Bien que la protéomique ait été largement appliquée pour l’étude du plasma humain, son application dans le domaine de la transfusion sanguine reste peu employée. En collaboration avec l’EFS, l’objectif de cette thèse a donc été de proposer des outils analytiques destinés à évaluer l’impact des traitements d’inactivation des agents pathogènes et des conditions de stockage sur les protéines plasmatiques. Le traitement au bleu de méthylène est le traitement d’inactivation virale le plus utilisé en France. Une approche globale et ciblée se sont intéressées aux modifications induites par ce traitement photochimique. Plusieurs modifications, notamment sur les sous-unités du fibrinogène, ont pu être identifiées, après analyse nanoLC-nanoESI-Qh-FT-ICR MS. L’origine de la diminution d’activité du fibrinogène a pu ainsi être expliquée. Une étude thermique a permis d’identifier un marqueur de dégradation plasmatique: la RBP4. Dans le plasma, elle forme un complexe avec la transthyrétine. Lors de la dégradation du plasma, ce complexe se dissocie. Une méthode de quantification absolue, basée sur des peptides AQUA, a été développée permettant de doser RPB4 libérée dans le plasma au cours de la conservation du plasma. Enfin, deux matrices innovantes pour l’électrophorèse sur gel ont été évaluées pour la séparation de protéines plasmatiques. L’une incorpore un polymère préformé, le dextran, à une solution d’acrylamide classique. L’autre fait appel à un polymère hydrophile, le NAT. Toutes deux présentent de bonnes propriétés optiques et mécaniques, augmentent significativement la résolution des spots protéiques et facilitent l’identification des protéines par MS. / Proteomics has been widely applied to study plasmatic proteins; its application to the field of transfusion medicine is s quite recent. In partnership with the French blood agency (EFS), the main objective of the Ph.D work was to provide analytical tools to evaluate the impact of pathogen inactivation treatments and storage conditions on plasmatic proteins. Photochemical treatment using methylene-blue is the most used for pathogen inactivation in France. Both a global and targeted studies were carried to determine the proteins modifications involved by this treatment. Based on nanoLC-nanoESI-Qh-FT-ICR MS analyses, several modifications were pointed out, especially targeting the sub-unit of fibrinogen. This allows the decrease in fibrinogen clottability after methylene-blue treatment to be explained.A study of thermal degradation on plasma sample pointed out a new marker of plasma degradation: the RBP4. It circulates associated to with transthyretin as a macromolecular complex: during degradation, this complex dissociates releasing RBP4 in plasma. An absolute quantification method was developed using AQUA peptides to assay the amount of the free form of RBP4 in plasma during storage.Two innovative matrices for gel electrophoresis were developed and evaluated for plasma protein separation. One of them relies on the use on a preformed polymer incorporated prior to acrylamide polymerization. The other one is based on a hydroxylated acrylamide monomer, the N-acryol-tris(hydroxymethyl)-amino methane. Both exhibited interesting optical and mechanical properties, enhanced spot resolution and outstanding protein/peptide recovery, which facilitates protein identification by MS.

L’utilisation fréquente de biomédicaments, composés principalement de protéines recombinantes, nécessite de développer les outils qui permettent de stabiliser les protéines. La lyophilisation est une technique couramment utilisée, dans domaine pharmaceutique, afin de convertir une formulation de protéine de l’état liquide à l’état sec, assurant ainsi une meilleure stabilité à la protéine. Une première partie de cette thèse a consisté à détailler, à l’échelle moléculaire, les mécanismes de bio-préservation des disaccharides pendant une procédure de lyophilisation, et comment une faible quantité de glycérol pouvait exacerber les propriétés bioprotectrices des disaccharides. L’analyse in-situ par imagerie Raman des trois étapes d’un cycle de lyophilisation a révélé que l’efficacité bioprotectrice résultait de la combinaison de propriétés physiques liées à la capacité du disaccharide de former un verre (valeur de Tg) et à celle de former un réseau de liaisons hydrogène rigide, caractérisé par des temps de vie des liaisons plus longs. Une seconde partie a été consacrée à l’étude d’un certain type de dérivé de β-cyclodextrines, qui sont des molécules permettant à la fois la libération contrôlée de molécules et d’inhiber des phénomènes d’agrégation. Les analyses Raman de la dénaturation chaude du lysozyme en présence de la HPβCD ont révélé des phénomènes originaux, en particulier un effet déstabilisateur ou stabilisateur suivant une vitesse de chauffe plus ou moins rapide de la solution. Cet effet cinétique a été relié à la capacité de la cyclodextrine à complexer les résidus de la protéine plus ou moins favorisée suivant la vitesse de chauffage. / The frequent use of biopharmaceuticals, mainly composed of recombinant proteins, requires the development of tools for stabilizing proteins. Freeze-drying is a commonly used technique in the pharmaceutical field to convert a liquid protein formulation into the dry state, thus providing better protein stability. A first part of this thesis focused in deciphering, on the molecular scale, the mechanisms of bio-preservation of disaccharides during a freeze-drying cycle, and in understanding how a small amount of glycerol could enhance the bio-protective properties of disaccharides. In situ Raman imaging analyzes, performed during the three steps of freeze-drying cycle revealed that the bio-protective efficiency resulted from the combination of physical properties related to the capacity of the disaccharide to be vitrified (Tg value) with that to form a rigid hydrogen-bond network, characterized by longer lifetime of the H-bonds. A second part has been devoted to the study of β-cyclodextrin derivatives, which can be used, both as drug delivery system and as inhibitor of protein aggregation. Raman analyzes performed during lysozyme thermal denaturation in presence of HPβCD, have revealed original results such as a destabilizing or stabilizing effect depending on the heating rate of the solution. This kinetic effect was related to the capability of cyclodextrins to include the protein residues inside their cage, probably favored by a slow heating.

Bibliography: leaves 187-202 / 204 leaves ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Organic Chemistry, 1991

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Orientador: Prof. Dr. Luiz Cláudio Fernandes / Dissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Educação Física. Defesa: Curitiba, 2004 / Inclui bibliografia / Resumo: Este estudo teve como objetivos analisar as respostas imunitárias da proliferação dos linfócitos obtidos dos linfonodos mesentéricos, as respostas fagocíticas, a produção do ânion superóxido e o volume lisossomal dos macrófagos peritoneais, retirados de ratos Wistar, machos, 60 a 90 dias de vida, divididos nos seguintes grupos: CTL - não treinados; GLN - não treinados e suplementados com L- glutamina; EX - treinados e, EX/GLN - treinados e suplementados com L- glutamina. Os ratos foram suplementados com L-glutamina (0,125g/kg de peso corporal) por gavagem, e nos não suplementados receberam água destilada. Os ratos EX e EX/GLN foram submetidos ao treinamento de natação de 6 semanas, em piscinas individuais com água aquecida a 32°C, com sobrecarga de chumbo colocada no dorso do animal, na carga de 6% da massa corporal do animal. Na primeira semana, os animais nadaram sem sobrecarga, para ambientação ao meio aquático. Nas outras semanas nadaram com sobrecarga, começando com 30 minutos de duração, e chegando ao final da sexta semana a nadar 1h30 minutos, sem intervalo. Dois dias após o último dia de treinamento, os animais foram ortotanasiados por deslocamento cervical e retirou-se o sangue, os linfonodos mesentéricos e os macrófagos peritoneais para análise. A glicemia e a glutaminemia não foram diferentes, p>0,05, entre os grupos. Aumento significativo (p<0,05), na proliferação linfocitária, na fagocitose, na produção do ânion superóxido e no volume lisossomal foram observados no grupo EX/GLN, comparados com o grupo EX. Portanto, nossos resultados sugerem que a suplementação de L-glutamina foi eficaz em reverter o papel imunossupressor do exercício físico de longa duração e alta intensidade / Abstract: This study aimed to investigate the immunitary response of lymphocytes from mesenteric limphonodes, phagocytosis, production of anion superoxide and lysosome volume from peritoneal macrophages, withdrawn from male Wistar rats, 60 - 90 days old, set up as follow: CTL - non exercised; GLN - non exercised and supplemented with L-glutamine; EX - exercised and EX/GLN - exercised and supplemented with L-glutamine. The rats received by gavage L-glutamine (0.125g/kg b.w.) and the controls distilled water. Rats from EX and EX/GLN groups trained during 6 weeks, in single swimming pools with warm water (32°C), bearing load of 6% of body weigth. In the first week they swan with no load for adaptation. Then, they swan with the load starting with 30 minutes long and at the enf of the 6 th week swan for 1hour and 30 minutes, no break. Two days later the animals were killed by cervical dislocation and blood, limphonodes e peritoneal macrophages were withdrawn. Glycemia and glutaminemia were not different (p>0.05) between the groups. In the EX/GLN group there was a significant increase (p<0,05), in the rate of lymphocyte proliferation, phagocytosis, production of anion superoxide and lysosome volume. Thus, our results suggest that supplementation with L- glutamine to these animals was able to reverse the immunosuppression effect of long term and high intensity exercise.

Bibliography : leaves 166-183. / v, 198 leaves ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Chemistry, 1993?

Bibliography: leaves 163-172. / vi, 172 leaves ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Organic Chemistry, 1993

Bibliography : leaves 198-204. / vi, 204 leaves ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Organic Chemistry, 1992

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