified as MLF+ phenotype if it usually stimulates the bacterial growth or MLF- in the opposite case. The known metabolites that stimulate or inhibit the MLF cannot always explain the phenotypic distinction. In this work, a multidisciplinary workflow combining non-targeted metabolomics, targeted analysis, statistics and network was developed. The main objective was to unravel diverse yeast metabolites involved in yeast-bacteria interaction via a direct comparison of exo-metabolomes of MLF+ and MLF- phenotypes.To that purpose, and for the first time in the research of interspecies microbial interactions, two metabolomics platforms, Fourier Transform Ion Cyclotron Resonance -Mass Spectrometry (FT-ICR-MS) and Liquid Chromatography coupled with Mass Spectrometry (UPLC-Q-TOF-MS) were used in combination. To better visualize the high-throughput data generated from the two platforms, a novel unsupervised statistical method, the MetICA was developed and validated. Compared to classical principal component analysis (PCA), the new method reduced the data dimension in a more robust and reliable way. To extract metabolic features involved in the phenotypic distinction, we have compared different statistical classifiers and selected the best one for each dataset. Putative structures of these biomarkers were validated via MS/MS fragmentation analysis and their physiological roles to bacteria were confirmed in vitro. The discovery of biomarkers was complemented by targeted HPLC (high performance liquid chromatography) analysis. The complementarities between different analytical techniques led to new biomarkers of distinct chemical families, such as phenolic compounds, carbohydrates, nucleotides, amino acids and peptides. Furthermore, metabolic network analysis has revealed connections between yeast biomarkers and suggested bacterial pathways influenced by yeast exo-metabolome.Our multidisciplinary workflow has shown its ability to find new and unexpected molecular evidence of wine yeast-bacteria interaction.

Made available in DSpace on 2016-12-23T14:41:52Z (GMT). No. of bitstreams: 1 Thieres Magaive Costa Pereira.pdf: 7173330 bytes, checksum: 5a2aee56b5048351ab0fc838877f8030 (MD5) Previous issue date: 2013-08-23 / O petróleo é uma mistura complexa, sendo uma das misturas mais desafiadoras para as análises químicas. Muitas técnicas são empregadas para a caracterização dos constituintes do óleo. Entretanto, quando combinamos o altíssimo poder de resolução e exatidão da espectrometria de massas, em especial a Ressonância Ciclotrônica de Íons com Transformada de Fourier, (FT-ICR MS) com fontes de ionização a pressão atmosférica, uma atribuição precisa de mais de 20 000 compostos orgânicos do petróleo pode ser obtida. Na indústria do petróleo existe um grande interesse na análise de ácidos naftênicos e asfaltenos devido a problemas como corrosão e formação de depósitos, que aumentam os custos de produção. Portanto este trabalho tem como objetivo a avaliação da termodegradação de ácidos naftênicos, além do emprego das fontes de ESI, APCI, APPI, LDI e MALDI para aquisição de novos dados relativos a caracterização de asfaltenos. Dois petróleos foram utilizados nos ensaios de termodegradação, o petróleo A com acidez de 2,38 mg KOH g-1 e o petróleo B com 4,79 mg KOH g-1 os quais foram tratados a 280, 300 e 350 ºC pelo período de 2, 4 e 6 horas. De uma maneira geral, as principais classes identificadas para ambas às amostras foram O2, N e NO2, respectivamente. Uma leve redução do NAT e da abundância relativa referente à classe O2 foram observado em função da temperatura e do tempo de envelhecimento (T = 280 → 300 oC e t = 2 → 6 h), sendo que a temperatura de tratamento térmico a 350 ºC demonstrou-se determinante na remoção de compostos da classe O2. O perfil químico de amostras de asfaltenos brasileiros foi avaliado utilizando-se cinco diferentes métodos de ionização em ambos os modos de ionização: positivo e negativo, depois, a distribuição de peso molecular, distribuição de classe, parcelas típicas de número de carbono contra DBE e diagramas van Krevelen foram obtidos e discutidos. Um comportamento atípico foi observado para a fonte de LDI e MALDI (±) FT-ICR MS. Um amplo perfil, de m/z 500-3000, apresentando espaçamento de 24 Da foi observado, e este corresponde a formas alotrópicas de fulereno, C60. Em geral, os asfaltenos apresentaram um elevada proporção de espécies heteroatómicas como: HC, HC [H], N, N [H], N2O, N2O [H], N2, N2 [H], O, O[H] para o modo (+) e N, N [H], NO, NO [H], NO, NO2[H], N2O, N2O[H] para o modo negativo. Nos diagramas de DBE contra o número de carbono, os asfaltenos mostraram para cada valor de DBE uma pequena amplitude de compostos com respeito à NC, proporcionando assim, imagens que formam uma linha de 45 ° entre os eixos CN e DBE. Isto indica que estes compostos não exibem grandes cadeias de alquílicas, sendo composta principalmente por anéis aromáticos. Em geral os diagramas de van Krevelen mostraram alta proporção de compostos com razão H/C ~0,7, corroborando com a existência de espécies altamente aromáticas / Petroleum is a complex mixture, being one of the most challenging mixtures for chemical analyzes. Many techniques are employed to characterize constituents of the oil. However , when we combine the highest resolving power and accuracy of mass spectrometry , especially Ion Cyclotron Resonance Fourier Transform ( FT - ICR MS ) with sources of atmospheric pressure ionization , an assignment need more than 20 000 organic compounds in the oil can be obtained. In the oil industry there is a great interest in the analysis of asphaltenes and naphthenic acids due to problems such as corrosion and deposit formation, which increases production costs. Therefore this study aims to evaluate the thermodegradation of naphthenic acids, besides the use of sources of ESI, APCI, APPI, MALDI and LDI for the acquisition of new data on the characterization of asphaltenes . Two test oils were used in thermodegradation of the oil with an acidity of 2.38 mg KOH g -1 and Oil B with 4.79 mg KOH g- 1 which were treated at 280 , 300 and 350 ° C for a period of 2 , 4 and 6 hours. Generally, the major classes identified for both samples O2, NO2, and C, respectively. A slight reduction of NAT and relative abundance refers to the class O2 were observed as a function of temperature and aging time (T = 280 → 300 ° C t = 2 → 6 h), and the heat treatment temperature at 350 ° C showed a determinant for the removal of class O2. The chemical asphaltenes Brazilian samples was evaluated using five different methods of ionization in both modes of ionization: positive and negative, then the molecular weight distribution, distribution class, typical plots versus carbon number and DBE van Krevelen diagrams were obtained and discussed. An unusual behavior was observed for the LDI and MALDI source ( ± ) FT - ICR MS . A comprehensive profile of m / z 500-3000, with spacing of 24 Da was observed, and this corresponds to allotropes of fullerene C60. In general, the asphaltenes had an heteroatómicas high proportion of species such as HC , HC [ H] , N, [ H] , N2O N2O [ H] N2, N2 [ M ] O O [ H] to mode (+) and N, [ H ] , NO [ H] , NO , NO2, [ H] , N2O N2O [ H ] for the negative mode . In the diagrams DBE against the carbon number , the asphaltenes shown for each value of DBE composed of a small amplitude with respect to NC, thus providing images that form a line of 45 ° between the axes CN and DBE. This indicates that these compounds do not exhibit large chain alkyl, composed mainly of aromatic rings. In general diagrams van Krevelen showed a high proportion of compounds ratio H / C ~ 0.7, confirming the existence of highly aromatic species

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2014-12-22T14:31:16Z No. of bitstreams: 1 Dissertação - Maria Juíva Marques de Faria - 2014.pdf: 3781718 bytes, checksum: 5a98e4c3d48dde6e80b58427a1a73012 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2015-01-26T13:13:18Z (GMT) No. of bitstreams: 1 Dissertação - Maria Juíva Marques de Faria - 2014.pdf: 3781718 bytes, checksum: 5a98e4c3d48dde6e80b58427a1a73012 (MD5) / Made available in DSpace on 2015-01-26T13:13:18Z (GMT). No. of bitstreams: 1 Dissertação - Maria Juíva Marques de Faria - 2014.pdf: 3781718 bytes, checksum: 5a98e4c3d48dde6e80b58427a1a73012 (MD5) Previous issue date: 2014-05-07 / Bovine mastitis is an inflammatory/infectious process of the mammary gland, its etiology is complex and multivariate. The most common mastitis has bacterial origin. Because of the high cost to treat this disease, and the bacterial resistance to antibiotics, beyond the losses due to the milk discard or the presence of drug residues in milk, studies are necessary to find out alternative methods to circumvent these problematic. The medicinal plants with antimicrobial activity have been making room in veterinary medicine with the objective to reduce the impact and the use of drugs indiscriminately, as well as maintain the integrity of the animal and control the dissemination of resistant bacterial lineage. Therefore, the aim of this work was characterize the oleoresin Copaifera spp., verify the antimicrobial activity in vitro of oleoresin and essential oil of Copaifera spp. in combating against aerobic and facultative isolated bacteria from cows’ milk with subclinical grade III mastitis diagnostic and evaluate preliminarily, in vivo, phytotherapic formulation composed of Copaifera spp. The tests for characterization of the oleoresin, confirmed its authenticity and quality, considered appropriate for use. The analyzes performed by gas chromatography coupled to mass spectrometry (GC/MS) and electrospray ionization Fourier transformed íonic cyclotron resonance mass spectrometry (ESI FT-ICR MS) identified sesquiterpenes and diterpenes present in the essential oil and oleoresin of Copaifera spp., respectively. The technique of High Performance Liquid Chromatography (HPLC) for sesquiterpenes, especially for β-caryophyllene, can be used in quality control of essential oil and oleoresin of Copaifera spp., as well as in phytotherapic formulation using oleoresin of Copaifera spp. The oleoresin showed better antimicrobial activity than the essential oil of Copaifera spp. (ρ<0,0001) in combating the 55 isolated bacteria. The antibiogram test of the three phytotherapic formulations demonstrated that the oleoresin had antimicrobial activity against the isolated microorganisms. The preliminary evaluation, in vivo, of a phytotherapic formulation containing 20% oleoresin of copaiba was neither effective nor safe, because it intensified the inflammatory process, requiring further tests, such as cytotoxicity and irritability of the raw material plant and of the vehicle used. Key words: Bovine Mastitis. Oleoresin. Essential Oil. Copaifera spp. Minimum Inhibitory Concentration. ESI FT-ICR MS. / Mastite bovina é um processo inflamatório/infeccioso da glândula mamária, de etiologia complexa e multivariada, sendo a de origem bacteriana a mais frequente. Devido aos elevados custos dos tratamentos dessa enfermidade, resistência bacteriana aos antibióticos, além dos prejuízos inerentes ao descarte do leite ou à presença de resíduos de medicamentos no mesmo, faz-se necessário estudos que busquem métodos alternativos para contornar essa problemática. As plantas medicinais com potencial antimicrobiano vêm ganhando espaço na medicina veterinária com o objetivo de reduzir o impacto e o uso de medicamentos de forma indiscriminada, bem como manter a integridade do animal e controlar a disseminação de linhagens bacterianas resistentes. Diante disso, o objetivo desse trabalho foi caracterizar a oleorresina de Copaifera spp., verificar atividade antimicrobiana in vitro da oleorresina e do óleo essencial de Copaifera spp. frente às bactérias aeróbias e facultativas isoladas de leite de vacas diagnosticadas com mastite subclínica grau III e avaliar preliminarmente, in vivo, formulação fitoterápica à base de Copaifera spp. Os testes de caracterização da oleorresina de Copaifera spp. demonstraram sua autenticidade e qualidade, a qual foi considerada adequada para o uso. As análises realizadas por cromatografia gasosa acoplada à espectrometria de massas (CG/EM) e por espectrometria de massas com ionização por electrospray de ressonância ciclotrônica de íons por transformada de Fourier (ESI FT-ICR MS) identificaram os sesquiterpenos e diterpenos presentes no óleo essencial e na oleorresina de Copaifera spp., respectivamente. A técnica Cromatografia Líquida de Alta Eficiência (CLAE) para sesquiterpenos, em especial para β-cariofileno, pode ser utilizada no controle de qualidade de oleorresina e óleo essencial de Copaifera spp., bem como em formulação fitoterápica utilizando oleorresina de Copaifera spp. como matéria-prima vegetal. A oleorresina apresentou melhor atividade antimicrobiana do que o óleo essencial de Copaifera spp. (ρ<0,0001) frente às 55 bactérias isoladas. O teste de antibiograma das três formulações fitoterápicas demonstrou que a oleorresina de copaíba reproduziu a sua atividade antimicrobiana frente aos micro-organismos isolados. A avaliação preliminar, in vivo, de uma formulação fitoterápica contendo 20% oleorresina de copaíba não foi eficaz e nem segura, pois intensificou o processo inflamatório, havendo necessidade de testes futuros, como de citotoxicidade e de irritabilidade dessa matéria-prima vegetal e do veículo utilizado.

Submitted by Cláudia Bueno (claudiamoura18@gmail.com) on 2016-08-17T21:37:32Z No. of bitstreams: 2 Dissertação - Géssica Adriana Vasconcelos - 2016.pdf: 2159448 bytes, checksum: a3e5f1ad74eff4a8b0472438d2a2bbf0 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Cláudia Bueno (claudiamoura18@gmail.com) on 2016-08-17T21:39:28Z (GMT) No. of bitstreams: 2 Dissertação - Géssica Adriana Vasconcelos - 2016.pdf: 2159448 bytes, checksum: a3e5f1ad74eff4a8b0472438d2a2bbf0 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-08-17T21:39:28Z (GMT). No. of bitstreams: 2 Dissertação - Géssica Adriana Vasconcelos - 2016.pdf: 2159448 bytes, checksum: a3e5f1ad74eff4a8b0472438d2a2bbf0 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-03-10 / Outro / Petroleomics by FT-ICR MS was able to characterize petroleum and its derivatives at molecular level. The most used ionization source on the analysis of these compounds is electrospray. However, species that are more basic or more acidic suppress the ionization of other compounds with less strength. This disadvantage consists of, especially in positive mode, only detecting compounds with higher pKa values preventing the mass spectrometer from reading less basic species that is also present in the sample. In view of this, the development of new methods whose purpose is to minimize these effects is essential. An alternative is to use fast techniques of extraction and separation followed by analysis by FT-ICR MS. This study pursued two goals: first, to perform an extraction of vacuum residue into basic and neutral nitrogen compounds using different methods, and second, to fractionate the basic nitrogen compounds present in RV according to their mass/ratio. Three methods were tested to achieve the first objective: solvent extraction (LLE), column chromatography (LC), and solid phase extraction (SPE). The results showed that LLE was the less selective technique among all three studied procedures. SPE and LC showed similar results, and they allowed to partially separate the basic nitrogen compounds from neutral species. To accomplish the second goal, a fractionation using SPE was applied to the vacuum residue sample. This procedure permitted to detect compounds that apparently were not present in the untreated sample as a consequence of a reduction on ion suppression effect. Electrospray ionization and high resolution mass spectrometry (FT-ICR MS) were employed on the analysis of each sample. / A petroleômica por FT-ICR MS foi capaz de caracterizar o petróleo e seus diferentes derivados a nível molecular. A fonte de ionização mais utilizada na análise destes compostos é o electrospray. Componentes mais básicos e mais ácidos, entretanto, suprimem a ionização de outras espécies com menor força. A grande desvantagem deste fato consiste, especialmente no modo positivo, detectar somente os compostos com elevado pKa impedindo a leitura das espécies menos básicas presentes na mesma amostra. À vista disso, é fundamental o desenvolvimento de novos métodos que tenham por finalidade minimizar estes efeitos. Uma alternativa é fazer o uso de técnicas rápidas de extração e separação seguida pelas análises por FT- ICR MS. Desta forma, este estudo teve dois objetivos principais: primeiro, realizar uma extração dos compostos nitrogenados de resíduo de vácuo em suas frações básica e neutra utilizando diferentes métodos e, segundo realizar um fracionamento dos compostos nitrogenados básicos presentes em RV de acordo com a sua massa. Para se alcançar o primeiro objetivo, três métodos foram testados: Extração líquido-líquido (LLE), cromatografia em coluna clássica (LC) e extração em fase sólida (SPE). Os resultados mostraram que o LLE foi a menos seletiva dentre os três procedimentos estudados. SPE e LC apresentaram resultados semelhantes e foi possível separar parcialmente os compostos básicos das espécies neutras. Para alcançar o segundo objetivo, um fracionamento utilizando extração em fase sólida foi empregado na amostra de resíduo de vácuo. Este procedimento permitiu detectar compostos que aparentemente não estavam presentes na amostra não tratada como consequência da redução do efeito de supressão iônica. A ionização por electrospray e por espectrometria de massa de alta resolução (FT-ICR MS) foram utilizados na análise de cada amostra.