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L’objectif principal de cette thèse était de comprendre comment la variabilité temporelle et spatiale de l’oxygénation de l’eau de fond est enregistrée dans le test des foraminifères benthiques, à travers le rapport Mn/Ca et les paramètres des pores. Pour atteindre cet objectif, Ammonia tepida, un groupe d’espèces pseudocryptiques, du Grevelingenmeer (Pays-Bas) a été principalement étudié. A ce site, le gradient bathymétrique est accompagné par une augmentation de la durée et de l’intensité de l’hypoxie/anoxie saisonnière qui influence indirectement le rapport Mn/Ca et directement la porosité d’A. tepida. Notre étude de la Zone à Oxygène Minimum de la Mer d’Arabie suggère la présence d’un important biais diagénétique dans le signal Mn/Ca lors de la transition d’un foraminifère vivant vers un test fossile. En conclusion de cette thèse, la composition Mn/Ca d’un test des foraminifères benthiques dans notre zone d’étude semble être influencée par trois facteurs temporelles 1) l’oxygénation du milieu benthique et la position de la zone redox enrichie en Mn2+ dans l’eau interstitielle, 2) l’activité des bactéries câblées et 3) la période de calcification, ainsi que par trois facteurs spatiaux qui déterminent la position du foraminifère par rapport à la zone de Mn2+ lors de la calcification 4) le microhabitat, 5) la migration verticale des foraminifères et 6) la bioturbation de la macrofaune. Dans le Grevelingenmeer, la porosité d’A. tepida montre une relation avec l’oxygénation. Le couplage entre porosité et oxygénation semble être direct, une plus grande porosité permettant des échanges de gaz plus intensifs. Au contraire, la relation entre le rapport Mn/Ca et l’oxygénation serait plus indirecte, puisque celle-ci est également influencée par l’activité microbienne. L’utilisation simultanée de ces deux proxies totalement indépendants nous semble prometteuse, spécialement parce que ces proxies réagissent à différentes échelles de temps. / The main objective of this thesis was to investigate how the temporal and spatial variability of bottom water oxygenation is recorded in the benthic foraminiferal test, by the Mn/Ca ratio, and by the pore parameters. To achieve this objective, Ammonia tepida, a group of pseudocryptic species, of the Lake Grevelingen (Netherlands) was mainly studied. At this site, the depth gradient is accompanied by an increase in duration and intensity of seasonal hypoxia/anoxia which indirectly influences the Mn/Ca ratio and directly the porosity of A. tepida. Our study of the Oxygen Minimum Zone in the Arabian Sea suggests the presence of a significant diagenetic effect during the transition of a Mn/Ca signal from a living foraminifer to a fossil signal. In conclusion of this PhD research, the Mn/Ca signal of a benthic foraminiferal test seems to be influenced by three temporal factors: 1) the oxygenation of the benthic ecosystem and the position of the Mn2+ zone in the interstitial water, 2) the cable bacteria activity, and 3) the period of calcification, as well as by three spatial factors which determine the position of the foraminifer with respect to the Mn2+ zone during calcification 4) the microhabitat, 5) the vertical migration of the foraminifer and 6) the bioturbation of macrofauna. In the Lake Grevelingen, the porosity of A. tepida shows a relationship with oxygenation of the benthic ecosystem. The coupling between porosity and oxygenation could be rather direct, a greater porosity allowing more intensive gas exchanges. In contrast, the relationship between the Mn/Ca ratio and the oxygenation would be more indirect, being influenced in particular by the microbial activity. The simultaneous use of these two totally independent proxies seems promising, especially because these proxies react at different time scales.

Ce mémoire décrit l'utilisation de la spectrométrie de masse en mode électrospray pour l'étude mécanistique de réactions catalytiques.Dans le premier chapitre, le mécanisme de la réaction de Wacker est étudié ; l'implication d'espèces dinucléaires en palladium, dans le cas de l'utilisation de la benzoquinone comme oxydant, est mise en évidence. La réoxydation du palladium avant la décomplexation du produit a également été établie.Le deuxième chapitre est consacré à l'étude mécanistique de l'oxydation de l'allylphénole en milieu aqueux. La détection d'un intermédiaire epoxyde confirme un mécanisme en cascade : isomérisation, epoxydation et solvolyse.L'étude du mécanisme de réactions de type Heck déshydrogénant représente le dernier chapitre, quatre intermédiaires clefs du cycle catalytique proposé, ont été détectés. / This thesis describes the use of electrospray spectrometry for mechanistic studies of catalytic reactions. The mechanism of the Wacker reaction has been studied in the first chapter ; the implication of dinuclear palladium species, using benzoquinone as oxidant, has been demonstrated. The reoxidation of the palladium before the decoordination of the product has also been established. The second chapter focuses on the mechanism study of the oxidation of allylphenol in aqueous media. The detection of an epoxyde as intermediate corroborates the proposed mechanism : isomerisation, epoxydation solvolysis. The mechanism of a Dehydrogenative Heck reaction is described in the last chapter. Four key intermediates of the catalytic cycle have been identified.

A constante preocupação com o aumento do uso de agrotóxicos nas lavouras e os riscos gerados pelos resíduos destes compostos fazem com que os órgãos responsáveis pela fiscalização de alimentos no Brasil controlem a presença dessas substâncias nos produtos que chegam à mesa do consumidor. Atualmente, um dos grandes problemas na produção de alimentos é a utilização de substâncias proibidas em lavouras, muitas das quais não possuem estudos nem limites máximos de resíduos (LMR) estabelecidos, assim como a utilização de substâncias já registradas, mas em quantidades ou métodos de manejo incorretos. Ambos os casos podem resultar em sérios problemas à saúde humana. O objetivo deste estudo foi a avaliação da determinação de morfolina em amostras de manga utilizando técnicas como a Extração em Fase Sólida e a Cromatografia Gasosa acoplada à Espectrometria de Massas (SPE-GC-MS), assim como a Microextração em Sorvente Empacotado e Cromatografia Gasosa acoplada à Espectrometria de Massas (MEPS-GC-MS). Um segundo objetivo deste estudo consistiu em desenvolver, validar e avaliar uma metodologia analítica capaz de identificar quantitativamente a morfolina em amostras de manga por Cromatografia Líquida de Ultra Eficiência acoplada a Espectrometria de Massas em tandem (UHPLC-MS/MS). Para análise por GC-MS fez-se necessária a etapa de derivatização do analito, de forma que o mesmo aumentasse sua volatilidade e diminuísse a polaridade. A comparação entre as técnicas SPE e MEPS não foi possível devido ao efeito de matriz causado pela contaminação do liner e da coluna cromatográfica. Já a metodologia validada por UHPLC-MS/MS seguiu os critérios exigidos pelo Manual de Garantia da Qualidade Analítica, do Ministério da Agricultura Pecuária e Abastecimento (MAPA). O método foi aplicado em mangas de diferentes variedades obtidas no comércio local. Não foram encontrados resíduos de morfolina em nenhuma das amostras investigadas, de acordo com a metodologia proposta. Os resultados apresentados neste trabalho estabelecem metodologias eficientes, rápidas e de baixo custo na determinação de morfolina em amostras de manga. / The recurrent increasing of the use of pesticides on crops and the consequent risks due the exposure to chemical residues have urged the food regulation agencies to control the levels of these substances in products that reach consumer\'s table. A major problem nowadays for the production of food is the use of banned substances in crops and the extrapolation of the limit dosages of substances, which may result in serious problems to human health. Furthermore, many of these substances commonly used in crops still lack substantial information about the maximum residue levels (MRLs). The aim of this study was the evaluation of morpholine levels in mango samples using distinct techniques such as Solid Phase Extraction followed by Gas Chromatography coupled to Mass Spectrometry (SPE-GC-MS) and Microextraction by Packed Sorbent followed by Gas Chromatography Mass Spectrometry (MEPS-GC-MS). Another main goal of this work is the development, validation and evaluation of an analytical methodology to identify and quantitfy the presence of morpholine in mango samples, using Ultra-High Performance Liquid Chromatography coupled to tandem Mass Spectrometry (UHPLC-MS/MS). For the analysis of samples by GC-MS, it was required an initial step of derivatization of the analyte, in order to increase its volatility and reduce polarity. The comparison between SPE and MEPS techniques could not be performed due to matrix effects caused by contamination of the liner and the chromatographic column. On the other hand, the validated methodology for UHPLC-MS/MS presented herein followed all requirements proposed by the Analytical Quality Assurance Manual, accordingly to the Ministry of Agriculture, Livestock and Food Supply from Brazil. This method was applied in mango sample belonging to a wide variety of species found in the city of São Carlos, SP. Nevertheless, no significant levels of morpholine residues were found in any of the samples. In this work, we established a methodology efficient, fast and low cost for the determination of morpholine in mangos.

A constante preocupação com o aumento do uso de agrotóxicos nas lavouras e os riscos gerados pelos resíduos destes compostos fazem com que os órgãos responsáveis pela fiscalização de alimentos no Brasil controlem a presença dessas substâncias nos produtos que chegam à mesa do consumidor. Atualmente, um dos grandes problemas na produção de alimentos é a utilização de substâncias proibidas em lavouras, muitas das quais não possuem estudos nem limites máximos de resíduos (LMR) estabelecidos, assim como a utilização de substâncias já registradas, mas em quantidades ou métodos de manejo incorretos. Ambos os casos podem resultar em sérios problemas à saúde humana. O objetivo deste estudo foi a avaliação da determinação de morfolina em amostras de manga utilizando técnicas como a Extração em Fase Sólida e a Cromatografia Gasosa acoplada à Espectrometria de Massas (SPE-GC-MS), assim como a Microextração em Sorvente Empacotado e Cromatografia Gasosa acoplada à Espectrometria de Massas (MEPS-GC-MS). Um segundo objetivo deste estudo consistiu em desenvolver, validar e avaliar uma metodologia analítica capaz de identificar quantitativamente a morfolina em amostras de manga por Cromatografia Líquida de Ultra Eficiência acoplada a Espectrometria de Massas em tandem (UHPLC-MS/MS). Para análise por GC-MS fez-se necessária a etapa de derivatização do analito, de forma que o mesmo aumentasse sua volatilidade e diminuísse a polaridade. A comparação entre as técnicas SPE e MEPS não foi possível devido ao efeito de matriz causado pela contaminação do liner e da coluna cromatográfica. Já a metodologia validada por UHPLC-MS/MS seguiu os critérios exigidos pelo Manual de Garantia da Qualidade Analítica, do Ministério da Agricultura Pecuária e Abastecimento (MAPA). O método foi aplicado em mangas de diferentes variedades obtidas no comércio local. Não foram encontrados resíduos de morfolina em nenhuma das amostras investigadas, de acordo com a metodologia proposta. Os resultados apresentados neste trabalho estabelecem metodologias eficientes, rápidas e de baixo custo na determinação de morfolina em amostras de manga. / The recurrent increasing of the use of pesticides on crops and the consequent risks due the exposure to chemical residues have urged the food regulation agencies to control the levels of these substances in products that reach consumer\'s table. A major problem nowadays for the production of food is the use of banned substances in crops and the extrapolation of the limit dosages of substances, which may result in serious problems to human health. Furthermore, many of these substances commonly used in crops still lack substantial information about the maximum residue levels (MRLs). The aim of this study was the evaluation of morpholine levels in mango samples using distinct techniques such as Solid Phase Extraction followed by Gas Chromatography coupled to Mass Spectrometry (SPE-GC-MS) and Microextraction by Packed Sorbent followed by Gas Chromatography Mass Spectrometry (MEPS-GC-MS). Another main goal of this work is the development, validation and evaluation of an analytical methodology to identify and quantitfy the presence of morpholine in mango samples, using Ultra-High Performance Liquid Chromatography coupled to tandem Mass Spectrometry (UHPLC-MS/MS). For the analysis of samples by GC-MS, it was required an initial step of derivatization of the analyte, in order to increase its volatility and reduce polarity. The comparison between SPE and MEPS techniques could not be performed due to matrix effects caused by contamination of the liner and the chromatographic column. On the other hand, the validated methodology for UHPLC-MS/MS presented herein followed all requirements proposed by the Analytical Quality Assurance Manual, accordingly to the Ministry of Agriculture, Livestock and Food Supply from Brazil. This method was applied in mango sample belonging to a wide variety of species found in the city of São Carlos, SP. Nevertheless, no significant levels of morpholine residues were found in any of the samples. In this work, we established a methodology efficient, fast and low cost for the determination of morpholine in mangos.

Hereditary Hemochromatosis (HH) is an inherited recessive autosomal disorder characterized by accumulation of excess iron. When iron binding proteins become saturated, concentrations of free, or non-transferrin-bound iron (NTBI) rise, a condition thought to be responsible for the adverse effects associated with HH. To investigate that disturbing iron homeostasis plays a role in free radical injury in HH, protein carbonyls were found to be 1-7 times higher in patients with HH than in controls, with the greatest increases being observed in untreated HH patients with high ferritin and >90% transferrin saturation with iron. An Unpaired t test revealed a P value of 0.0278 (P< 0.05), which is considered to be statistically significant. Our data showed a significant positive correlation (linear relationship) between the level of carbonyl content and ferritin concentration in plasma samples from patients with HH. In vitro oxidation of transferrin and ferritin standards with hydrogen peroxide and excess iron, followed by immobilized trypsin digestion (Poroszyme), high-resolution LC-MS/MS analysis (Q-TOF Ultima, Waters) and MS/MS data processing (PEAKS, Bioinformatics Solution), identified several tryptic peptides containing oxidized Met,Trp and His residues. Mapping of the oxidized ferritin residues showed them to be located on the inner face of each sub-unit, the face directed toward the ferritin core where iron is normally stored. Using the same methodology, oxidized residues were subsequently detected in ferritin and transferrin isolated from plasma samples of patients severely affected with HH. Comparing of MS/MS spectra of in vitro oxidized samples that have most fragment ion peaks in common with oxidized peptide MS/MS spectra from samples of patients with HH revealed a significant correlation between the two. These data show that elevated NTBI may be involved in oxidative modification of the iron binding proteins, ferritin and transferrin, and that such modifications may play a significant role in the pathophysiology of HH.

Hereditary Hemochromatosis (HH) is an inherited recessive autosomal disorder characterized by accumulation of excess iron. When iron binding proteins become saturated, concentrations of free, or non-transferrin-bound iron (NTBI) rise, a condition thought to be responsible for the adverse effects associated with HH. To investigate that disturbing iron homeostasis plays a role in free radical injury in HH, protein carbonyls were found to be 1-7 times higher in patients with HH than in controls, with the greatest increases being observed in untreated HH patients with high ferritin and >90% transferrin saturation with iron. An Unpaired t test revealed a P value of 0.0278 (P< 0.05), which is considered to be statistically significant. Our data showed a significant positive correlation (linear relationship) between the level of carbonyl content and ferritin concentration in plasma samples from patients with HH. In vitro oxidation of transferrin and ferritin standards with hydrogen peroxide and excess iron, followed by immobilized trypsin digestion (Poroszyme), high-resolution LC-MS/MS analysis (Q-TOF Ultima, Waters) and MS/MS data processing (PEAKS, Bioinformatics Solution), identified several tryptic peptides containing oxidized Met,Trp and His residues. Mapping of the oxidized ferritin residues showed them to be located on the inner face of each sub-unit, the face directed toward the ferritin core where iron is normally stored. Using the same methodology, oxidized residues were subsequently detected in ferritin and transferrin isolated from plasma samples of patients severely affected with HH. Comparing of MS/MS spectra of in vitro oxidized samples that have most fragment ion peaks in common with oxidized peptide MS/MS spectra from samples of patients with HH revealed a significant correlation between the two. These data show that elevated NTBI may be involved in oxidative modification of the iron binding proteins, ferritin and transferrin, and that such modifications may play a significant role in the pathophysiology of HH.

The aim of presented diploma thesis was to characterize recent knowledge in the field of beer proteomics. The main part of this work was focused on modern instrumental methods of protein analysis, especially on protein identification by mass spectrometry. In experimental part proteins from selected beer samples were isolated, purified and separated by 2-D electrophoresis. The identification was performed by MALDI MS/MS and LC-MS/MS. Identified proteins were divided into 6 groups - serpines and protein Z, trypsine/-amylase inhibitors, yeast proteins, LTP protein, hordeins and other proteins. Proteomic analysis provided identification of proteins important for final analytical and sensory characteristics of the beer - for final beer quality and taste