La cosmétique valorise des extraits de plantes en les intégrant dans des produits de soin. Ces extraits constituent les ingrédients actifs de la formule cosmétique. Les plantes sont diverses, riches et contiennent de nombreux composés d’intérêt biologique. La phytochimie s’intéresse à décrire le contenu métabolique d’extraits de plantes. Mais la caractérisation moléculaire de ces matrices complexes reste un défi majeur aujourd’hui. En effet, les étapes de traitements de données sont longues et laborieuses. Un outil de profilage métabolique, GAINS, a ainsi été développé afin de traiter de façon automatisée les données issues d’analyses en chromatographie liquide couplée à la spectrométrie de masse haute résolution. Il constitue un support au phytochimiste car le traitement automatisé des données permet de gagner du temps par rapport à un traitement manuel. Cet outil associé à une vaste base de données de produits naturels permet d’attribuer des candidats potentiels aux pics détectés. GAINS fait aussi appel à un module de fragmentation in silico afin d’étayer les propositions de candidats. Cela permet de comparer le spectre de fragmentation modélisé des candidats avec le spectre de fragmentation expérimental.L’ensemble des études phytochimiques réalisées pour identifier ou isoler des composés va de pair avec l’étude du potentiel d’effet biologique des extraits sur la peau, organe ciblé par les cosmétiques. Cela sert à découvrir les actions bénéfiques que peut avoir l’extrait. En connaissant le contenu phytochimique, on peut expliquer et rationaliser les tests d’activités biologiques. Le développement d’un actif anti-âge à partir de la salicaire, une plante de la région Centre-Val de Loire, en est l’exemple. / The cosmetic field valorizes plant extracts by integrating them in care products. These extracts constitute the active ingredients of the cosmetic formulation. Plants are diverse, rich and contain numerous compounds of biological interest. Phytochemistry is a way to describe the metabolic content of plant extracts. But molecular characterization of these complex matrices remains a major challenge nowadays. Indeed,steps of data treatment are time-consuming and laborious. In this way, a tool of metabolic profiling, GAINS, has been developed in order to treat in an automatized way data from analyses performed in liquid chromatography coupled with high-resolution mass spectrometry. It constitutes a real support for phytochemists because automatized data treatment allows gaining time compared to manual treatment. This tool, associated with a large database of natural compounds make possible to assign potential candidates to detected peaks. GAINS appeals a module of in silico fragmentation for holding candidates assignments up.This permits to compare modeled spectrum of fragmentation of candidates with experimental spectrum off ragmentation.The whole set of phytochemical studies realized to identify or isolate compounds goes hand in hand with the study of potential biological effects of extracts to the skin, targeted organ by skin-care products. This allows the discovery of beneficial actions that the extract could have. By knowing the phytochemical content, it is possible to explain and rationalize assays about biological activities. The development of an anti-aging ingredient from purple loosestrife, a plant occurring in the region Centre-Val de Loire, is an example of it.

No description available.

A película adquirida (PA) é um filme formado pela adsorção seletiva de proteínas, glicoproteínas e lipídeos à superfície dentária. A presença de proteínas na PA forma uma interface protetora sobre a superfície do dente, participando em todos os eventos interfaciais que ocorrem na cavidade bucal, tais como des- e remineralização, lubrificação das superfícies dos dentes, e aderência bacteriana. Com o advento da proteômica, tem havido um aumento considerável no conhecimento acerca do perfil proteico de PAs adquiridas formadas sobre o esmalte dentário, em diferentes situações, mas nenhum trabalho até o momento descreveu o perfil proteômico de PAs formadas sobre a dentina. Este estudo foi pioneiro em comparar o perfil proteico de PAs formadas in situ sobre o esmalte e a dentina, nos tempos de 10 minutos e 2 horas, utilizando análise proteômica quantitativa livre de marcadores. Os experimentos foram realizados por três dias consecutivos. Em cada dia, os 9 voluntários receberam profilaxia dentária e em seguida utilizaram um aparelho vestibular com 6 blocos de esmalte e 6 de dentina humanos por 10 minutos ou 2 horas. Após esses períodos, a PA formada era coletada com auxílio de um papel filtro de eletrodos embebido em ácido cítrico 3%. Para as análises foi realizado um pool com os papéis dos 9 voluntários de todos os dias, para cada substrato e tempo de formação. Após a extração e digestão das proteínas, a separação dos peptídeos foi realizada por nano-HPLC (nano-Cromatografia Líquida de Alta Performace), interligada a um espectrômetro de massa (nLC-ESI-MS/MS). Os dados MS/MS obtidos foram processados e pesquisados em bancos de dados de proteínas humanas (UniProt e TrEMBL), utilizando o algoritmo SEQUEST no software Proteome Discoverer 1.3. Para a PA formada sobre o esmalte, foram identificadas 160 e 64 proteínas, nos tempos de formação de 10 minutos e 2 horas, respectivamente. Os respectivos números de proteínas identificadas para a dentina foram 86 e 52, respectivamente. Nos tempos de 10 minutos e 2 horas, respectivamente, 25 e 11 proteínas foram comuns a ambos os substratos e foram submetidas à quantificação livre de marcadores (SIEVE), revelando que a maioria das proteínas com diferença de expressão entre os dois substratos teve sua expressão aumentada na dentina. Foram identificadas ainda, no tempo de 10 minutos de formação da PA, 135 e 61 proteínas exclusivas ao esmalte ou à dentina, respectivamente. O número correspondente de proteínas exclusivas para o tempo de 2 horas foi de 53 e 41 proteínas, para o esmalte e dentina, respectivamente. Dentre as proteínas exclusivas da dentina, foram identificadas várias proteínas relacionadas ao complexo cálcio/calmodulina, assim como proteínas associadas à tumorigênese e à fosforilação/desfosforilação de proteínas. Em adição, muitas das proteínas identificadas no presente estudo, tanto para o esmalte quanto para a dentina, ainda não foram caracterizadas e, portanto, não têm função conhecida na PA. Sua caracterização e estudos funcionais futuros poderão trazer novos horizontes no entendimento da importância da PA para a proteção da estrutura dentária, bem como do papel da PA como sítio de biomarcadores para doenças bucais e sistêmicas. / The acquired pellicle (AP) is a film that results from selective adsorption of proteins, glicoproteins and lipids on the tooth surface. The presence of proteins in the AP forms a protective interface on the tooth surface that participates in all the surface events occurring in the oral cavity, such as de- and remineralization, lubrification of the tooth surfaces and bacterial adherence. With the advent of Proteomics, considerable increase in the knowledge of the protein profile of the AP formed on tooth enamel, under different circunstances, has been observed. However, so far the proteomic profile of the AP formed on dentin has not been described. This is the first study to compare the proteomic profile of APs formed in situ for 10 minutes and 2 hours, on enamel and dentin, using quantitative label-free proteomics. The experiments were conducted for 3 consecutive days. Each day, 9 volunteers were submitted to dental prophylaxis and in sequence wore a vestibular device containing 6 human enamel and 6 human dentin blocks for 10 minutes or 2 hours. After these periods, the PA formed was collected with an electrode filter paper soaked in 3% citric acid. The papers from the 9 volunteers, for each substrate and time of pellicle formation were pooled and used for analysis. After protein extraction and digestion, peptides were separated by nano-HPLC (High-performance liquid chromatography) coupled to a mass spectrometer (nLC-ESI- MS/MS). The obtained MS/MS spectra were searched against human protein databases (UniProt and TrEMBL) using SEQUEST algorithm in Proteome Discoverer 1.3 software. For the AP formed on enamel, 160 and 64 proteins were identified for the times of pellicle formation of 10 minutes and 2 hours, respectively. The respective numbers of identified proteins for dentin were 86 and 52, respectively. For the times of 10 minutes and 2 hours, respectively, 25 and 11 proteins were common to both substrates. They were submitted to label-free quantification, which revealed that most of the proteins with differential expression were overexpressed in the dentin. For APs formed for 10 minutes, 135 and 61 proteins were identified exclusively for enamel or dentin, respectively. The corresponding number for the 2-hour APs was 53 and 41 proteins, respectively. Among the proteins identified exclusively in dentin, many proteins related with calcium/calmodulin complex, as well as proteins associated with tumorigenesis and protein phosphorylation/dephosphorylation were found. In addition, many of the identified proteins, both for enamel and dentin, remain uncharacterized and, therefore have no described function in the AP. In the future, their characterization and functional studies might open new avenues for the understanding of the importance of the AP for the protection of the dental structure, as well as for the use of the AP as a site for biomarkers of oral and systemic diseases.

O arsênio é um dos mais tóxicos elementos químicos e reconhecidamente carcinogênico. Ele pode ser encontrado em alimentos basicamente em 5 formas: arsenobetaína (AsB), dimetil-arsênio (DMA), monometil-arsênio (MMA), arsenato (As5+) e arsenito (As3+), sendo estas duas últimas (As-i) as mais tóxicas. Assim, é de suma importância a utilização da especiação química de As para avaliação dos reais riscos associados à ingestão de alimentos contaminados. Neste sentido, o presente trabalho teve como objetivos o desenvolvimento de um método para separação das espécies de As por LC e detecção por ICP-MS; extrações quantitativas das espécies de As de tecidos animais e em grãos de arroz; aplicação dos métodos em amostras de alimentos consumidos no Brasil; e estudo do metabolismo do As em diferentes cultivares de arroz. O método desenvolvido para a extração das espécies de As em tecidos animais (ovo, músculos de ave, peixe e boi, etc.) utilizou apenas metanol (10%v/v) e ácido nítrico (2%v/v) como extratores e 2 minutos de sonicação, mostrando recuperação quantitativa do analito (>88%, n=3) pela análise dos materiais de referência (CE278, DOLT-3, DORM-3 e SRM NIST 1577). No entanto, para a análise de arroz, apenas ácido nítrico 2%v/v foi utilizado como extrator, possibilitando uma recuperação quantitativa (>94%, n=6), quando da análise do material de referência (NIST Rice Flour 1568a). Para a separação cromatográfica foram avaliadas diversas colunas, das quais a troca aniônica (Hamilton PRP-X100®) foi utilizada em todos os experimentos. A aplicação dos métodos na análise de alimentos diversos consumidos no Brasil mostrou uma grande variação nas concentrações das diferentes espécies do arsênio em músculos, ovos e, especialmente, no arroz. Devido as altas concentrações de As encontradas em amostras de arroz comercializadas no Brasil (em vários casos com predominância das espécies mais tóxicas, As3+ e As5+) e ao grande consumo deste alimento no país e no mundo, foi também realizado um estudo de metabolismo do As em 6 diferentes cultivares. O foco deste estudo foi a síntese de fitoquelatinas (PCs), compostos não peptídicos produzidos por plantas expostas a elementos tóxicos, e sua influência no fator de transferência (TF) do As solo-grãos. O TF do As, assim como a concentração das PCs, variaram em relação aos cultivares (genótipos) estudados. Além disso, as PCs mostraram ter uma forte correlação positiva entre si e com a concentração de As-i nos grãos, bem como uma correlação negativa do TF do As raízes-grãos. Portanto, os métodos desenvolvidos demonstraram fácil aplicação em rotina para avaliação toxicológica dos alimentos em relação às espécies de As e, finalmente, o estudo de metabolismo do As pela planta do arroz pode contribuir para escolha de cultivares que o absorvam menos, reduzindo sua ingestão frente ao consumo de arroz. / Arsenic is one of the most toxic chemicals and known carcinogen. It can be found in food basically in 5 different forms: arsenobetaine (AsB), dimethyl arsenic (DMA), monomethyl arsenic (MMA), arsenate (As5+) and arsenite (As3+), the latter two forms (i-As) are the most toxic. Thus, it is of extreme importance the use of chemical speciation for the evaluation of the real risks associated to arsenic intake from contaminated food. In this sense, this study aimed develop a simple method for separation of arsenic species by LC and detection by ICP-MS, quantitative extraction of arsenic species from animal tissues and rice grains, the application of the proposed method in the analysis of food samples commercialized in Brazilian markets, and the study of arsenic metabolism in different rice cultivars. The method developed for the extraction of arsenic species in animal tissues (egg, muscle of chicken, fish and cattle, etc.) used only methanol (10%v/v) and nitric acid (2%v/v) as extractant and 2 minutes of sonication, showing quantitative recovery of the analyte (>88%, n=3) when analyzing reference materials (CE278, DOLT-3, DORM-3 e SRM NIST 1577). However, for the analysis of rice grains, only nitric acid (2%v/v) was used as extractant, allowing a quantitative recovery (>94%, n=6), when analyzing the reference material (NIST Rice Flour 1568a). For the chromatographic separation several columns were evaluated and an anion exchange column (Hamilton PRP X-100) was used in all experiments. The analysis of several foods consumed in Brazil, showed a wide variation in the concentrations of different arsenic species in muscle, eggs, and especially in rice. Due to the high concentrations of arsenic found in rice samples (in several cases with predominance of more toxic species, As3+ and As5+) and the large consumption of this food in the country and abroad, it was also carried out a study on arsenic metabolism in 6 different rice cultivars. The focus of this study was the synthesis of phytochelatins (PCs), non-peptide compounds produced by plants exposed to toxic elements, and its influence on the transfer factor (TF) soil-grains. The TF of arsenic as well as the concentration of PCs varied in relation to the cultivars (genotype) studied. Furthermore, the PCs have shown a strong positive correlation between themselves and with the concentration of i-As in the grains, and a negative correlation with TF roots-grains. Therefore, the developed method demonstrated feasibility for routine use in toxicological studies of arsenic species in food samples and, finally, the study of arsenic metabolism in rice plant can contribute to the selection of cultivars that absorb less arsenic, thereby reducing the intake of this toxic element by rice consumption.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-05-09T14:52:34Z No. of bitstreams: 1 adrianeantoniapereira.pdf: 4227832 bytes, checksum: c75e5a3802fd9faef6e64104b4d14e9d (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-05-17T14:26:31Z (GMT) No. of bitstreams: 1 adrianeantoniapereira.pdf: 4227832 bytes, checksum: c75e5a3802fd9faef6e64104b4d14e9d (MD5) / Made available in DSpace on 2017-05-17T14:26:31Z (GMT). No. of bitstreams: 1 adrianeantoniapereira.pdf: 4227832 bytes, checksum: c75e5a3802fd9faef6e64104b4d14e9d (MD5) Previous issue date: 2015-03-27 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / As azalactonas são basicamente aminoácidos protegidos que podem ser utilizados na síntese de produtos naturais ou sintéticos. Apesar de serem excelentes pró-nucleófilos, este esqueleto apresenta dois sítios eletrofílicos podendo se comportar como eletrófilo em reações com nucleófilos. Neste trabalho são descritas reações de abertura de azalactonas catalisadas por um ácido de Brønsted, o ácido canforssulfônico (ACS). Para esse fim, as azalactonas foram preparadas em duas etapas, sendo que a primeira consistiu na acilação dos aminoácidos com cloreto de benzoíla em meio alcalino levando aos precursores azalactônicos com até 75% de rendimento e subsequentemente uma reação de ciclização intramolecular mediada por um ativador de ácido carboxílico, o EDC, conduzindo aos compostos desejados com rendimentos que variaram de 8298%. As condições otimizadas para a reação de abertura de azalactonas consistiu no emprego de 10 mol % de ácido canforssulfônico como catalisador, diclorometano como solvente, sem agitação a temperatura ambiente. Avaliou-se o escopo para diversas azalactonas e também para diversos nucleófilos. Os rendimentos foram satisfatórios variando de 43-96%, onde mesmo utilizando substratos impedidos do ponto de vista estéreo, como é o caso do terc-butanol, conduziu ao produto de abertura com 57% de rendimento. Os produtos foram caracterizados por RMN de 1H, 13C, IV e EMAE. Após o preparo e caracterização, voltou-se a atenção para a compreensão do mecanismo de reação envolvido em reações de abertura de azalactonas por nucleófilos catalisadas por ACS. O estudo por ESI(+)-MS/MS evidenciou que o catalisador participa do ciclo catalítico protonando a azalactona em uma etapa anterior ao ataque do nucleófilo, contribuindo assim para diminuição da energia do sistema. / Azlactones are basically protected amino acids which can be used in the synthesis of natural and synthetic products. Despite of being excellent pro-nucleophiles, their structures have two electrophilic sites which could be involved in reactions in the presence of nucleophiles. In this work, azlactone ring opening reactions catalyzed by a Brønsted acid, camphorsulfonic acid (CSA), are described. First we prepared azlactone rings in two steps, amidation using benzoyl chloride in basic conditions following by intramolecular ciclization using EDC. Azlactones were isolated with good to excellent yields (82-98%). The optimized reaction condition consists in the use of 10 mol% of camphorsulfonic acid as catalyst, dichloromethane as solvent, at room temperature without stirring. Next, the scope of various azlactones and nucleophiles were evaluated. Chemical yields were good to excellent, and even by using high sterically bulky substrates such as tert-butanol, leads to the product with a good yield (57%). All synthesized compounds were fully characterized by 1H NMR, 13C NMR, IR and HRMS. Finally, we turned our attention to understand the reaction mechanism. The study by ESI-MS revealed the catalyst participates in the catalytic cycle as a proton donor in a previous step to nucleophilic attack, thereby contributing for a decreased energy system.

Les oestrogènes font partie de la famille des neurostéroïdes. Notamment, l’Estradiol (17β-E2) pourrait avoirdes effets bénéfiques sur certaines anomalies liées à l'âge (déclin mnésique et cognitif, maladie d'Alzheimer). Le dosagede très faibles teneurs du 17β-E2 et de ces métabolites (Estrone, Estriol), dans les tissus cérébraux et le plasma, constitueun outil indispensable pour estimer la variation de leurs niveaux avec l'âge et dans différentes pathologies.Tout d'abord, nous avons élaboré une méthode en RP-HPLC-ESI(-)-MS/MS de quantification des oestrogènes sansdérivation chimique. La sélectivité de la séparation et la sensibilité de la détection de ces molécules ont été amélioréesaprès optimisation des paramètres MRM et chromatographiques.Puis, différents agents de dérivation ont été synthétisés et évalués afin d’augmenter le taux d’ionisation pour améliorerla sensibilité de détection. Après l’analyse ESI-MS et MS/MS de onze dérivés du 17β-E2, nous nous sommes intéressésplus particulièrement au dérivé 17β-E2-Q8S.Afin de réduire les interférences, une méthode de préparation de l’échantillon biologique (tissus cérébral, plasma desouris) a été développée. Les deux dosages (sans et avec dérivation) ont été comparées en termes de sensibilité, limitesde détection et de quantification (LD et LQ), linéarité et reproductibilité. Puis, elles ont été appliquées à l’analyse deséchantillons de cerveau, d'hippocampe et de plasma prélevés sur des animaux jeunes et âgés traités avec des quantitésconnues de 17β-E2. La méthode qui nous est apparue la plus robuste est la méthode sans dérivation avec une LD de0,5 fmole.μL-1. / Estrogens are neurosteroids, especially Estradiol (17β-E2) which is considered to be the most biologicallyactive form. 17β-E2 could have positive effects on some age-associated anomalies (memory and cognitive impairment,Alzheimer's disease). For elucidating and better understanding the molecular and cellular mechanisms that underliethe effects within the brain, it is necessary to quantitate 17β-E2 and its metabolites (estrone and estriol) in brain andplasma.First, the RP-HPLC-ESI(-)-MS/MS method without derivatization was developed. The selectivity of the separationand the sensitivity of detection of the estrogens has been improved after optimization of MRM and chromatographicparameters.Secondly, the various derivatization agents were evaluated after their synthesis in order to improve the sensitivity,selectivity and signal enhancement. After studying the eleven synthesized derivatives of 17β-E2 in ESI-MS andMS/MS, promising results were obtained with the 17β-E2-Q8S derivative.A simple purification method using liquid-liquid extraction followed by C18 solid phase extraction has been optimizedin order to minimize assay interferences. The two assays (with and without derivatization) were then compared interms of efficiency, detection and quantification limits (LOD/LOQ), calibration linearity and reproducibility. Then,both methods were validated on biological samples (brain, hippocampus and plasma) collected from animals treatedwith known amounts of 17β-E2. Finally, the more robust method was the method without derivatization with a LODof 0.5 fmol.μL-1.

Orientador: Fernando Antonio Santos Coelho / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-14T14:01:00Z (GMT). No. of bitstreams: 1 Amarante_GiovanniWilson_D.pdf: 9724952 bytes, checksum: a133d434d4953b2d2f726a07b6f23d0d (MD5) Previous issue date: 2009 / Resumo: Esse trabalho de doutorado visou explorar a reação de Morita-Baylis- Hillman (MBH) nos aspectos relacionados ao seu mecanismo e a utilização de seus adutos, como substratos para síntese de moléculas com propriedades biológicas. Realizamos estudos mecanísticos dessa reação, utilizando ESI-MS, sendo possível, interceptar e caracterizar, em fase gasosa, intermediários dessa reação. Outro estudo nos permitiu re-avaliar o comportamento de (tio)uréias como catalisadores desta reação. Neste trabalho, propomos um mecanismo com a participação de (tio)uréias na reação de MBH. Na segunda parte deste trabalho de tese visamos a utilização de adutos de MBH na síntese de moléculas de interesse biológico. Sendo assim, realizamos a síntese de esqueletos 4-fluoroquinolônicos. Para tal, utilizamos adutos de MBH derivados do o-nitrobenzaldeído. Em particular, estudamos o mecanismo de uma etapa desta síntese, uma ciclização em TFA. Neste estudo, um novo intermediário chave foi caracterizado por ESI-MS. Após 4 etapas sequenciais preparamos a Norfloxacina. Outro alvo foi a síntese enantiosseletiva de um análogo do DFP. Neste trabalho, utilizamos um aduto de MBH que após 3 etapas obtivemos um álcool alílico derivado, que foi submetido às condições de epoxidação de Sharpless. Estes epóxidos foram submetidos a uma reação de abertura de anel e oxidação benzílica mediada por Pd(II). Após 2 etapas, concluímos a síntese dos dois enantiômeros análogos do DFP. A última parte deste trabalho consistiu no emprego do rearranjo de Curtius em adutos de MBH onde preparamos aciloínas, que ao serem tratadas com diferentes aminas, forneceram 1,2-amino-alcoóis em altas seletividades. A primeira estratégia foi aplicada na síntese do (+/-)-bupropion e a segunda estratégia permitiu a síntese diastereosseletiva da (+/-)-espisulosina / Abstract: This work explored the Morita-Baylis-Hillman (MBH) reaction. Mechanistic aspects are explored as well as the uses of its adducts as substrates for the synthesis of biologically active compounds. Using ESI-MS, we describe mechanistic studies of this reaction, intercepted and characterized intermediates under gas phase conditions. Using the same method, we re-evaluated the behavior of (thio)ureas as organocatalysts in the MBH reaction. In this work we propose a new mechanistic view for the role of (thio)ureas as Brønsted-acid catalysts in the MBH reaction. In the second part of this thesis, we used the MBH adducts to prepare substances with pharmaceutical properties. Thus, we synthesized a 4- fluorquinolone skeleton. As starting materials, we used MBH adducts from onitrobenzaldehyde. In particular, we studied one step of this synthesis, an intramolecular cyclization in presence of TFA. We used ESI-MS to intercept and characterize a new key intermediate for this reaction. After 4 steps, the antibiotic Norfloxacine was prepared. We also propose the enantioselective synthesis of DFP (5,5-dimethyl-3-(2- propoxy)-4-(4-methanesulfonylphenyl)-2-(5H)-furanone) analogues. We used a MBH adduct as a starting material, and after 3 steps we prepared an allylic alcohol derivative. This compound was then used in the asymmetric Sharpless epoxidation methodology. In the final steps, we used palladium to catalyze stereoselective ring opening and a sequential benzylic oxidation. After 2 steps, we concluded the synthesis of both enantiomers of the DFP analogues. In the last part of this work, we used the Curtius rearrangement on Morita- Baylis-Hillman adducts to prepare acyloins. They were treated with different types of amines to give diastereoselective 1,2-aminoalcohols. The first strategy was applied in the synthesis of the (+/-)-bupropion and the second was used in the diastereoselective synthesis of the (+/-)-spisulosine / Doutorado / Quimica Organica / Doutor em Ciências

The main objective of this diploma thesis is to describe simultaneous electroencephalography-correlated functional magnetic resonance imaging analysis using EEG data processing. It includes basic characteristics of EEG and fMRI recording and analysis and their combination as simultaneous EEG/fMRI analysis and deals with obstructions during its processing. The thesis includes a design of an experiment used for recording and analysis of simultaneous EEG/fMRI data using EEG source reconstruction for regressor construction. Thesis incorporates a software solution used for extraction of signal describing a source activity interpolated by EEG source reconstruction. The signal is then processed and used to construct a basic regressor. The thesis also deals with the software solution being used for a study focused on intracranial epileptic discharges localization using a simultaneous EEG/fMRI analysis in which it reveals source activity during ongoing epileptic spike and summarizes the results.

Vacuum deposition techniques like thermal evaporation and CVD with their precise layer control and high layer purity often cannot be applied for the deposition of chemical or biological molecules. The molecules are usually decomposed by heat. To overcome this problem, the Electrospray ionization (ESI) process known from mass spectroscopy is employed to transfer molecules into vacuum and to deposit them on a substrate. In this work, a homemade ESI tool was used to deposit BSA (Bovine serum albumin) layers with high deposition rates. Solutions with different concentrations of BSA were prepared using a methanol:water (MeOH:H2O) mixture (1:1) as solvent. The influence of the substrate distance on the deposition rate and on the transmission current was analyzed. Furthermore, the layer thickness distribution and layer adhesion were investigated.

Les phytochélatines (PC) sont des polypeptides ayant la structure générale, (alpha-Glu-Cys)n-Gly, où n = 2 à 11. Leur synthèse est induite par un grand nombre de végétaux en réponse à une élévation de la concentration du milieu en métaux, en particulier le cadmium (ci-après, Cd). Le but de cette étude a été de développer un outil pour évaluer la biodisponibilité du Cd dans les eaux douces. Pour ce faire, une méthode analytique a été réalisée afin de déterminer les phytochélatines induites dans les algues C. reinhardtii. Celle-ci consiste à utiliser la chromatographie liquide couplée à la spectrométrie de masse en tandem (LCHP-SM/SM) "on-line". L’ionisation des molécules est celle faite par électronébulisation (IEN) (traduction de electrospray ionisation). L’objectif principal de ce mémoire est la validation de cette méthode : la détermination des courbes de calibration et des limites de détection et l'identification d'interférences potentielles. L’utilisation de dithiothreitol (DTT) à une concentration de 25 mM a été nécessaire à la conservation de la forme réduite des phytochélatines. En effet, suite à la validation de la méthode d’analyse des phytochélatines il a été démontré qu’elle représente un potentiel d’application. Ceci dans la mesure où l’induction des phytochélatines (PC2, PC3 et PC4) dans les algues C. reinhardtii a été possible à deux concentrations de Cd (1 x 10-7 M et 1 x 10 6 M) et ce, après plusieurs temps d'induction (1, 2, 4, et 6 h). Ainsi, l’étude de la stabilité des phytochélatines a été réalisée et toutes les températures examinées ont démontré une diminution des phytochélatines analysées par HPLC-ESI-MS/MS. Il se pourrait que la cause de la dégradation des phytochélatines soit physique ou chimique plutôt que bactérienne. Toutefois, un approfondissement au niveau de la purification de la solution d’extraction serait nécessaire à la mise au point de la dite méthode analytique afin de quantifier les phytochélatines dans l’algue C. reinhardtii. / Phytochelatins (PC) are polypeptides having the general structure, (alpha-Glu-Cys)n-Gly, where n = 2 to 11. Many plants respond to an elevated concentration of metals in environment, particularly Cd, by synthesizing PC. The purpose of this study was to develop a tool to assess the bioavailability of the Cd in fresh water by determining phytochelatins in algae, C. reinhardtii, by online HPLC-ESI-MS/MS. The gold of this work was the validation of the analytical method i.e. the determination of the calibration curves and the limits of detection. The addition of dithiothreitol (DTT), 25 mM, was found to be necessary to maintain the PC in their reduced form for analysis. It was shown that the liquid chromatography coupled to tandem mass spectrometry (HPLC-ESI-MS/MS) technique has excellent potential for PC analysis, however, it will still requires some more work with respect to sample purification. Furthermore, the stability of the PC was evaluated for different sample storage temperatures. At all temperatures studied, some degradation of PC was observed possibly due to physical rather than chemical or bacterial reasons. Finally, the induction of phytochelatins (PC2, PC3 and PC4) was observed in C. reinhardtii for two Cd concentrations (10-7 M and 10-6 M) and for several induction times (1, 2, 4, and 6 h).