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Development of novel mass spectrometry-based approaches for searching for low-mass tyrosinase inhibitors in complex mixtures / Développement de nouvelles approches basées sur la spectrométrie de masse pour le criblage d’inhibiteurs de la tyrosinase en milieu complexeSalwiński, Aleksander 24 April 2014 (has links)
Ce manuscrit de thèse présente le développement de méthodes basées sur la spectrométrie de masse consacrées à la recherche d'inhibiteurs d'enzymes en milieux complexes, tels que les extraits de plantes. L’enzyme Tyrosinase a été utilisé comme principale cible biologique du fait de son implication dans les processus d’hyperpigmentation cutanée. De ce fait, la recherche d’inhibiteurs de cette enzyme, présente un grand intérêt pour l'industrie cosmétique. La première partie de ce manuscrit décrit la mise en place de la chromatographie d'affinité frontale (FAC), permettant d’obtenir le classement simultané des inhibiteurs présent dans un mélange complexe en fonction de leurs affinités avec la cible biologique. Deux capillaires hydrophiles de phase monolithiques ont été évalués afin de réduire au maximum les interactions non spécifiques indésirables entre les analytes et le support solide d’immobilisation. De plus, nous avons étudié la faisabilité de l’utilisation de phases à base de silice comme support solide d’immobilisation des enzymes dans le cadre de ces analyses par chromatographie d'affinité frontale. La seconde partie du manuscrit de thèse est consacrée au développement et à l’optimisation de l’approche nommée ENALDI-MS (Enzyme-coupled Nanoparticles-Assisted Laser Desorption/Ionisation Mass Spectrometry) permettant d’accéder à une gamme des faibles masses (m/z 500 Da). Elle est déclinée en une première approche dite par ‘extinction d’ions’ (Ion Fading, IF-ENALDI), basée sur l’identification directe de la liaison des inhibiteurs vis-à-vis de l’enzyme sans pré-traitement de l’échantillon végétal. Une seconde déclinaison de l’ENALDI-MS concerne une approche dite par ‘Ion Hunting’ (IH - ENALDI MS), basée sur une méthode de pré-concentration sélective des inhibiteurs présents dans l'échantillon. / This thesis report presents the development of mass spectrometry-based methods for searching for inhibitors of enzymes in complex mixtures, such as plant extracts. Tyrosinase enzyme was used as the main biological target for the reason of a significant importance of its inhibitors in the cosmetic industry as the skin whitening agents. The first part of this report describes Frontal Affinity Chromatography (FAC), an approach enabling simultaneous ranking the inhibitors within the complex mixture according to their affinities to the biological target. Two hydrophilic capillary-scale polymer-based bioaffinity stationary phases were evaluated in the context of the presence of undesirable nonspecific interactions between the analyte and the solid immobilisation support. In addition, we explored the usability of two types of silica-based particles as a solid support for enzyme immobilisation for FAC. The second part of the thesis manuscript is devoted to Enzyme-coupled Nanoparticle-Assisted Laser Desorption/Ionisation Mass Spectrometry (ENALDI MS) as a low-mass compatible extension of the Intensity ion Fading MALDI MS (IF-MALDI MS) method for high-throughput screening of the inhibitors in the complex mixtures. Two variations of ENALDI MS were evaluated: 'Ion Fading' (IF-ENALDI MS), based on on-the-spot binding of inhibitors by enzyme molecules and 'Ion Hunting' (IH-ENALDI MS), based on selective pre-concentration of inhibitors present in the sample.
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Analysis Of Protein Purification By Affinity ChromatographySridhar, P 05 1900 (has links) (PDF)
No description available.
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Identificação e validação de um novo alvo funcional de um peptídeo com atividade anti-hipertensiva do veneno da Bothrops jararaca / Identification and validation of a novel functional target of a peptide from Bothrops jararaca venom with antihypertensive activityJuliano Rodrigo Guerreiro 21 May 2009 (has links)
O BPP-10c é um decapeptídeo bioativo, rico em resíduos de prolina e é expresso em uma proteína precursora no cérebro e na glândula de veneno da Bothrops jararaca. Recentemente demonstramos que o BPP-10c tem um potente e sustentado efeito anti-hipertensivo em ratos espontaneamente hipertensos (SHR), sem, no entanto, causar qualquer efeito em ratos normotensos, por um mecanismo farmacológico independente da inibição da enzima conversora de angiotensina (ECA), levando à hipótese de que outro mecanismo poderia estar envolvido na atividade do peptídeo. Neste trabalho, usamos cromatografia de afinidade para isolar e identificar as proteínas renais com afinidade pelo BPP-10c e demonstramos que a argininosuccinato sintase (AsS) é a principal proteína a se ligar ao peptídeo. Além disso, mostramos que essa interação promove um aumento na atividade catalítica da enzima, de forma dose-dependente. A AsS é reconhecida como uma peça chave na regulação do ciclo da citrulina-óxido nítrico (NO), e sua ação é passo limitante na síntese de NO. A interação funcional do BPP-10c com a AsS foi evidenciada pelos seguintes efeitos promovidos pelo peptídeo: i) estimulação da produção de NO por células HUVEC e da produção de arginina por células HEK 293, ii) aumento da concentração plasmática de arginina em SHR. Corroborando esses achados, mostramos a reversão dos efeitos do peptídeo, inclusive sobre a pressão arterial em SHR, quando o MDLA, um inibidor específico da AsS, foi co-administrado. Em conjunto, os resultados apresentados neste trabalho sugerem que a AsS é fundamental para o efeito anti-hipertensivo do BPP-10c. Tais resultados nos levaram a propor a AsS como um novo alvo terapêutico, e o BPP-10c como molécula-líder para a geração de medicamentos para tratamento de doenças relacionadas à hipertensão arterial / BPP-10c is a bioactive proline-rich decapeptide, part of the C-type natriuretic peptide precursor, expressed in the brain and in the venom gland of Bothrops jararaca. We recently showed that BPP-10c displays a strong, sustained anti-hypertensive effect in spontaneous hypertensive rats (SHR), without causing any effect in normotensive rats, by a pharmacological mechanism independent of angiotensin converting enzyme inhibition; therefore, we hypothesized that another mechanism should be involved in the peptide activity. Here we used affinity chromatography to search for kidney cytosolic proteins with affinity for BPP-10c and demonstrate that argininosuccinate synthetase (AsS) is the major protein binding to the peptide. More importantly, this interaction activates the catalytic activity of AsS in a dose-dependent manner. AsS is recognized as an important player of the citrulline-nitric oxide (NO) cycle that represents a potential limiting step in NO synthesis. Accordingly, the functional interaction of BPP-10c and AsS was evidenced by the following effects promoted by the peptide: i) increase of NO production in human umbilical vein endothelial cell culture, and of arginine in human embryonic kidney cells; ii) increase of arginine plasma concentration in SHR. Moreover, MDLA, a specific AsS inhibitor, significantly reduced the anti-hypertensive activity of BPP-10c in SHR. These results led us to suggest AsS as a new therapeutically useful target for the development of activators, such as BPP- 10c, useful to treat hypertension related diseases
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Comparison of Lectins and their suitability in Lectin Affinity Chromatography for isolation of GlycoproteinsAndersson, Pontus January 2020 (has links)
Virtually all extracellular proteins in humans are glycoproteins and likewise are many biopharmaceuticals. The glycosylation is directly correlated to biological function and stability of these proteins. The ability to isolate glycoproteins is thus of great importance in many applications. The most common isolation method for glycoproteins is affinity chromatography using lectins, a ubiquitous and versatile group of carbohydrate-binding proteins. The lectin Concanavalin A (ConA) has long been used for this purpose but suffers from undesired leakage into the eluate, causing an inquiry of alternative chromatography ligands or optimization of the ConA resin.In this study, a total of 20 different lectins, including ConA, were evaluated and compared in terms of suitability as ligands in affinity chromatography for glycoprotein isolation. The lectins’ binding to glycoproteins were studied, mainly through microtiter plate binding assays using a monoclonal IgG1 antibody and Conalbumin (Ovotransferrin). Further, sugar-specificities and potential eluting sugars for the lectins were examined through inhibition with eight different carbohydrates. Additionally, the glycoprotein binding and leakage of ConA columns were examined, and a potential leakagereducing treatment of ConA resin evaluated.ConA was found to be superior in binding to the investigated glycoproteins but exhibited a limited binding when immobilized to an agarose resin. This discrepancy is likely a consequence of structurally hidden glycans on the used glycoproteins and requirements of long residence time when used in a chromatographic setting. Binding competition with several sugars were investigated with a similar microtiter plate binding assay. This method displayed potential to predict the behaviour of sugars and their suitability as eluting agents in a chromatography column. The best eluting sugar for ConA was showed to be methylmannoside, ideally in combination with methylglucoside. Lastly, evaluation of ConA columns with a crosslinking glutaraldehyde-treatment showed that the ConA ligand leakage may be significantly reduced, although further studies and optimizations are needed.This study thus presents a repertoire of lectins and their differences in terms of glycoprotein-binding and sugar-specificity, as well as evaluations of ConA columns’ efficiency and potential leakage-prevention.
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Biochimie analytique de complexes de réparation de l'ADN : élaboration d'un système analytique intégré / Biochemistry of DNA double-strand breaks repair complexes : elaboration of an analytical systemBerthelot, Vivien 12 December 2014 (has links)
Dans les cellules eucaryotes, les cassures double-brin sont réparée selon deux voies principales : la recombinaison homologue et la jonction des extrémités non homologues, toutes deux bien connues dans la littérature. Cependant quelques zones d'ombres persistent quant à deux aspects singuliers de leur mise en œuvre :- Si ces deux mécanismes peuvent opérer dans les cellules, quels sont les déterminismes qui président au choix d'une voie de réparation plutôt que de l'autre ?- Dans le cas où les cassures double-brin sont induites dans l'ADN par des rayonnements ionisants – comme ceux employés en radiothérapie anticancéreuse – coment s'opère la réparation lorsque les extrémités générées ne sont pas compatibles avec une ligation immédiate ? Connaître les protéines impliquées dans ce cas permettrait d'élaborer des adjuvants à la thérapie anticancéreuse.Afin de contribuer à répondre à ces questionnements, nous avons voulu élaborer un système analytique intégré qui permît 1) le recrutement spécifique de complexes de réparation des cassures double-brin de l'ADN sur des phases chromatographiques constituées au laboration, 2) la résolution de ces complexes sur gel d'acrylamide non-dénaturants et leur visualisation et 3) la caractérisation biochimique fine des complexes séparés. La méthodologie élaborée au cours de cette thèse a concerné chacun des trois points ci-dessous : 1) nous avons conçu et mis en œuvre un système chromatographique nous permettant de distinguer les protéines recrutées spécifiquement sur des oligonucléotides duplexes d'ADN dotés d'extrémités libres de l'ADN (mimant des cassures double-brin) des autres protéines se fixant sur la séquence interne des oligonucléotides ; 2) nous avons adapté à notre problématique une méthodologie d'électrophorèse non-dénaturante permettant la résolution des complexes purifiés tout en garantissant leur intégrité au cours de la migration ; 3) grâce à la visualisation directe des complexes résolus dans le gel, nous avons pu déterminer leur composition en protéines par spectrométrie de masse.L'étude biochimique des complexes purifiés a démontré que les complexes purifiés étaient fonctionnels, c'est à dire capable de liguer deux oligonucléotides entre eux. La fouille des données de spectrométrie de masse, obtenues à partir d'un grand nombre d'expériences indépendantes, nous a permis de montrer qu'ils étaient de la physiologie de l'ADN et particulièrement représentatifs de la diversité des mécanismes de réparation.De manière intéressante, nous avons pu observer que certaines protéines recrutées spécifiquement sur les mimes de cassures double-brin de l'ADN, ne sont pourtant pas connues pour intervenir dans les processus de réponse aux dommages de l'ADN (synthèse de nucléotides, checkpoint, topologie de l'ADN, cytosquelette).Le rôle des protéines évoquées ci-dessus sera prochainement caractérisé in cellulo notamment avec des stratégies de type RNAi. D'autre part, nous utiliserons les développements méthodologiques décrits ci-dessus pour étudier les mécanismes de réparation des cassures double-brin radio-induites, tels qu'ils sont mis en jeu dans les cellules tumorales en constituants de nouvelles phases chromatographiques avec des oligonucléotides irradiés. / In eucaryotic cells, DNA double-strand breaks are repaired through two main pathways : the homologous recombination and the non homologous end joining . Altough these pathways are well characterized, two particular aspects of the repair remain poorly understood :- If two separated pathways may occur in the cells, which mechanism(s) govern the choice of the pathway that will ultimately lead to the repair ?- If the double-strand break is induced by ionizing radiations – as those employed in anti-cancerous radiotherapy – how does the repair occur if the DNA ends at the edge of the break are not compatible with a direct ligation ? A proper knowledge of the proteins involved in this repair would allow the development of additives, useful to increase the efficiency of the radiotherapy.To investigate these questions, we designed a new analytical system allowing : 1) the specific recruitment of DNA double-strand break repair complexes on home-made chromatographic phases, 2) the separation of these complexes in a non-denaturing polyacrylamide gel and their subsequent visualization and 3) their biochemical characterization.The methodology developped in this work has been focused on the following points : 1) we designed and implemented a chromatographic system allowing the distinction between proteins recruited onto duplex DNA oligonucleotide with free DNA ends (mimicking DNA double-strand breaks) and proteins fixed onto the internal sequence of the same oligonucleotides ; 2) we adapted to our problematic a methodology of non-denaturing electrophoresis thus allowing the separation of the purified complexes while guaranteeing their integrity during the migration, 3) we also determined their composition by mass spectrometry after their visualization.The biochemical study has shown that the purified complexes were still functionnal, that is they were able to efficiently ligate two oligonucleotides. The study of the data provided by the mass spectrometry analysis of independant experiences proved that the complexes belonged to the DNA physiology and were especially representative of the diversity of the DNA repair pathways.Interestingly, we observed that some of the protein specifically recruited onto the the double-strand breaks were not known to be involved in the DNA repair (nucleotide synthesis, checkpoint, DNA topology, cytoskeleton).The rôle of these proteins should be characterized in cellulo especially with siRNA. On the other hand we will also use the methodological development described above to study the repair mechanisms of radio-induced DNA double-strand breaks occuring in the irradiated tumorous cells. To achieve this study we will elaborate new chromatographic phases with pre-irradiated oligonucleotides.
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Développement et mise en oeuvre de colonnes monolithiques d’affinité boronate pour des techniques séparatives miniaturisées / Boronate affinity monolithic columns for miniaturized separation techniquesEspina Benitez, Maria Betzabeth 08 October 2018 (has links)
Une partie des recherches actuelles dans le domaine de l’analyse chimique concerne la miniaturisation et l’intégration d’étapes analytiques afin de répondre, entre autres, à des besoins de portabilité, d’automatisation mais aussi d’apporter des solutions pour analyser des échantillons de plus en plus petits. Le développement et la mise en œuvre de colonnes monolithiques d’affinité boronate (µBAMC) couplées « in-line » à des techniques séparatives miniaturisées s’inscrit dans cette démarche. Ce travail de thèse s’est focalisé sur (1) une compréhension des mécanismes de rétention en chromatographie d’affinité boronate (interactions spécifiques avec les composés cis-diols, conditions de reconnaissance, interactions secondaires), (2) le développement de supports monolithiques d’affinité boronate miniaturisés et (3) leur couplage «in-line» avec une séparation électrocinétique et détection conventionnelle dans un format capillaire. Différentes voies d’élaboration de colonnes monolithiques ont été comparées (en termes d’affinité, de nombre de sites boronate actifs et de stabilité). La faisabilité du couplage en ligne de ces supports µBAMC avec une étape de séparation électrophorétique (par CZE et CIEF) a été démontrée vis-à-vis de la purification/préconcentration et séparation de 3 catécholamines contenant des groupements cis-diols (Adrénaline, Noradrénaline et Dopamine) dans l’urine. Les couplages ont été optimisés avec succès permettant l’analyse automatisée et miniaturisée de ces neurotransmetteurs dans l’urine (volume échantillon < 10 µL) avec des limites de détection de l’ordre de la dizaine de ppb et des taux de récupération proches de 100 % / Part of the current research in the field of chemical analysis concerns the miniaturization and the integration of analytical steps in order to meet, among other things, the need of portability and automation but also to provide solutions for analyzing small samples. The development and implementation of monolithic boronate affinity columns (µBAMC) in-line coupled to miniaturized separation techniques is part of this approach. This thesis work focused on (1) an understanding of the retention mechanisms in boronate affinity chromatography (specific interactions with cis-diol compounds, recognition conditions and secondary interactions), (2) the development of miniaturized boronate affinity monolithic supports and (3) their in-line coupling with electrokinetic separation and conventional detection in a capillary format. Different ways of elaboration of monolithic columns were compared (in terms of affinity, number of actives sites and stability). The feasibility of in-line coupling of these µBAMC supports with an electrophoretic separation step (by CZE and CIEF) has been demonstrated in terms of purification / preconcentration and separation of 3 catecholamines containing cis-diol groups (adrenaline, noradrenaline and dopamine) in urine. The couplings have been successfully optimized allowing the automated and miniaturized analysis of these neurotransmitters in urine (sample volume <10 µl) with limits of detection of about the tens of ppb and recovery yields close to 100 %
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Synthesis of a biotin-functionalized biguanide for the identification of the tumor growth inhibition mechanism of metforminMohebali, Farzaneh 08 1900 (has links)
No description available.
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Untersuchungen zur molekularen Ursache der Multiplen Sulfatase-Defizienz: Reinigung, Funktions- und Strukturanalyse von varianten Proteinen des Formylglycin-generierenden Enzyms / The molecular cause of multiple sulfatase deficiency: cleaning, functional and structural analysis of variant proteins of formylglycine-generating enzymeMühlhausen, Helene 14 January 2015 (has links)
No description available.
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Analýza glykoproteinů ze slinných žláz klíštěte \kur{Ixodes ricinus} / Analyses of glycoproteins from the salivary glands of the tick \kur{Ixodes ricinus}BUČINSKÁ, Lenka January 2010 (has links)
I characterized several potential glycoproteins in salivary gland extracts from unfed and partially fed females of ticks Ixodes ricinus using enzyme deglycosylation and lectin labeling. Affinity-based (chromatografic) analysis was applied for isolations of glycoproteins with specificity for GNA (mannose), HPA (N-acetylgalactosamine) and MAA II (sialic acid) lectins. GNA specific 120 kDa glycoprotein was isolated from partially fed females and is modified with N-linked glycans containing {$\alpha$}1,3-mannose. Mass spectrometry analyses confirmed the presence carboxypeptidase M in elution fraction gain with GNA affinity chromatography. GNA specific proteins were purified from unfed female salivary gland extracts. MS analyses identified them as proteins similar to arylsulfatase B and cytoskeletal Sojo protein. Proteins (85 and 56 kDa) isolated with HPA affinity chromatography were characterized as Trappin 12, which is a host protein. MAA II lectin was used for labelling and isolation of 100 kDa protein. N-terminal sequence of the MAA II specific protein predicted similarity with a host protein, Siglec 1. Fucose in salivary gland extract was detected with the labelling of AAA, AAL, UEA I and LTL lectins. Results showed that salivary gland extracts contain {$\alpha$}1,2-; {$\alpha$}1,3- and {$\alpha$}1,6- N-linked fucose and O-linked fucose probably as well. GNA specific proteins were detected in partially fed salivary glands acini type II and III using electron transmission microscopy. Fucose was detected on gut and salivary gland structures using fucose-specific lectin AAL.
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Pharmacochimie antiprotozoaire en séries quinazoline et quinoléine : synthèse, évaluation biologique et recherchedu mécanisme d'action. / ANTIPROZOAL PHARMACOCHEMISTRY IN QUINAZOLINE AND QUINOLINE SERIES : SYNTHESIS, BIOLOGICAL EVALUATION AND RESEARCH FOR THE MECHANISM OF ACTIONKieffer, Charline 11 December 2014 (has links)
Le paludisme et la leishmaniose sont les deux plus importantes infections parasitaires au monde, en termes de mortalité. La recherche de nouvelles molécules actives contre les protozoaires responsables de ces « maladies tropicales négligées », Plasmodium sp et Leishmania sp, est un enjeu majeur de santé publique. Après une première partie dressant un état des lieux des connaissances disponibles en matière de chimiothérapie antiplasmodiale, une seconde partie s’est intéressée à l’étude des propriétés anti-infectieuses du noyau 2-trichlorométhyl-quinazoline, en introduisant en position 4 des motifs alcynyles par couplage de Sonogashira, optimisés par LC/MS. Une troisième partie a porté sur la pharmacomodulation en positions 2 et 4 du même noyau, notamment par réactions de SNAr. Une quatrième partie a consisté à rechercher le mécanisme d'action des meilleures quinazolines antiplasmodiales, via une approche de chromatographie d'affinité sur inhibiteur immobilisé. La fonctionnalisation multi-étapes des molécules les plus puissantes, par un bras espaceur, a été suivie de leur ancrage sur divers supports solides, pour constituer des matrices biocompatibles spécifiques. L’une d’entre-elles a permis la mise en évidence de 2 cibles plasmodiales protéiques originales : la GTPase Pfrab6 et la pyruvate-kinase PfpyrK1. Enfin, une cinquième partie relate la pharmacomodulation antileishmanienne du noyau 8-nitroquinoléin-2(1H)-one. Les travaux se sont intéressés à l'étude de la substitution de la position 4 de ce noyau par des réactions de SNAr, ainsi que des réactions de couplages pallado-catalysés optimisées à l’aide de la technologie micro-ondes. / Malaria and leishmaniasis are the two most important parasitic infections worldwide, in terms of mortality. Thus, the research for new molecules targeting the protozoa parasites responsible for these “neglected tropical diseases”, Plasmodium sp and Leishmania sp, constitute a major challenge in public health. Our work focused first on a current state of knowledge about antiplasmodial chemotherapy. In a view to develop the study of the anti-infective properties of the 2-trichloromethylquinazoline scaffold, a second part presented antiplasmodial pharmacomodulation at position 4 using Sonogashira cross-coupling reaction, optimized with the LC/MS technology. A third part concerned other pharmacomodulation reactions, especially at positions 2 and 4, using especially SNAR reactions. A fourth part consisted in the research of the mechanism of action of the best antiplasmodial quinazolines by using the affinity chromatography on immobilized inhibitor approach. The multi-step functionalization of the most potent derivatives by a spacer side chain was followed by their anchoring onto various solid supports, so as to generate different biocompatible specific matrices. One of them, put in contact with a parasitic lysate, allowed the identification of two original plasmodial targets: the GTPase Pfrab6 and the pyruvate-kinase PfpyrK1. Finally, a fifth part presented the antileishmanial pharmacomodulation of the 8-nitroquinolin-2(1H)-one scaffold, especially at position 4 of the quinoline ring, involving SNAr reactions (with amines, phenols or thiophenols) or pallado-catalyzed coupling reactions (in particular Suzuki-Miyaura), some of them being optimized under microwave irradiation.
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