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 fungus Cunninghamella elegans has in many studies shown to have abiotransformation similar to the metabolism of mammals. If the biotransformation isgeneral, it enables the production of metabolites by the fungus and the use asreference material. The purpose of the project were to examine whether themetabolic process of C. elegans is general, with respect to the formation ofglucosides, and can be applied to different classes of drugs. During the project, theanalyses were performed on a UPLC Q-TOF, run in both MSE and MSMS mode. Themobile phase used consisted of MeOH and 0.1 % formic acid in MQ water. Toincrease the concentration of possible glucosides, the samples were subjected to anacidic or alkaline SPE. Glucosides were detected in the fungal incubates of diclofenac,buprenorphine, norbuprenorphine and oxazepam. For diclofenac, besides twodifferent glucosides (diclofenac glucoside and hydroxylated diclofenac glucoside), ahydroxylated metabolite and a hydroxylated metabolite conjugated with sulfate werediscovered. In the samples containing buprenorphine, the phase I metabolitenorbuprenorphine was also encountered. Further, in the fungal incubates ofdexamethasone a defluorinated metabolite was identified, which is a metabolicpathway never before described for C. elegans.ISSN: 1650

O sequenciamento genético do DNA humano permitiu maior compreensão da funcionalidade dos seres vivos e principalmente a causa de muitas doenças. Entretanto, os estudos em genética têm se limitado a resolverem os problemas da ciência, e atualmente, a solução para o avanço nessa área tem se atribuído à proteômica. Dessa forma, a pesquisa em química analítica intensificou-se na busca de estratégias melhores para a caracterização de proteomas, em três aspectos principais: preparo de amostra, desenvolvimento da instrumentação analítica e bioinformática. Verifica-se a possibilidade da aplicação de muitas técnicas, atualmente, destaca-se a análise de peptídeos (proteômica shotgun) por cromatografia líquida multidimensional acoplada à espectrometria de massas sequencial (LC/LC-MS/MS), devido à possibilidade de automatização, minimização dos problemas e resultados satisfatórios na análise de amostras biológicas complexas. Portanto, neste trabalho desenvolveu-se um método LC/LC-MS/MS (modalidade on-line column switching) o qual se constitui de coluna trocadora catiônica (homemade), trap de aprisionamento e limpeza, coluna capilar hidrofóbica e separação e detecção por espectrometria de massas sequencial automatizada. Com a proposta de uma instrumentação analítica aperfeiçoada, realizamos a confecção de um trap com partículas de elevada retenção dos peptídeos, o que permite ótima recuperação de amostra. Por se tratar de uma técnica de elevada complexidade instrumental, devido a difícil compatibilidade entre as dimensões cromatográficas, possíveis perda de analito no processo e elevado tempo de análise, propomos uma nova abordagem de otimização, por meio de estudos quimiométricos. Assim, neste trabalho, foram avaliados doze parâmetros instrumentais e destes houve uma simplificação de apenas dois fatores, responsáveis por 95% da resposta ótima do método. Este fato permitiu valores da cobertura da proteína de BSA (82,54%), número de peptídeos (65) e score (2134,05) superiores aos reportados na literatura, os quais apresentam tempos de análise maiores. Este estudo fornece informações do comportamento químico dos peptídeos em relação ao método proposto, por meio de uma superfície de resposta e equação matemática que pode contribuir para a aplicação em diferentes proteomas. / The genetic sequence of human DNA has helped the comprehension of life and principally the cause of various diseases. However, genetic studies have limited to resolve science problems and currently solutions to advance in this field have been attributed to proteomics. Thus, the analytical chemistry has intensified on the search for a better strategy to proteomic characterization in three principal aspects: sample preparation, development of analytical instrumentation, and bioinformatics. Proteomics involves the application of many techniques, currently; the peptide analyses (shotgun proteomics) by multidimensional liquid chromatography coupled to tandem mass spectrometry (LC/LC-MS/MS) is the state of the art. The main reasons are because it allows full system automation, less problems in repeatability, and adequate results in analysis of highly complex biological samples. Therefore, this dissertation developed the method LC/LC-MS/MS (modality on-line column switching), this has a cation exchange column (homemade), trap column to clean, hydrophobic capillary column and separation and detection by tandem mass spectrometry. We have proposed an improved analytical instrumentation, with a homemade trap that particles have high retention of peptide, which permit great recuperation of samples. Because it is an instrumental technique difficult, such as, obtain compatibility between the chromatography dimensions, can lose samples in the process and long time analysis, we have proposed a new optimization approach with chemometric analysis of data. On that, were evaluated twelve instrumental parameters and there was a simplification only two factors, these were responsible for 95% of greater response of method. As a result, the coverage of BSA protein was 82,54%, number of peptides 65 and score of 2134,05 values of high significance if compare from that there are in literature that presented greater time analysis. This work describes information about chemistry of peptides to method proposed through a surface of response and math equation that can contribute to different proteomes.

O sequenciamento genético do DNA humano permitiu maior compreensão da funcionalidade dos seres vivos e principalmente a causa de muitas doenças. Entretanto, os estudos em genética têm se limitado a resolverem os problemas da ciência, e atualmente, a solução para o avanço nessa área tem se atribuído à proteômica. Dessa forma, a pesquisa em química analítica intensificou-se na busca de estratégias melhores para a caracterização de proteomas, em três aspectos principais: preparo de amostra, desenvolvimento da instrumentação analítica e bioinformática. Verifica-se a possibilidade da aplicação de muitas técnicas, atualmente, destaca-se a análise de peptídeos (proteômica shotgun) por cromatografia líquida multidimensional acoplada à espectrometria de massas sequencial (LC/LC-MS/MS), devido à possibilidade de automatização, minimização dos problemas e resultados satisfatórios na análise de amostras biológicas complexas. Portanto, neste trabalho desenvolveu-se um método LC/LC-MS/MS (modalidade on-line column switching) o qual se constitui de coluna trocadora catiônica (homemade), trap de aprisionamento e limpeza, coluna capilar hidrofóbica e separação e detecção por espectrometria de massas sequencial automatizada. Com a proposta de uma instrumentação analítica aperfeiçoada, realizamos a confecção de um trap com partículas de elevada retenção dos peptídeos, o que permite ótima recuperação de amostra. Por se tratar de uma técnica de elevada complexidade instrumental, devido a difícil compatibilidade entre as dimensões cromatográficas, possíveis perda de analito no processo e elevado tempo de análise, propomos uma nova abordagem de otimização, por meio de estudos quimiométricos. Assim, neste trabalho, foram avaliados doze parâmetros instrumentais e destes houve uma simplificação de apenas dois fatores, responsáveis por 95% da resposta ótima do método. Este fato permitiu valores da cobertura da proteína de BSA (82,54%), número de peptídeos (65) e score (2134,05) superiores aos reportados na literatura, os quais apresentam tempos de análise maiores. Este estudo fornece informações do comportamento químico dos peptídeos em relação ao método proposto, por meio de uma superfície de resposta e equação matemática que pode contribuir para a aplicação em diferentes proteomas. / The genetic sequence of human DNA has helped the comprehension of life and principally the cause of various diseases. However, genetic studies have limited to resolve science problems and currently solutions to advance in this field have been attributed to proteomics. Thus, the analytical chemistry has intensified on the search for a better strategy to proteomic characterization in three principal aspects: sample preparation, development of analytical instrumentation, and bioinformatics. Proteomics involves the application of many techniques, currently; the peptide analyses (shotgun proteomics) by multidimensional liquid chromatography coupled to tandem mass spectrometry (LC/LC-MS/MS) is the state of the art. The main reasons are because it allows full system automation, less problems in repeatability, and adequate results in analysis of highly complex biological samples. Therefore, this dissertation developed the method LC/LC-MS/MS (modality on-line column switching), this has a cation exchange column (homemade), trap column to clean, hydrophobic capillary column and separation and detection by tandem mass spectrometry. We have proposed an improved analytical instrumentation, with a homemade trap that particles have high retention of peptide, which permit great recuperation of samples. Because it is an instrumental technique difficult, such as, obtain compatibility between the chromatography dimensions, can lose samples in the process and long time analysis, we have proposed a new optimization approach with chemometric analysis of data. On that, were evaluated twelve instrumental parameters and there was a simplification only two factors, these were responsible for 95% of greater response of method. As a result, the coverage of BSA protein was 82,54%, number of peptides 65 and score of 2134,05 values of high significance if compare from that there are in literature that presented greater time analysis. This work describes information about chemistry of peptides to method proposed through a surface of response and math equation that can contribute to different proteomes.

Les tanins condensés et les pigments polymérisés sont deux grandes familles de macromolécules qui jouent des rôles importants sur la qualité organoleptique du vin rouge. Leurs structures oligo-polymériques ainsi que leurs évolutions durant le vieillissement du vin rouge sont mal connues. L’objectif de cette étude est d’approfondir notre connaissance sur leurs structures et leurs évolutions durant le vieillissement. Durant ce travail, nous avons caractérisé, pour la première fois dans le règne végétal, une nouvelle sous-famille de tanins condensés nommés : les tanins condensés couronnes, qui ont des propriétés spécifiques. Ils sont plus polaires que des tanins condensés de type B. Durant le vieillissement, les concentrations des tanins de type B diminuent alors que celles des tanins couronnes restent plutôt stables. Leurs concentrations sont plus élevées dans les vins issus de cépage Syrah que dans les vins issus de cépage Cabernet Sauvignon et Merlot. Au niveau sensoriel, une forte corrélation entre la concentration des tanins couronnes et l’intensité d’astringence ressentie par les dégustateurs a été montrée. Un premier test d’activité biologique du tétramère couronne montre une activité inhibitrice intéressante de l’agrégation du peptide β-amyloïde impliquée dans la maladie d’Alzheimer. En même temps, une nouvelle méthode de quantification des tanins condensés liés par pont éthylidène via un marqueur spécifique en utilisant une détection par fluorescence a été développée. Concernant les pigments polymérisés avec différents types de liaisons inter-flavonoïdes, leurs mécanismes de dépolymérisation chimique en milieu acide ont été clarifiés et une méthode de quantification sur un système UPLC-Q-TOF a été développée. Les pigments oligo-/polymériques contribuent plus à la couleur du vin vieux que les pigments mono-/dimériques. La concentration des structures oligo-polymériques ayant des liaisons de type A est plus stable que celle des pigments polymérisés par des liaisons de type B ou par le pont éthylidène durant le vieillissement du vin. De plus, durant nos investigations sur les pigments polymérisés, les structures de type A-F(type A)-F(n) et de type F(n)-A-F(type A) ainsi que les formes acétylées d’anthocyane trimère et les formes acétylées des anthocyanes dimères liées par pont éthylidène ont été montrées pour la première fois. / Condensed tannins and polymeric pigments are two families of macromolecules which play important roles on the organoleptic quality of red wine. Their oligo-polymeric structures and their evolutions during red wine aging are poorly known. The objective of this study is to deepen our knowledge of their structures and their evolutions during red wine aging. During our work, we characterized, for the first time in plant kingdom, a new family of condensed tannins named : crown condensed tannins, which have specific properties. They are more polar than B-type tannins. During red wine aging, the concentration of B-type tannins decreased while the crown tannins remained stable. Their concentrations are higher in Syrah wines than in wines made from Cabernet Sauvignon and Merlot grapes. From a sensory point of view, a strong correlation between the concentration of crown tannins and the intensity of astringency rated by the tasters was obtained. The first biologic activity assay of the crown tetramer showed an interesting inhibitory activity on aggregation of β-amyloid peptide involved in Alzheimer's disease. At the same time, a new quantification method of ethylidene bridge linked condensed tannins via a specific marker using fluorescence detection has been developed. Concerning polymeric pigments with different types of inter-flavonoids linkages, their acidic depolymerization mechanisms have been clarified for the first time and a quantification method using a UPLC-Q-TOF system has been developed. Oligo-/polymeric pigments contributed more to the color of old wine than mono-/dimeric pigments. The concentration of the pigmented oligo-polymers with A type linkages is more stable than that of polymeric pigments by B-type linkages or by ethylidene bridge during red wine aging. In addition, during our study of polymeric pigments, A-F(A type)-F(n) and F(n)-A-F(A type) structures as well as the acetylated form of trimeric anthocyanin and acetylated forms of dimeric anthocyanin linked by ethylidene bridge have been shown for the first time.

Perfluoroalkyl substances (PFASs) are anthropogenic compounds that have been classed as persistent organic pollutants (POPs) and are found in both commercial and industrial products. PFASs have been detected in different environmental matrices and have been found to bioaccumulate in all trophic levels. The adverse effects that are associated with PFAS exposure include reduced body weight, increased liver weight, hepatocellular hypertrophy, a decrease in serum cholesterol and triglycerides. This project aims to characterize the metabolic changes in lipid metabolism in the liver after exposure to one of the well-studied PFASs, the perfluorooctane sulfonate (PFOS), during the embryo development using the domestic chicken as a model organism. The characterization of the metabolic changes was done by conducting both quantitative lipidomic analysis and semi-quantitative global profiling on extracted lipids from liver homogenates from a former related project looking at fatty acid profiles. The extracted lipids were analyzed using UHPLC/Q-TOF-MS. In the quantitative analysis, the PFOS-treated groups (0.1 ug/g and 1.0 ug/g)exhibited higher lipid concentrations when compared with the solvent control group (5% DMSO) and the untreated group leading to the conclusion that PFOS exposure disrupts the lipid metabolism. When comparing the lipid concentrations between the two PFOS-treated groups (0.1 ug/g and 1.0 ug/g), the majority of the lipids exhibited higher lipid concentrations in the 1.0 ug/g PFOS-treated groups leading to the conclusion that the effect PFOS has on the lipid metabolism is dose dependent. In the global profiling analysis, 63 lipids showed significant differences when comparing the solvent control group with samples either treated with 0.1 ug/g PFOS or 1.0 ug/g PFOS.

Perfluorooctane sulfonate (PFOS) is widely used in industrial products and is potentially dangerous to the aquatic environment due to not being broken down whether by chemical or biological means, having a half-life of more than 41 years and disrupting hormones. Hexabromocyclododecane (HBCD) is the third most used brominated flame retardant and is of environmental concern as it bioaccumulates and magnifies in the food chain and is highly toxic to aquatic organisms. The purpose of this study was to examine the effect of PFOS and HBCD on the embryos of rainbow trout (Onchorhynchus mykiss) by analyzing lipid profiles with UHPLC/Q-TOF-MS. The fish embryos were treated with various concentrations of PFOS and HBCD (0.058-58 μg/l and 0.014-14 μg/l respectively) with DMSO as carrier solvent and then extracted after homogenization with 0.9% NaCl-solution followed by addition of ISTD mixture, methanol, methyl tert-butyl ether (MTBE) and MQ-water. The raw data was processed with MZmine-2.32. 153 lipids were identified with the main lipids consisting of glycerophospholipids and triacylglycerols. A two-tailed t-test was used to study the impact of the chemical exposure on the embryos, where p-values below 0.05 were lipids considered as significant change. The HBCD exposure caused significant change in various triacylglycerols, whereas PFOS exposure caused significant change in triacylglycerols as well as in glycerophospholipids such as PC(O-38:5) and LPC(20:4). The results were in alignment with previous studies.

The aim of this master thesis project was to develop methods for sample preparation and analysis by LC-MS suitable for global metabolomics of human plasma samples. In this thesis six different methods was tested, based on precipitation, solid phase extraction (SPE), and ultrafiltration. The methods differ in their mechanisms of action, but the end goal is the same, to remove the proteins and other high molecular weight compounds from the samples and to retain as many metabolites as possible in a reproducible manner. The LC-MS analysis were performed on a C18 and a HILIC type column using electrospray ionization (ESI) with both positive and negative ionization to cover as much of the metabolome as possible. The MarkerLynx software was then used to extract features from the chromatograms. The coefficient of variation (CV) was calculated and the features with CV &gt; 30 % were removed. The features were then used to compare the methods with each other in order to see whether any of the methods yielded the same capture of features. The largest amount of features was detected for precipitation with MeOH when using a C18 type column. For HILIC type columns precipitation with MeOH with a small addition of H3PO4 resulted in most unique features detected. The ionization mode was found to be less important, compared whit the choice of column, but more features was detected using positive mode than negative mode.

Le développement des outils analytiques pour l'analyse de la matière organique complexe en géochimie organique a connu de nombreuses avancées ces dernières années. Ce développement a permis de répondre à un grand nombre de questions quant à la composition de la matière organique. Cependant, beaucoup des points restent encore à élucider comme notamment la caractérisation des fractions de hauts poids moléculaires ainsi que le suivi de la réactivité de la matière organique. Ce travail de thèse a eu pour objectif (i) d'adapter les techniques de spectrométrie de masse déjà existantes pour l'analyse de la matière organique fossile (notamment par la sélection de la source d'ionisation atmosphérique la plus adaptée) mais également (ii) de développer un nouveau type de couplage entre la chromatographie d'exclusion stérique (SEC) et la spectrométrie de masse APPI-QTOF pour l'analyse des fractions peu polaires de hauts poids moléculaires. L'adaptation du l'APPI-QTOF a tout d'abord permis de mieux comprendre la réactivité de contaminants organiques polyaromatiques en présence de phases minérales. Le couplage SEC-APPI-QTOF a, quant à lui, permis d'améliorer les connaissances sur la structure des asphaltènes. Cependant, malgré la « simplification » rendue possible par la SEC, la très grande quantité d'informations reste difficile à interpréter et prend beaucoup de temps. Un modèle mathématique a donc été développé basé sur des analyses numériques et statistiques des spectres de masse, permettant de les comparer entre eux afin de distinguer l'origine des échantillons et de suivre l'impact de processus physico-chimiques (altérations naturelles - traitements de remédiation). / The development of analytical tools for organic geochemistry analysis has increased these past years. This development has allowed answering many questions about organic matter composition. However, many issues remain to be clarified including the characterization of high molecular weight fractions and monitoring the reactivity of organic matter. This thesis has focused on both (i) existing method improvements for fossil organic geochemistry analysis but also on (ii) developing a new type of coupling between the size exclusion chromatography (SEC) and the APPI-QTOF mass spectrometry for high molecular weight weakly polar fractions. Adjustments on APPI-QTOF mass spectrometry have allowed a better understanding of polyaromatic organic contaminant reactivity in presence of mineral matrices. The success of this coupling has allowed a better understanding of the structure of asphaltenes. However despite the "simplification" obtained by the SEC, the large amount of information remains difficult to interpret and time-consuming. A mathematical model has been developed based on numerical and statistical analysis of mass spectra, allowing direct comparison of mass spectra and being able to identify several types of information such as origins of samples, monitoring of physico-chemical processes and also the efficiency of soil recovery treatments as well as the identification of analytical protocols.