Mass spectrometry (MS) has played a major role in the proteomic analysis of an array of biological samples. Even so, inherent limitations exist such as sample complexity and the dynamic range. In an attempt to overcome these limitations, prefractionation is typically performed followed by reversed phase liquid chromatography coupled with MS. Pre-fractionation can be performed in several formats including chromatographic or electrophoretic based methods. Solution-based isoelectric point (pI) fractionation, specifically isoelectric trapping (IET), provides an attractive alternative for pre-fractionation in bottom-up proteomic studies. A recently developed device, membrane separated wells for isoelectric focusing and trapping (MSWIFT), provides rapid separation on the basis of pI and resulting solutions are MS compatible without the need for extensive sample cleanup. Initial experiments demonstrate fractionation using MSWIFT, of peptide mixtures ranging from standards to a yeast lysate where resulting fractions are analyzed using matrixassisted laser desorption/ionization (MALDI) – MS or further separated using reversed phase liquid chromatography followed by tandem MS (MS/MS) analysis. Identified yeast proteins range in size, pI and copy number illustrating an ability to increase the depth of proteome coverage when using MSWIFT. Extensive studies were also performed using MSWIFT in a multi-stage fractionation platform to improve peptide and protein identifications for the first large-scale proteomic study of the model fungus, Neurospora crassa. A second focus of this work is the development of a new sample preparation method for proteolytic digestion and high-throughput separations using MSWIFT. Histidine is used as a neutral pH, isoelectric, sample buffer for tryptic digestion of proteins and also assists in rapid separations using MSWIFT owing to the low conductivity. Tryptic digests of individual standard proteins and a mixture of standard proteins are used to illustrate these advantages. Finally, the histidine buffer sample preparation method is incorporated into a two-dimensional separation strategy. Tryptic peptides are fractionated using MSWIFT and resulting solutions are further separated using capillary electrophoresis (CE) coupled with MALDI-MS/MS. Performing the two-dimensional strategy allows for increased confidence in peptide and protein assignment owing to experimentally determined in-solution charge states and estimated pI values.

There is described a electromigration separation method for the analysis of low concentrations of copper with the possibility to detect a Wilson disease in my thesis. There were developed a methodology and propitious electrolyte systems, which can pre-concentrate copper prior to its on-line analysis. The developed electrolyte system for analysis had the following composition: LE1 (1.10-2 M CH3COOH, 2.10-2 M CH3COONH4, 2.10-4 M SPADNS), LE2 (1.10-2 M CH3COONH4, 1.10-2 M NH4OH, 1.10-4 M BKP, 5.10-5 M PAR, 5.10-3 M C6H17N3O7) and DE (7.10-5 M Cu(CH3COO)2, 1.10-3 M CH3COONH4 a 1.10-2 M CH3COOH). With a suggested electrolyte was achieved almost seventyfold accumulation of copper during 42 minutes, which lowered the concentration detection limit on the level convenient for the detection of Wilson disease. Complete analysis took about 98 minutes. There was accomplished a conductometric detection of copper in my thesis, however the developed electrolyte system can also analyze a copper by photometric detection, which is more sensitive.

L’objectif de cette thèse porte sur l’étude de la compartimentalisation cellulaire et de la prise en charge de l’uranium (U) par les protéines cytosoliques des cellules branchiales du poisson zèbre (Danio rerio, espèce modèle en toxicologie aquatique) après exposition contrastées (chronique vs. aiguë, 20 et 250 µg.L-1) par voie directe. Cette étude a nécessité le développement, l’utilisation et le couplage d’outils analytiques de pointe (SEC, IEF hors-gel, RP-UHPLC pour la séparation, ICP-SFMS, ESI-FTMS/MS pour la détection) avec comme défis majeurs la conservation des interactions non-covalentes U biomolécule et une sensibilité maximale pour travailler à des niveaux d’exposition proches de ceux rencontrés dans l’environnement. Après extraction, 24 à 32% de la charge branchiale totale en U est contenue dans le cytosol dans lequel la distribution de l’U sur les biomolécules (en fonction de leur PM mais aussi de leur pI) diffère selon le niveau d’exposition. Enfin, une cartographie des biomolécules cibles de l’U a permis (i) de mettre en évidence une affinité particulière de l’U pour les protéines à caractère acide et/ou contenant du phosphore et (ii) d’identifier 24 protéines candidates pour lier U. / The objective of this thesis is to study the cellular compartmentalization and the chelation of uranium (U) by cytosolic proteins of gill cells of the zebrafish (Danio rerio, model species in aquatic toxicology) under different direct exposure conditions (chronic vs. acute, 20 and 250 µg.L 1). This study required the development of hyphenated techniques (SEC, IEF off-gel, RP-UHPLC for the separation, ICP-SFMS, ESI-FTMS/MS for the detection) with the main challenges of maintaining the non-covalent U-biomolecule interactions and enhancing sensitivity for the analysis of environmentally relevant samples. After extraction, 24% to 32% of the total U detected in the gills were present in the cytosolic fraction, in which the U distribution on the biomolecules (as a function of their MW and pI) varied depending on the exposure level. Finally, U target biomolecules mapping allowed us (i) to highlight a particular affinity of U for acidic and/or P-containing proteins and (ii) to identify 24 protein candidates for U binding.

Die phosphorylierungsabhängige Bindung von Arrestin an G-Protein-gekoppelte Rezeptoren (GPCRs) ist ein weit verbreiteter Mechanismus, um aktive Rezeptoren zu inhibieren. Für die Bindung von Arrestin an lichtaktiviertes, phosphoryliertes Rhodopsin (pRho), einem GPCR, der sich in der Diskmembran der Stäbchenzellen der Netzhaut befindet, ist in Lipidmembranen die Phosphorylierung von 2 der insgesamt 7 Phosphorylierungsstellen im Rezeptor-C-Terminus nötig, wohingegen 3 Phosphate das Arrestin quantitativ aktivieren. Welchen Einfluss höhere Phosphorylierungsniveaus auf die Rezeptor-Arrestin-Interaktion haben, ist bisher ungeklärt. In dieser Arbeit wurde daher eine Methode zur quantitativen Bestimmung der Rhodopsin-Phosphorylierung etabliert, die unterschiedlich pRho-Spezies präparativ getrennt und mit den isolierten pRho-Spezies verschiedene Parameter der Rezeptor-Arrestin-Interaktion, wie z.B. die Bindungsstöchiometrie der resultierenden pRho-Arrestin-Komplexe, untersucht. Für die Charakterisierung der Arrestinbindung wurden Titrationen mit 3 verschiedenen fluoreszenzmarkierten Arrestinmutanten und pRho in gemischten Phospholidipd/Detergens-Mizellen durchgeführt. Die in dieser Arbeit gewonnenen Daten zeigen, dass der Phosphorylierungsgrad von Rhodopsin neben der Affinität von Arrestin auch die Bindungsstöchiometrie beeinflusst. So haben die Komplexe bei geringem Phosphorylierungsgrad eine 2 : 1 (pRho : Arrestin) Stöchiometrie, während bei einem sehr hohen Phosphorylierungsgrad bevorzugt 1 : 1 pRho-Arrestin-Komplexe gebildet werden. Zudem weisen ein unterschiedliches Elutionsverhalten bei der säulenchromatographischen Trennung und Abweichungen in der pRho-Arrestin-Stöchiometrie verschiedener pRho-Präparationen mit ähnlicher Gesamtanzahl an Phosphaten auf einen zusätzlichen Einfluss unterschiedlicher Phosphorylierungsmuster hin. Insgesamt deuten die Daten auf eine komplexe Beziehung zwischen dem Phosphorylierungsgrad der Rezeptoren und dem Arrestin-Bindungsmodus hin. / The phosphorylation-dependent binding of arrestin to G protein-coupled receptors (GPCRs) is a widely used mechanism to inhibit active receptors. In lipid membranes, the binding of arrestin to light-activated, phosphorylated rhodopsin (pRho), a GPCR located in the disc membranes of retinal rod cells, requires the phosphorylation of 2 of the 7 phosphorylation sites in the receptor C-terminus, whereas 3 phosphates are required to completely activate arrestin. The influence of higher phosphorylation levels on the receptor/arrestin interaction is still unclear. In this thesis, a method for the quantitative determination of rhodopsin phosphorylation was established, rhodopsin species with varying degrees of phosphorylation were separated preparatively and different parameters of the receptor/arrestin interaction, such as the binding stoichiometry of the resulting pRho/arrestin complexes, were investigated for each isolated pRho species. For the characterization of arrestin binding, titrations with 3 different fluorescently labeled arrestin mutants and pRho in mixed phospholipid/detergent micelles were performed. The data obtained in this work show that the phosphorylation level of rhodopsin influences the binding stoichiometry in addition to the affinity of arrestin binding to rhodopsin. Thus, complexes with a low level of phosphorylation have a 2 : 1 (pRho : arrestin) stoichiometry, whereas 1 : 1 pRho/arrestin complexes are preferably formed at a very high degree of phosphorylation. In addition, a different elution behaviour during chromatographic separation and variances in the pRho/arrestin stoichiometry of different pRho preparations with similar overall number of phosphates indicate an additional influence of distinct phosphorylation patterns. Overall, the data indicate a complex relationship between receptor phosphorylation level and arrestin binding mode.

PsbO (manganese-stabilizing protein) is the largest extrinsic protein of photosystem II, located on the lumen side of photosystem. It is present in all known oxyphototrophic organisms. PsbO facilitates photosynthetic water splitting, which takes place in an oxygen evolving center (Mn4CaO5 cluster) of photosystem II. This work is focused on PsbO of higher plants and its isoforms, particularly their evolution and functions. Bioinformatic analyses revealed that majority of higher plants express exactly two psbO isoforms. A phylogenetic tree of PsbO sequences has an unusual topology. The two paralogous isoforms do not diverge at the base of the phylogenetic tree, as anticipated, but rather at the end of particular branches, at the level of family or lower taxonomic unit. In this work we propose and discuss several hypotheses concerning evolution of PsbO isoforms. The work further includes detailed analysis and identification of protein spots assigned to PsbO on 2D IEF-SDS PAGE gels of potato thylakoid proteins. We identified predominant version of PsbO isoform in most of the spots. We did not succeed to find any posttranslational modification. We optimized a method of psbO expression in E. coli and subsequent purification, which yielded relatively big amount of properly folded recombinant protein. Analysis of...

Alors que le protéome de l'ovocyte isolé, aux stades VG et MII a déjà été étudié, celui du follicule en croissance n'a jamais été décrit.Dans cette étude, nous avons cherché à identifier, comparer et caractériser les profils protéiques de follicules ovariens de souris à trois stades de leur développement in vitro distincts morphologiquement : follicules secondaires en début de culture - stade initial (IS), follicules avec une rupture complète de la membrane de Slavjanski (RMS) et follicules avec une cavité similaire à l'antrum (FA).Après un préfractionnement par IEF et une analyse LC-MS/MS en deux configurations (1D et 2D), 1403 protéines ont pu être identifiées dans le follicule ovarien de souris. 43,4 % (609) des protéines identifiées étaient communes aux trois stades et d'autres ont été identifiées uniquement à un stade : 71 au stade IS, 182 au stade RMS et 193 au stade FA. De plus, on a identifié 365 protéines qui n'avaient pas été décrites antérieurement dans le protéome de l'ovocyte ce qui pourrais indiquer qu'elles sont exprimées dans les cellules somatiques du follicule. Des analyses qualitatives et quantitatives complémentaires ont démontré une surreprésentation pour 44 fonctions biologiques par rapport aux fonctions biologiques des gènes constituant le génome de Mus musculus et mis en évidence des différences d'expression et d'abondance des protéines liées au cycle cellulaire, à la fixation des ions de calcium et à la glycolyse selon le stade de développement. Ces résultats représentent un point de départ pour beaucoup d'autres études de caractérisation moléculaire du développement folliculaire. / Until now only the proteome of isolated oocyte from fully grown follicle were described with the aim of oocyte maturation characterization. However the ability of oocyte to mature is acquired during its development within the follicle. Thus in this study we proposed a protein identification and characterization of whole mice ovarian follicle at three morphological stages during in vitro development: early secondary stage, described as initial stage (IS), follicles with a complete Slavjanski membrane rupture (RMS) and follicles with an antrum like cavity formation (FA). Using an IEF pre fractionation before protein digestion and two configurations of LC-MS/MS analysis (1D and 2D), 1403 proteins were successfully identified in the murine ovarian follicle. From those, 43.4 % (609) were commonly identified in the three stages and some were identified only at one single stage: 71 at IS stage, 182 at RMS stage and 193 at FA stage. Compared to the proteomes of isolated oocyte previously described, 365 proteins were only identified in our samples indicating that those ones were probably expressed in the somatic cells of the follicle. Additional qualitative and quantitative analysis highlighted 44 biological processes over represented in our samples when compared to Mus musculus gene database and different expressions and protein abundance implicated in cell cycle, calcium ion binding and glycolysis, throughout in vitro follicle development. This report represents so far the most complete catalogue of follicle proteins and could be an important milestone in the proteomic study of the follicle metabolism throughout in vitro development.

Alors que le protéome de l'ovocyte isolé, aux stades VG et MII a déjà été étudié, celui du follicule en croissance n'a jamais été décrit.Dans cette étude, nous avons cherché à identifier, comparer et caractériser les profils protéiques de follicules ovariens de souris à trois stades de leur développement in vitro distincts morphologiquement : follicules secondaires en début de culture - stade initial (IS), follicules avec une rupture complète de la membrane de Slavjanski (RMS) et follicules avec une cavité similaire à l'antrum (FA).Après un préfractionnement par IEF et une analyse LC-MS/MS en deux configurations (1D et 2D), 1403 protéines ont pu être identifiées dans le follicule ovarien de souris. 43,4 % (609) des protéines identifiées étaient communes aux trois stades et d'autres ont été identifiées uniquement à un stade : 71 au stade IS, 182 au stade RMS et 193 au stade FA. De plus, on a identifié 365 protéines qui n'avaient pas été décrites antérieurement dans le protéome de l'ovocyte ce qui pourrais indiquer qu'elles sont exprimées dans les cellules somatiques du follicule. Des analyses qualitatives et quantitatives complémentaires ont démontré une surreprésentation pour 44 fonctions biologiques par rapport aux fonctions biologiques des gènes constituant le génome de Mus musculus et mis en évidence des différences d'expression et d'abondance des protéines liées au cycle cellulaire, à la fixation des ions de calcium et à la glycolyse selon le stade de développement. Ces résultats représentent un point de départ pour beaucoup d'autres études de caractérisation moléculaire du développement folliculaire.

Antibodies are Y-shaped proteins. In the human body, antibodies areproduced by plasma cells, mainly T and B cells which are included inthe adaptive immune system. The production of antibodies is stimulatedby antigens. The binding between an antigen-specific antibody and itsantigen can be like the interconnect between a lock and a key.Therefore, antibodies are widely used as diagnostic tools for avariety of diseases but most importantly cancer. Some rapid diagnostictests are completely dependent on the specificity and reactivity ofantibodies such as UBC® Rapid produced by IDL Biotech AB. Therefore,the quality of these antibodies is important. This master thesis at IDL Biotech aimed to validate six anticytokeratinantibodies that are currently used in several rapid cancerdiagnostic tests produced by IDL. Antibody validation is a processwhere specificity, selectivity and reproductivity of an antibody isdemonstrated through specific laboratory investigations. During thisthesis, two laboratory methods were used to validate antibodies,namely, isoelectric focusing electrophoresis and the Attana QuartzCrystal Microbalance based biosensor. Isoelectric focusing electrophoresis (IEF) is a method that determinesproteins pI-values which can then reveal information about posttranslationalmodifications and protein sustainability during storage.IEF revealed changes in pI-values in two antibodies: AB2 and AB4. Attana biosensor analysis on AB1-5 showed that all antibodies havehigh specificity, reactivity and relatively high affinity to theircytokeratin targets. It also revealed that 4 antibodies (AB1 and AB3-5) have lower cross-reactivity with other cytokeratins than theirtarget cytokeratins compared to AB2. Keywords: Antibody validation, Isoelectric focusing, QCM, Attanabiosensor, biosensors, rapid diagnostics, epithelial carcinomas.

No presente estudo, foram selecionados 33 pacientes infectados com HCV genótipo 3a, tratados com IFN e Ribavirina, incluindo pacientes cirróticos (C) e não-cirróticos (NC), respondedores (R) e não-respondedores (NR). Foi realizado o seqüenciamento das regiões E2 e NS5A do genoma do HCV. As seqüências geradas foram analisadas quanto a presença de mutações para correlacionarmos com a resposta virológica sustentada ao tratamento e com a presença de cirrose. Na análise estatística as mutações na região E2 não apresentaram diferença significante. As mutações conservadoras encontradas nas regiões NLS e V3 da NS5A apresentaram diferença significante. Estudos funcionais envolvendo a proteína NS5A são necessários para que possamos avaliar o valor preditivo das mutações conservadoras e não-conservadoras encontradas na NS5A / The aim of this study was to analyse the sequences of fragments of E2 and NS5A regions from 33 outpatients infected with HCV genotype 3, including cirrhotic (C) and non-cirrhotic (NC) patients that have responded (R) or not (NR) to treatment. In the E2 region, we did observe few amino acids changes between patients without statistical significance. In the NLS region and in V3 domain, we found conservatives mutations with statistical significance. In conclusion, our results confirm that the ISDR domain is not predictive for treatment success in patients infected with HCV genotype 3. The carboxy-terminal region and especifically V3 domain region showed most of variations. Structure-function studies are required to identify precisely how NS5A and IFN interact

Made available in DSpace on 2017-07-20T12:40:20Z (GMT). No. of bitstreams: 1 Mariana_N_Levandowski.pdf: 3613595 bytes, checksum: 6d13f04a1872ade8572c9eae481770c6 (MD5) Previous issue date: 2017-02-13 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / This study presents a theoretical and experimental analysis of the conformational equilibrium of phenylacetic acid and its derivatives, namely: o-fluorophenylacetic, ochlorophenylacetic, o-bromophenylacetic, o-nitrophenylacetic, hydroxyphenylacetic, m-fluorophenylacetic, p-fluorophenylacetic, p-nitrophenylacetic, p-hydroxyphenylacetic, p-mercaptophenylacetic and p-aminophenylacetic. Thetechniques used were computational calculations and nuclear magnetic resonance and infrared spectroscopy.The study made it possible to verify the conformational preference of each compound, determined by their energies and geometries, which were evaluated both for the isolated molecule, at distinct theory levels, and in different solvents, with the IEF-PCM solvation model. By using natural bond orbital calculations, it was possible to identify the main orbital interactions for each conformation. With the total deletion calculations, it was verified that there was a balance between the steric and hyperconjugative effects in the stabilization of the most stable structures. The majority conformers of halogenated compounds in ortho revealed the same spatial orientation between the carbonyl and the benzene ring, similar to that of the phenylacetic acid. In the derivatives, nitro and hydroxylated in ortho, there was the presence of an intramolecular hydrogen bond, but in none of these cases the interaction referred to one of the main conformers. The infrared experiments in solvents of different polarities, allowed the identification of more than one band, enabling the attribution of conformers present in the equilibrium as well as the observation of the same tendency as the one obtained theoretically. The coupling constant 1JCH was evaluated through nuclear magnetic resonance. No significant variation of this constant was observed, showing that the major contribution to the equilibrium is related to the majoritarian conformer, which prevails even with the change in the polarity of the medium, for each compound under evaluation. / Neste trabalho foi apresentado uma análise teórica e experimental do equilíbrio conformacional do ácido fenilacético e seus derivados, sendo eles: ofluorofenilacético, o-clorofenilacético, o-bromofenilacético, o-nitrofenilacético, ohidróxifenilacético, m-fluorofenilacético, p-fluorofenilacético, p-nitrofenilacético, phidróxifenilacético,p-mercaptofenilacético e p-aminofenilacético. As técnicas utilizadas foram os cálculos computacionais e as espectroscopias de ressonância magnética nuclear e infravermelho. Com este estudo foi possível verificar a preferência conformacional de cada composto, determinadas pelas suas energias e geometrias, as quais foram avaliadas tanto para a molécula isolada, em distintos níveis de teoria, quanto em diferentes solventes, com o modelo de solvatação IEFPCM. Através dos cálculos dos orbitais naturais de ligação foi possível identificar as principais interações orbitais para cada conformação. Com os cálculos de deleção total, verificou-se que houve um balanço entre os efeitos estérico e hiperconjugativo na estabilização das estruturas mais estáveis. Os confôrmeros majoritários dos compostos halogenados em orto mostraram a mesma orientação espacial entre a carbonila e o anel benzênico, semelhante ao ácido fenilacético. Nos derivados,nitrado e hidroxilado em orto, houve a presença de uma ligação de hidrogênio intramolecular, em nenhum dos casos esta interação era referente a um dos confôrmeros principais. Utilizando a técnica de infravermelho na região do fundamental da carbonila, em solventes de diferentes polaridades, permitiram identificar a presença de mais de uma banda, sendo possível a atribuição dos confôrmeros presentes no equilíbrio, bem como observar a mesma tendência obtida teoricamente. Com a espectroscopia de ressonância magnética nuclear foi avaliada a constante de acoplamento 1JCH. Não houve uma variação significativa desta constante, mostrando que a maior contribuição no equilíbrio está relacionada com o confôrmero majoritário, que prevalece mesmo com a mudança da polaridade do meio, de cada composto avaliado.