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Characterization of Pathogens for Potential Diagnostic TestsGallegos, Karen M. 23 September 2013 (has links)
No description available.
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Validation de la neuraminidase à titre de marqueur enzymatique pour la détection de virus dans l’airToulouse, Marie-Josée 10 July 2019 (has links)
Les méthodes connues de détection des virus dans l’air sont complexes et ne permettent pas de détecter rapidement plusieurs types de virus à la fois. De plus, peu d’informations sont disponibles concernant l’efficacité des échantillonneurs à récolter et à préserver l’infectivité des virus. Ce projet avait pour objectif d’étudier la détection des virus dans l’air basée sur la présence d’un marqueur enzymatique, soit la neuraminidase. Une enzyme purifiée et des souches de virus influenza ont été utilisés comme modèles. L’efficacité de trois échantillonneurs à récolter les virus dans l’air a été comparée et les effets de l’aérosolisation et de l’échantillonnage dans deux chambres d’aérosolisation sur l’acitivité de la neuraminidase et sur l’infectivité des virus ont été étudiés. Les résultats obtenus démontent la stabilité de la neuraminidase et valident son utilisation comme marqueur enzymatique pour la détection générique, simple, rapide et abordable des virus dans des échantillons d’air. / The known methods for the detection of viruses in the air are complex and do not allow to quickly detect several types of viruses at the same time. In addition, little information is available regarding the effectiveness of samplers to collect and preserve the infectivity of the viruses. This project aimed to investigate the detection of viruses in the air based on the presence of an enzymatic label, the neuraminidase. A purified enzyme and strains of influenza viruses were used as models. The effectiveness of three samplers to collect the viruses in the air and was compared and the effects of aerosolization and sampling in two aerosolization chambers on neuraminidase activity and viruses infectivity were studied. The results obtained demonstrate the stability of the neuraminidase and validate its use as an enzymatic marker for the generic, simple, fast and affordable detection of viruses in air samples.
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Étude de la résistance des virus influenza B contemporains aux inhibiteurs de la neuraminidase et son impact sur le fitness viralFage, Clément 12 September 2019 (has links)
Les virus influenza ont toujours eu un impact considérable sur l’humanité. Les virus influenza B (IB) ont longtemps été négligés et sous-estimés par rapport aux virus influenza A (IA). Ils ne représentent que 10 à 20% des infections annuelles par les virus influenza, mais peuvent devenir majoritaire certaines années et entrainer des symptômes similaires à ceux des virus IA. Les inhibiteurs de la neuraminidase (INA) sont les principaux traitements disponibles contre les virus influenza. Cependant, à la suite de mutations, certains virus influenza ont développé des mécanismes de résistance limitant dangereusement les options thérapeutiques. Actuellement, très peu de souches résistantes sont isolées en clinique car le fitness de ces virus résistants, c’est-à-dire leur capacité à se répliquer et se transmettre, est altéré. Or, il a été montré que des virus IA peuvent améliorer leur fitness et propager le phénotype de résistance. L’objectif de ce projet de thèse est de mieux comprendre la résistance des virus influenza B et son impact sur le fitness viral afin de mieux prévenir l’émergence de souches résistantes. Nous avons, dans un premier temps, étudié la résistance des virus influenza A et B au peramivir, le plus récent des INA. Nous avons confirmé que cette molécule est hautement active contre les souches saisonnières A et B. De plus, elle conserve une activité antivirale in vitro contre certaines souches virales ayant une sensibilité réduite ou hautement réduite contre d’autres INA (l’oseltamivir et le zanamivir). Dans un second temps, nous avons étudié le fitness de virus influenza B contemporains résistants in vitro et chez la souris. Nous avons montré que certains virus influenza B, ayant un phénotype de résistance croisée, peuvent maintenir un fitness similaire à celui du virus sauvage in vitro et in vivo. De plus, nous avons pu décrire et analyser un nouveau mécanisme de résistance chez les virus influenza B observé chez un cas clinique. Ces résultats soulignent la dangerosité de notre dépendance aux INA et nous encouragent à faire évoluer les stratégies thérapeutiques.
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Diversité des mécanismes de résistance aux inhibiteurs de la neuraminidase des virus influenza A : implications de résidus conservés dans le site actif de la neuraminidase et de la balance fonctionnelle entre la neuraminidase et l’hémagglutinine / Diversity of resistance mechanisms to influenza A neuraminidase inhibitors : implication of conserved residues in the neuraminidase active site and of the functional balance between the neuraminidase and the hemagglutininRichard, Mathilde 15 December 2010 (has links)
Chaque année, les épidémies de grippe, dont les principaux agents étiologiques sont les virus influenza de type A, ont un impact considérable sur la population en terme de morbidité et de mortalité. Le virus influenza A comporte à sa surface deux glycoprotéines, la neuraminidase et l’hémagglutinine. Ces deux protéines ont des fonctions antagonistes : l’hémagglutinine permet l’entrée du virus dans la cellule hôte et la neuraminidase, par son activité sialidase, libère les nouveaux virions formés. Bien que la prophylaxie du virus grippal repose essentiellement sur la vaccination, les antiviraux jouent un rôle important dans la lutte contre les épidémies de grippe et dans la stratégie développée en prévision d'une pandémie grippale. Les inhibiteurs de la neuraminidase (INAs) sont des antiviraux efficaces contre la grippe. Ils inhibent l’activité enzymatique de la neuraminidase et empêchent la libération des nouveaux virions formés. La démarche méthodologique qui a conduit à l’élaboration de molécules ciblant la neuraminidase laissait espérer une apparition limitée de résistance. Cependant, des cas de résistances aux INAs ont été mis en évidence lors d’études cliniques. Outre la nécessité d’une surveillance étroite, il est donc important d’étudier et de comprendre les diverses mécanismes susceptibles d’induire une résistance aux INAs. Le travail de cette thèse s’est ainsi porté sur la compréhension de la diversité des mécanismes de résistance. Dans un premier temps, nous avons étudié l’impact de mutations sur l’ensemble des résidus structuraux du site actif de la neuraminidase. Nous avons observé que la plupart de ces mutations n’altéraient pas les caractéristiques du virus et induisaient une légère baisse de sensibilité aux INAs. Par la suite, nous avons cherché à explorer les possibilités de synergie dans la résistance aux INAs par la combinaison de deux mutations structurales du site actif de la neuraminidase. Sur quatre virus produits, seul le virus possédant la double mutation E119V+I222L était viable, malgré une capacité réplicative in vitro altérée. La combinaison de ces deux mutations induit une synergie dans la résistance à l’oseltamivir. Enfin, nous avons voulu intégrer l’interaction fonctionnelle de la neuraminidase avec l’hémagglutinine. Nous avons montré que la combinaison d’une hémagglutinine de faible affinité pour les récepteurs sialylés permettait de restaurer un virus possédant une neuraminidase déficiente. Ainsi, un virus influenza peut se libérer de la fonction de la neuraminidase, cible des seuls antiviraux efficaces disponibles à l’heure actuelle. Les mécanismes de résistances aux inhibiteurs de la neuraminidase sont multiples. L’émergence durant les deux dernières saisons hivernales de virus résistants aux INAs sans pression de sélection a remis en question les hypothèses développées sur l’infectivité et la transmissibilité de souches résistantes, ouvrant de nouvelles perspectives quant à l’étude des mécanismes permettant l’obtention de virus épidémiogènes résistants aux INAs / Each winter, influenza epidemics have a considerable impact on the population in terms of morbidity and mortality. Influenza A virus is the main etiologic agents of influenza. They present at their surface two glycoproteins, the neuraminidase and the hemagglutinin. These two proteins have antagonist functions : the hemagglutinin allows the virus to enter the host cell and the neuraminidase, through its sialidase activity, releases progeny virions from host cells. Although prophylaxis of influenza is mainly based on vaccination, antiviral drugs play a very important role in the fight against epidemics of influenza and the strategy developed in anticipation of a flu pandemic. The neuraminidase inhibitors are effective antiviral against influenza. Through the inhibition of the neuraminidase enzymatic activity, they prevent the release of new virions formed. The introduction into clinical practice of new drugs requires monitoring in order to detect the potential emergence of resistance. Although the approach to the design of neuraminidase inhibitors has provided hope that resistance will be limited, resistance to NAIs already been observed in clinical, encouraging close monitoring. It is therefore important to continue to study and understand the various mechanisms of resistance to neuraminidase inhibitors. The work of this thesis has thus focused on understanding the diversity of resistance mechanisms. Initially, we studied the impact of mutations in all structural residues of the active site of neuraminidase. We observed that most of these mutations did not alter the characteristics of the virus and induced very limited resistance to antivirals. Subsequently, we then sought to explore opportunities for synergy in resistance by the combination of two structural mutations of the active site of neuraminidase. On four viruses produced, only the virus with the double mutation E119V+I222L in the active site of neuraminidase was viable, although its in vitro replicative capacity was impaired. The combination of these two mutations induced a synergistic resistance to oseltamivir. Finally, we wanted to integrate the functional interaction of neuraminidase with hemagglutinin. We have shown that the combination of a hemagglutinin low affinity for sialylated receptors allowed to rescue a virus with a deficient neuraminidase. Thus an influenza virus may discharge the function of neuraminidase, the target of the only available effective antivirals. The mechanisms of resistance to neuraminidase inhibitors are numerous. Plus, the circulation in the last two seasons of resistant viruses without selective pressure challenges the assumptions developed on the possible emergence of resistance in clinic. This opens new issues to consider in order to understand the mechanisms that allowed this emergence and transmission
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Functional expression of influenza neuraminidase in Pichia pastoris. / 流行性感冒病毒神經氨酸酶於巴斯德畢赤酵母中的功能性表達 / Liu xing xing gan mao bing du shen jing an suan mei yu Baside bi chi xiao mu zhong de gong neng xing biao daJanuary 2009 (has links)
Tse, Yuk Tin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 141-149). / Abstracts in English and Chinese. / Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- The influenza virus --- p.1 / Chapter 1.1.1 --- Influenza NA and its inhibitors --- p.3 / Chapter 1.1.2 --- Follow-up on the use of NAIs --- p.9 / Chapter 1.2 --- Sources of NA for experimental studies --- p.11 / Chapter 1.2.1 --- Viral sources --- p.11 / Chapter 1.2.2 --- NA isolation --- p.12 / Chapter 1.2.3 --- Recombinant NA expressed in cell lines --- p.12 / Chapter 1.2.4 --- Glycosylation on NA functionality --- p.13 / Chapter 1.2.5 --- Recombinant NA expressed in yeast --- p.15 / Chapter 1.3 --- Research objectives --- p.16 / Chapter 2 --- Cloning of Influenza Neuraminidase and Expression in P. pastoris --- p.17 / Chapter 2.1 --- Background --- p.17 / Chapter 2.1.1 --- Full-length cloning of the A/HongKong/483/97(H5N 1) NA --- p.17 / Chapter 2.1.2 --- Identification of the H274 equivalent --- p.19 / Chapter 2.1.3 --- The experiment --- p.22 / Chapter 2.2 --- Materials and methods --- p.23 / Chapter 2.2.1 --- Preparation of chemically competent Escherichia coli --- p.23 / Chapter 2.2.1.1 --- Reagents --- p.23 / Chapter 2.2.1.2 --- Reagent setup --- p.23 / Chapter 2.2.1.3 --- Equipment --- p.23 / Chapter 2.2.1.4 --- Procedure --- p.23 / Chapter 2.2.2 --- Amplification of N1 NA and EGFP genes --- p.24 / Chapter 2.2.2.1 --- Reagents --- p.24 / Chapter 2.2.2.2 --- Reagent setup --- p.25 / Chapter 2.2.2.3 --- Equipment --- p.25 / Chapter 2.2.2.4 --- Procedure --- p.26 / Chapter 2.2.2.4.1 --- Amplification of the full-length N1 NA gene from cDNA --- p.26 / Chapter 2.2.2.4.2 --- Amplification of the EGFP gene --- p.26 / Chapter 2.2.3 --- TA cloning of PCR products --- p.27 / Chapter 2.2.3.1 --- Reagents --- p.27 / Chapter 2.2.3.2 --- Reagent setup --- p.27 / Chapter 2.2.3.3 --- Equipment --- p.28 / Chapter 2.2.3.4 --- Procedure --- p.28 / Chapter 2.2.3.4.1 --- TA cloning of PCR products --- p.28 / Chapter 2.2.3.4.2 --- Site-directed mutagenesis by overlapping PCR --- p.30 / Chapter 2.2.4 --- Construction of P. pastoris expression vectors --- p.31 / Chapter 2.2.4.1 --- Reagents --- p.31 / Chapter 2.2.4.2 --- Reagent setup --- p.31 / Chapter 2.2.4.3 --- Procedure --- p.32 / Chapter 2.2.4.3.1 --- Generation of N1 NA expression vectors --- p.32 / Chapter 2.2.4.3.2 --- Generation of EGFP expression vectors --- p.34 / Chapter 2.2.5 --- Transformation of P. pastoris --- p.37 / Chapter 2.2.5.1 --- Reagents --- p.37 / Chapter 2.2.5.2 --- Reagent setup --- p.37 / Chapter 2.2.5.3 --- Equipment --- p.38 / Chapter 2.2.5.4 --- Procedure --- p.38 / Chapter 2.2.5.4.1 --- Preparation and transformation of electrocompetent P. pastoris --- p.38 / Chapter 2.2.5.4.2 --- PCR analysis of P. pastoris transformants (colony PCR) --- p.39 / Chapter 2.2.6 --- Expression of N1 NA and EGFP in P. pastoris --- p.40 / Chapter 2.2.6.1 --- Reagents --- p.40 / Chapter 2.2.6.2 --- Reagent setup --- p.40 / Chapter 2.2.6.3 --- Procedure --- p.41 / Chapter 2.2.6.3.1 --- Small-scale protein expression in P. pastoris --- p.41 / Chapter 2.2.6.3.2 --- Sequence alignment --- p.42 / Chapter 2.2.6.3.3 --- Data processing --- p.42 / Chapter 2.3 --- Results and Discussion --- p.43 / Chapter 2.3.1 --- Cloning of NA and EGFP into the pPICZB expression vector --- p.43 / Chapter 2.3.2 --- Growth of P. pastoris transformants --- p.51 / Chapter 3 --- Physical Characterization of Influenza Neuraminidase Expressed in P. pastoris --- p.53 / Chapter 3.1 --- Background --- p.53 / Chapter 3.1.1 --- Structural significance of disulphide bonds in NA --- p.53 / Chapter 3.1.2 --- Localization of recombinant N1 NA in P. pastoris --- p.55 / Chapter 3.1.3 --- The experiment --- p.56 / Chapter 3.2 --- Materials and methods --- p.57 / Chapter 3.2.1 --- Differential centrifugation --- p.57 / Chapter 3.2.1.1 --- Reagents --- p.57 / Chapter 3.2.1.2 --- Reagent setup --- p.57 / Chapter 3.2.1.3 --- Equipment --- p.57 / Chapter 3.2.1.4 --- Procedures --- p.58 / Chapter 3.2.1.4.1 --- Cell harvesting and lysis --- p.58 / Chapter 3.2.1.4.2 --- Preparation of crude membrane --- p.58 / Chapter 3.2.1.4.3 --- Preparation of plasma membrane --- p.58 / Chapter 3.2.2 --- Sodium dodecyl sulphate polyaciylamide gel electrophoresis (SDS-PAGE)… --- p.59 / Chapter 3.2.2.1 --- Reagents --- p.59 / Chapter 3.2.2.2 --- Reagent setup --- p.60 / Chapter 3.2.2.3 --- Equipment --- p.61 / Chapter 3.2.2.4 --- Procedure --- p.61 / Chapter 3.2.3 --- Immunoblotting --- p.61 / Chapter 3.2.3.1 --- Reagents --- p.61 / Chapter 3.2.3.2 --- Reagent setup --- p.62 / Chapter 3.2.3.3 --- Equipment --- p.62 / Chapter 3.2.3.4 --- Procedure --- p.62 / Chapter 3.2.3.4.1 --- Electroblotting --- p.62 / Chapter 3.2.3.4.2 --- Blocking and probing --- p.63 / Chapter 3.2.3.4.3 --- Immunodetection --- p.63 / Chapter 3.2.3.4.4 --- Molecular weight determination --- p.63 / Chapter 3.2.4 --- Confocal microscopy --- p.64 / Chapter 3.2.4.1 --- Equipment --- p.64 / Chapter 3.2.4.2 --- Procedure --- p.64 / Chapter 3.2.4.2.1 --- Image acquisition --- p.64 / Chapter 3.2.4.2.2 --- Image processing --- p.65 / Chapter 3.3 --- Results --- p.66 / Chapter 3.3.1 --- Localization of recombinant N1 NA in P. pastoris sub-cellular fractions --- p.66 / Chapter 3.3.2 --- Molecular weight determination for the N1 NA expressed in P. pastoris --- p.69 / Chapter 3.3.3 --- Cellular localization of recombinant N1 NA in P. pastoris --- p.71 / Chapter 3.4 --- Discussion --- p.77 / Chapter 3.4.1 --- Molecular weight determination for N1 NA expressed in P. pastoris --- p.77 / Chapter 3.4.2 --- Disulphide bond formation in N1 NA expressed in P. pastoris --- p.78 / Chapter 3.4.3 --- Cell-surface association of recombinant N1 NA in P. pastoris --- p.79 / Chapter 3.5 --- Conclusion --- p.81 / Chapter 4 --- Functional Characterization of Influenza Neuraminidase Expressed in P. pastor --- p.is / Chapter 4.1 --- Background --- p.82 / Chapter 4.1.1 --- Fluorometric NA activity assay --- p.82 / Chapter 4.1.2 --- Colorimetric assay of NA activity --- p.84 / Chapter 4.1.3 --- The experiment --- p.85 / Chapter 4.2 --- Materials and methods --- p.86 / Chapter 4.2.1 --- Fluorometric assay of N1 NA expressed in P. pastoris --- p.86 / Chapter 4.2.1.1 --- Reagents --- p.86 / Chapter 4.2.1.2 --- Reagent setup --- p.86 / Chapter 4.2.1.3 --- Equipment --- p.86 / Chapter 4.2.1.4 --- Procedure --- p.87 / Chapter 4.2.1.4.1 --- Calibrating cell density with viable cell counts --- p.87 / Chapter 4.2.1.4.2 --- End-point measurement of NA activity --- p.87 / Chapter 4.2.1.4.2.1 --- Determination of expression yield --- p.89 / Chapter 4.2.1.4.2.2 --- End-point assay of NAI sensitivity --- p.89 / Chapter 4.2.1.4.3 --- Kinetic measurement of NA activity --- p.90 / Chapter 4.2.1.4.3.1 --- Derivation ofV0 --- p.92 / Chapter 4.2.1.4.3.2 --- Graphical determination of KM --- p.93 / Chapter 4.2.1.4.3.3 --- Graphical determination of KI --- p.94 / Chapter 4.2.2 --- Colorimetric assay of N1 NA expressed in P. pastoris --- p.96 / Chapter 4.2.2.1 --- Reagents --- p.96 / Chapter 4.2.2.2 --- Reagent setup --- p.96 / Chapter 4.2.2.3 --- Equipment --- p.96 / Chapter 4.2.2.4 --- Procedure --- p.96 / Chapter 4.3 --- Results --- p.98 / Chapter 4.3.1 --- CFU determination --- p.98 / Chapter 4.3.2 --- Fluorescent NA activity assay for N1 NA expressed in P. pastoris --- p.98 / Chapter 4.3.2.1 --- End-point measurement of NA activity --- p.98 / Chapter 4.3.2.1.1 --- Course of N1 NA expression in P. pastoris --- p.102 / Chapter 4.3.2.1.1.1 --- NA activity per unit cell mass --- p.102 / Chapter 4.3.2.1.1.2 --- Yield of NA --- p.102 / Chapter 4.3.2.1.2 --- End-point assay for NAI sensitivity --- p.105 / Chapter 4.3.2.2 --- Kinetic measurement of NA activity and NAI sensitivity --- p.107 / Chapter 4.3.2.2.1 --- Graphical determination of KM --- p.107 / Chapter 4.3.2.2.2 --- Graphical determination of KI --- p.107 / Chapter 4.3.2.3 --- Colorimetric NA activity assay --- p.111 / Chapter 4.4 --- Discussion --- p.114 / Chapter 4.4.1 --- Fluorescent NA activity assay of N1 NA expressed in P. pastoris --- p.115 / Chapter 4.4.1.1 --- End-point measurement of NA activity --- p.115 / Chapter 4.4.1.1.1 --- Time course of expression --- p.115 / Chapter 4.4.1.1.2 --- Effect of H275Y mutation on NA activity and NAI sensitivity --- p.117 / Chapter 4.4.1.1.3 --- Effect of C-terminal tags on NA activity and NAI sensitivity --- p.117 / Chapter 4.4.1.2 --- Kinetic measurement of NA activity --- p.118 / Chapter 4.4.1.2.1 --- Graphical determination of KM --- p.119 / Chapter 4.4.1.2.2 --- Graphical determination of KI --- p.120 / Chapter 4.4.1.3 --- Comparison of fluorometric NA activity assays for use with whole P pastoris cells --- p.122 / Chapter 4.4.2 --- Colorimetric NA activity assay --- p.124 / Chapter 4.5 --- Conclusion --- p.126 / Chapter 5 --- Conclusions and Discussions --- p.127 / Chapter 5.1 --- General conclusions --- p.127 / Chapter 5.2 --- Follow-up --- p.127 / Chapter 5.2.1 --- Studies of influenza NA with enhanced activity --- p.128 / Chapter 5.2.2 --- NAI screening using yeast-expressed NA --- p.132 / Appendix --- p.134 / References --- p.141
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Sialic acid modulation of cardiac voltage-gated sodium channel gating throughout the developing myocardium /Stocker, Patrick J. January 2005 (has links)
Dissertation (Ph.D.)--University of South Florida, 2005. / Includes vita. Includes bibliographical references. Also available online as a PDF document.
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Influence des peptides d'élastine dans le diabète de type 2 et la thrombose et caractérisation biochimique et fonctionnelle de la sous-unité Neuraminidase-1 du complexe récepteur de l'élastine / Role of elastin peptides in type 2 diabetes and thrombosis, and functionality and biochemical characterization of Neuraminidase-1, subunit of elastin receptor complexKawecki, Charlotte 16 December 2015 (has links)
L’élastine est la protéine de la matrice extracellulaire (MEC) responsable des propriétés de résilience et d'élasticité des tissus élastiques. Durant le vieillissement, les protéines de la MEC vasculaire sont exposées à des réactions délétères qui altèrent leurs propriétés structurales et fonctionnelles. Une des caractéristiques principales des protéines de la MEC est leur longue demi-vie, associée à un renouvellement très lent, comme pour l'élastine. Ainsi, tout dommage survenant sur l'élastine est essentiellement irréparable. La fragmentation des fibres élastiques génère des peptides d’élastine (EDP) bioactifs capables de modifier le comportement des cellules environnantes en se liant au complexe récepteur de l’élastine (CRE), composé de trois sous-unités dont la neuraminidase-1 (Neu-1), sous-unité catalytique du CRE. Cette thèse a consisté en l'étude, chez la souris, du rôle des EDP dans le développement du diabète de type 2 et dans la thrombose, deux pathologies vasculaires liées à l'âge, et s'est également focalisée sur la sous-unité Neu-1 du CRE. Dans un premier temps, nous avons montré que les EDP favorisent le développement d’une insulinorésistance et d’un diabète de type 2. Cet effet implique l'interaction de Neu-1 avec la sous-unité β du récepteur à l'insuline qui diminue son niveau de sialylation altérant ses voies de signalisation. Dans un second temps, nous avons identifié un mécanisme d'action des EDP à deux niveaux (matriciel et plaquettaire) et mis en évidence la présence d'un CRE fonctionnel dans les plaquettes régulant la thrombose. Enfin, nous avons étudié la topologie membranaire de Neu-1 par différentes approches technologiques et identifié un domaine transmembranaire potentiel jouant un rôle important pour sa dimérisation et son activité sialidase. En conclusion, les EDP sont des acteurs clefs du remodelage vasculaire physiopathologique et des pathologies vasculaires associées et de contribuer à faire avancer nos connaissances sur l'organisation de Neu-1 à la membrane plasmique. / Elastin is the extracellular matrix (ECM) protein responsible for resilience and elasticity of tissues such as arteries. During ageing, vascular ECM proteins are subjected to deleterious reactions that alter their structural and functional properties (addition reactions, proteolysis). One of the main features of ECM proteins is their long half-life, associated with a low, or even, inexistent turnover. This is the case for elastin with an estimated half-life at 70 years. Therefore, any damage occurring on elastin will be mostly irreparable. Fragmentation of elastic fibers produces bioactive elastin-derived peptides (EDP) able to modify the behavior of surrounding cells by binding to the elastin receptor complex (ERC). This receptor is composed of three subunits, among which neuraminidase-1 (Neu-1) is the catalytic subunit. The aim of this thesis was to study, in mice, the role of EDP in the development of type 2 diabetes and in thrombosis, two age-related vascular diseases, and to focus on the Neu-1 subunit of the ERC. In a first time, we have shown that EDP promote the development of insulin resistance and type 2 diabetes. This effect involves Neu-1 interaction with the β subunit of the insulin receptor and leads to its reduced sialylation level and signaling. In a second time, we have demonstrated that EDP are regulators of thrombosis. We identified a two-level mechanism (matrix and platelet) and the presence of a functional ERC in platelets. Finally, we have studied the membrane topology of Neu-1 by different biophysical, biochemical and molecular biology approaches, and identified a potential transmembrane domain involved in the dimerization and sialidase activity of Neu-1. In conclusion, this thesis consolidates the concept that EDP are crucial actors of pathophysiological vascular remodeling and related vascular diseases, and expands our knowledge on the plasma membrane organization of Neu-1.
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Structural studies on the sialidases from Streptococcus pneumoniae and Pseudomonas aeruginosaXu, Guogang January 2009 (has links)
The sialidases are a group of glycosyl hydrolases that specifically remove terminal sialic acid (Neu5Ac) residues from various glycans. In the two common human pathogenic bacteria Streptococcus pneumoniae and Pseudomonas aeruginosa, these enzymes have been shown to be key virulence factors directly involved in bacterial colonization and infection. However, little is known about their detailed structural and mechanistic features and lack of this information significantly slows down the progress of new drug discovery targeting these enzymes. Therefore, we embarked structural and kinetic studies towards the three distinct sialidases (designated as NanA, NanB and NanC) from S. pneumoniae, as well as the putative sialidase (designated as PaNA) from P. aeruginosa. Full-length NanA failed to crystallize due to the presence of some natively disordered regions. The catalytic domain of NanA (CNanA) was therefore subcloned, which was crystallized and the structure was determined to 1.5 Å. CNanA exists as a dimer with close contacts between the two monomers. The second pneumococcal sialidase NanB only shares 24% sequence identity with NanA. Crystal structure of NanB was also determined to 1.7 Å, which exhibits a multi-domain monomeric architecture. In general, the core catalytic domain of both CNanA and NanB adopts the classic six- bladed β-propeller fold (or called sialidase fold), with a set of highly conserved residues stacking around the proposed active sites. NanC is a close homologue of NanB, sharing over 50% sequence identity. However, NanC crystallization is not successful so far. To compare the three sialidases in more detail, a computational NanC model was made based on the structure of NanB. Mapping of the active sites of CNanA and NanB was achieved using Neu5Ac2en, a general sialidase inhibitor as the probe. Although sharing many common features, NanA, NanB and NanC present different topologies around the catalytic centre, give these enzymes a high level of diversity in enzymatic kinetics, substrate specificity and catalytic properties. NMR studies show that NanA acts as a classic hydrolytic sialidase; while NanB is found to be an intermolecular trans-sialidase like the leech sialidase; NanC, however, handles multiple catalytic roles efficiently, which include releasing Neu5Ac2en from α2,3- sialyllactose and hydration of Neu5Ac2en to Neu5Ac with high efficiency. S. pneumoniae thus expresses NanA, NanB and NanC for disparate but cooperative roles. Such a working pattern of three sialidases in one microbe is unusual in nature, which might be essential for pneumococcal pathogenesis at various stages. Based on the crystal structures of CNanA and NanB, preliminary work towards S. pneumoniae sialidases inhibitor design is under way, in which, a variety of techniques, such as the fluorescence-based thermal shift assay, NMR spectroscopy, computational docking and X-ray crystallography, are incorporated in. The crystal structure of PaNA was determined to 1.9 Å. This protein appeared to be a unique trimer in crystal that is associated, in part, by the immunoglobulin-like trimerization domain around a three-fold crystallographic axis. The core catalytic domain of PaNA also presents the conserved sialidase fold. Surprisingly, no sialidase activity was detected with this enzyme. In addition, two key catalytic residues including one of the arginine in the arginine triad and the acid/base catalyst aspartic acid are missing in PaNA. In silico docking suggests that Phe129 may confer substrate selectivity towards pseudaminic acid, which is a specific carbohydrate superficially similar to Neu5Ac, but with different stereochemistry at the C-5 position. Site-directed mutagenesis further confirmed that mutation of Phe129 to alanine could turn PaNA into a poor sialidases. Moreover, the crystal structure of PaNA also indicates that His45, Tyr21 and Glu315 may form a charge relay to compensate the missing aspartic acid. Subsequent mutagenesis and NMR kinetic studies proved His45-Tyr21-Glu315 to be a novel charge relay taking the role of the acid/base catalyst. Therefore, PaNA could be a pseudaminidase with structural and mechanistic variations. This enzyme, together some other uncharacterized fellow proteins, might form a novel subclass in the sialidase superfamily. The various findings in the current projects provide meaningful insights towards several sialidases that have been linked to bacterial virulence, which may contribute to a more intensive understanding of S. pneumoniae and P. aeruginosa pathogenesis.
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The search for allosteric inhibitorsBrear, Paul January 2013 (has links)
This thesis describes the development of chemical tools that inhibit the sialidases NanA and NanB from Streptococcus pneumonia. The primary focus was on the discovery of allosteric inhibitors of NanA and NanB, however, promising inhibitors that act by binding at the active site of these enzymes were also investigated. Chapter 1 gives an overview of the use of chemical tools in the field of chemical biology. It focuses in particular on chemical tools that function by the allosteric regulation of their target proteins. The uses, advantages and methods of discovery of allosteric tools are discussed. Finally this chapter introduces the use of serendipitous binders for the discovery of allosteric sites. In particular, the use of CHES to identify novel allosteric sites on the sialidase NanB is proposed. Chapter 2 describes how the ‘hits' from a series of high throughput screens were reanalysed using a wide range of secondary assays to eliminate any false positives that were contaminating the results. This process removed eight of the eleven ‘hits'. Two of the remaining three compounds were then analysed further in an attempt to characterise their binding mode to NanA and/or NanB using modelling and X-ray crystallographic studies. Whilst, it was not possible to confirm the binding mode by X-ray crystallography modelling studies using the modelling software GOLD generated possible binding modes for these inhibitors. A structure activity relationship study was conducted for both compounds in an attempt to generate more potent inhibitors. Chapter 3 moves from the use of high throughput screens to identify hits against NanA and NanB to the use of the serendipitous binding of N-cyclohexyl-2-aminoethanesulfonic acid in the active site of NanB for the development of selective NanB inhibitors. First taurine was identified as the minimum unit of N-cyclohexyl-2-aminoethanesulfonic acid required to bind to the active site of NanB. Taurine was then used as the basis of an optimisation study. This chapter concludes with the identification of 2-(benzylammonio)ethanesulfonate as the next key intermediate in the development of N-cyclohexyl-2-aminoethanesulfonic acid based active site inhibitors of NanB. Chapter 4 follows on from Chapter 3 with the optimisation of 2-(benzylammonio)ethanesulfonate describing the design and synthesis of a wide range of analogues. From these compounds 2-[(3-chlorobenzyl)ammonio]ethanesulfonate was identified as the most potent and selective inhibitor. Detailed analysis of the binding of 2-[(3-chlorobenzyl)ammonio]ethanesulfonate to NanB gave a rationale for its improved inhibitory activity. The increase in inhibition occurred because on binding of 2-[(3-chlorobenzyl)ammonio]ethanesulfonate to the active site of NanB a well coordinated water molecule was displaced. The displacement of this water caused an increase in the flexibility of the enzyme's 352 loop. A detailed study of the flexibility of this loop in response to various N-cyclohexyl-2-aminoethanesulfonic acid based chemical tools was then conducted. The research in chapters 2 and 3 has recently been published. In Chapter 5 a molecule of N-cyclohexyl-2-aminoethanesulfonic acid that binds serendipitously in a previously unmentioned secondary site is elaborated into a ligand, known as Optactin, that binds strongly and selectively at this secondary site. It was then shown that Optactin inhibited NanB by binding at this secondary site. It was therefore concluded that this secondary site was in fact an allosteric site that could be used for the regulation of NanB. Chapter 6 describes the development of a rationalisation for the inhibition of NanB by Optactin. This study included the X-ray crystallographic analysis of the apo-NanB structure and the NanB-Optactin complex under a range of conditions. This was followed by mechanistic studies that identified the point in the catalytic cycle at which Optactin was inhibiting NanB. This chapter concludes with a hypothesis for the mechanism of inhibition of NanB by Optactin.
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Synthesis of sialyl mimetics as biological probesPhan, Tho Van January 2004 (has links)
Abstract not available
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