Submitted by Cássia Santos (cassia.bcufg@gmail.com) on 2017-07-25T13:32:13Z No. of bitstreams: 2 Dissertação - Junaine Vasques da Silva - 2002.pdf: 3727327 bytes, checksum: 4cb8c1db4d3fb95798779f39aae78673 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-07-26T12:05:00Z (GMT) No. of bitstreams: 2 Dissertação - Junaine Vasques da Silva - 2002.pdf: 3727327 bytes, checksum: 4cb8c1db4d3fb95798779f39aae78673 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-07-26T12:05:00Z (GMT). No. of bitstreams: 2 Dissertação - Junaine Vasques da Silva - 2002.pdf: 3727327 bytes, checksum: 4cb8c1db4d3fb95798779f39aae78673 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2002-12-19 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The main function of the most superficial layer of the epidermis, the Stratum Corneum (SC), is to provide a physical barrier that controls the transepidermal water loss as well as the permeation of another substances in both directions across the skin. The SC is formed by anabolically dead cells, the terminally differentiated corneocyte, and its function is essentially accomplished by forming a highly insoluble protein structure on the surface of the corneocytes, termed the cornified cell envelope, and by impeding water diffusion across the SC by mortaring the corneocytes together by layers of skin-specific lipids, essentially ceramide, cholesterol and fatty acid. In this work the cell envelope of the SC was spin labeled with a sulfhydryl-specific nitroxide reagent to investigate the water content effects upon the protein dynamics directly in the intact tissue. A two-state model for the nitroxide side chain described the coexistence of two spectral components in the electron paramagnetic resonance (EPR) spectra. The so-called strongly immobilized component, S, is associated with the EPR signal of a motionally restricted nitroxide fraction having its N-O group hydrogen bonded to protein (rigid structure) while the weakly immobilized component, W, corresponds to the signal provided by the spin labels with higher mobility (~10 times greater) exposed to the aqueous environment. The relative populations between these two mobility states, S and W, are in thermodynamic equilibrium. The standard Gibbs free energy, enthalpy and entropy changes for transferring the nitroxide side chain from the state contacting the solvent, W, to the one contacting protein, S, indicated that the reduction of the SC water content to below ~h 0.69, g H2O per g dry SC, stabilizes the protein interacting state, S. Upon decreasing the SC hydration level below ~h 0.69 the segmental motion of the polypeptide chains and the rotational motion of the spin-labeled side chain were also constrained. To test our methodology in a pure and very well known protein, we also studied the effects of two types of detergents on the bovine serum albumin (BSA). Both detergents, the anionic sodium dodecyl sulfate (SDS) and the zwitterionic N-hexadecyl-N,N-dimethyl-3-ammonium-1-propanesulfonate (HPS) increase the mobility of the protein backbone and of the nitroxide side chain. The thermodynamic parameters indicated that these detergents destabilize the protein favoring less compact conformations. This work can also be useful to improve the spectral analysis of site-directed spin labeling, especially for a more quantitative description in terms of thermodynamic parameters. / A camada mais superficial da epiderme, o Estrato Córneo (EC), tem como função principal a formação de uma barreira física que controla a perda de água do corpo bem como a permeação de outras substâncias em ambas as direções da pele. O EC é formado por células anabolicamente mortas, os corneócitos, os quais sofreram diferenciação celular terminal, e sua função é realizada formando uma estrutura de proteínas altamente insolúveis na superfície do corneócito, chamada de envelope celular, e também uma matriz lipídica, essencialmente ceramídios, colesterol e ácidos graxos, que dificultam a difusão da água. Neste trabalho, o EC foi marcado com marcadores de spin específicos para reagir com os grupos sulfidrilas das proteínas, para investigar os efeitos do conteúdo de água na dinâmica de proteínas diretamente no tecido intacto. Um modelo de dois estados para a cadeia lateral do nitróxido descreveu a coexistência de duas componentes espectrais de ressonância paramagnética eletrônica (RPE). A componente denominada fortemente imobilizada (S), surge de uma fração de marcadores com o átomo de oxigênio do nitróxido ligado à proteína (estrutura rígida) enquanto a componente fracamente imobilizada é gerada pelos marcadores com mobilidade mais alta (~10 vezes maior) e expostos ao ambiente aquoso. As populações relativas entre estes dois estados de mobilidade, S e W, estão em equilíbrio termodinâmico. Os parâmetros da termodinâmica: energia livre padrão de Gibbs, entalpia e entropia, envolvidos na transferência da cadeia lateral do nitróxido do estado W, contatando ao solvente, para o estado S, contatando a proteína, indicaram que a redução do conteúdo de água para abaixo de ~0.69g de H2O por g de EC seco, estabiliza o estado S (cadeia lateral do nitróxido dobrada sobre a cadeia principal da proteína). Ao diminuir o nível de hidratação para abaixo de ~ h 0.69 (g H2o/g EC seco) o movimento local da cadeia polipeptídica e o movimento rotacional da cadeia lateral do marcador de spin foram ambos reduzidos. Para testar nossa metodologia em uma proteína pura e bem conhecida, estudamos os efeitos de dois tipos de detergentes sobre a albumina do soro bovino (BSA). Ambos os detergentes, o aniônico dodecil sulfato de sódio (SDS) e o ziteriônico N-hexadecil-N,N-dimetil-3-amônio-1-propanosulfonato (HPS) aumentaram a mobilidade da cadeia principal da proteína e da cadeia lateral do nitróxido. Os parâmetros termodinâmicos indicaram que estes detergentes desestabilizam a proteína favorecendo conformações menos compactas. Os resultados do presente trabalho também podem contribuir para aprimorar a

Cette thèse est le résultat d’une cotutelle entre l'Université de Pau et des Pays de l'Adour (UPPA) à Pau, en France et l'Université Christian Albrecht (CAU) à Kiel, en Allemagne. Dans le cadre de cette collaboration internationale, des méthodes bio-analytiques sont développées pour analyser quantitativement et qualitativement des peptides et protéines marquées par le couplage de la chromatographie avec la spectrométrie de masse. Les peptides et les digestats des protéines sont marquées selon un protocole optimisé par des lanthanides en utilisant des composés à base de DOTA. La séparation des peptides est réalisée par IP-RP-nanoHPLC. Des données complémentaires sont acquises par MALDI-MS pour l'identification et par ICP-MS pour la quantification. Dans ce contexte, une étape de pré-nettoyage en ligne est développée et mise en œuvre dans le protocole de séparation par nanoHPLC. Cette étape permet l'élimination efficace des réactifs appliqués en excès et ainsi la diminution du bruit de fond lié à la présence de métaux lors des analyses par ICP-MS. Les données obtenues sont alors plus facile à interpréter, la sensibilité des signaux des peptides n’étant par ailleurs pas modifié. L'extraction en phase solide (SPE) appliquée comme alternative entraîne des pertes importantes de peptides et peut être considérée comme inadaptée pour l'analyse quantitative. Des additifs pour éluants de nanoHPLC, tels que l'EDTA et le HFBA sont testés et jugés non bénéfiques pour l'analyse des échantillons peptidiques normaux. HFBA peut être reconsidéré pour une application spéciale sur des peptides très hydrophiles. Des peptides marqués sont développés. Leur utilisation en quantité connue pourrait permettre la quantification rapide et simple d'un échantillon de digestat à faible complexité. De plus, cet ensemble de peptides permet la superposition fiable des chromatogrammes, et ainsi de comparer des données complémentaires obtenues par l’analyse d’échantillon par ICP-MS et MALDI-MS. Expériences d'application avec le couplage laser femtoseconde avec ICP-MS sont effectuées sur des plaques métalliques de MALDI MS et montrent des résultats très prometteurs. Pour cela, les échantillons préalablement identifiés par MALDI-MS sont analysés par fsLA-ICP-MS. Les premières tentatives de quantification sur la plaque en acier modifiée sont satisfaisantes et donnent des résultats répondant aux attentes. L’optimisation des paramètres de MALDI-MS facilite l’identification des peptides. / This PhD thesis was a Cotutelle between the Université de Pau et des Pays de l’Adour (UPPA) in Pau, France and the Christian-Albrechts University (CAU) in Kiel, Germany. In the course of this international collaboration, bio-analytical methods for the quantitative and qualitative analysis of labelled peptides and proteins were developed, which were based on the hyphenation of chromatography with mass spectrometry. Peptides and protein digests were lanthanide labelled using DOTA-based compounds according to an optimised protocol. Separation on the peptide level was performed using IP-RP-nanoHPLC. Complementary data sets were acquired using MALDI-MS for identification and ICP-MS for quantification. In this context, an online precleaning step was developed and implemented in the nanoHPLC separation routine, which allowed for effective removal of excess reagents. This lead to lowered metal backgrounds during ICP-MS measurements and thus better data interpretability, while guarding peptide recovery at a maximum level. An alternative offline purification using solid phase extraction (SPE) resulted in important peptide losses and can be considered unsuitable for quantitative analysis. Additives to the nanoHPLC eluents, such as HFBA and EDTA were tested and not deemed beneficial for the analysis of normal peptide samples. HFBA can be reconsidered for special application on very hydrophilic peptide species. A set of labelled peptides was developed, which due to application of known quantities could be employed for quick and simple quantification of a low complexity digest sample. In addition this peptide set allowed for the reliable superposition of chromatograms, enabling sample comparability especially for complementary ICP-MS and MALDI-MS data. Experiments for application of fsLA-ICP-MS on MALDI-MS target plates were conducted and showed very promising results. For this purpose, samples that were already identified using MALDI-MS were supposed to be remeasured using fsLA-ICP-MS. First quantification attempts on the modified steel target plate were successful and in the range of expectance. Adjusted parameters for MALDI-MS allowed for proper peptide identifications.