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Produção e caracterização da porção Fab do anticorpo anti-digoxina utilizando a tecnologia de phage display. / Production and characterization of the Fab portion of anti-digoxin antibody by phage display technology.Viviane Midori Murata 27 March 2012 (has links)
A digoxina é um medicamento usado para tratar distúrbios cardíacos, com janela terapêutica muito estreita. Para combater seu efeito tóxico, fragmentos Fab do anticorpo policlonal anti-digoxina estão disponíveis comercialmente. Nosso objetivo foi a obtenção de variantes de fragmentos Fab do anticorpo monoclonal anti-digoxina usando a tecnologia phage display, que permite gerar fragmentos de anticorpos de alta afinidade e especificidade. Uma biblioteca combinatória de fragmentos Fab anti-digoxina foi construída no vetor pComb3X a partir do RNA total do hibridoma anti-digoxina. Seis clones foram isolados, todos com sequência idêntica na cadeia pesada. A cadeia leve apresentou 2 clones idênticos, um pseudogene e um clone com um aminoácido distinto no CDR2. Quatro clones apresentando variações na sequência do framework1 da cadeia leve foram expressos como fragmentos Fab solúveis. Todos apresentaram ligação à digoxina-BSA por ELISA e Western blotting. A ligação específica do anticorpo também foi confirmada pelo BIAcore, que permitiu ranqueamento entre os clones. / Digoxin is a pharmaceutical used in the control of cardiac dysfunction. Its therapeutic window is very narrow. To counteract the toxic effect, polyclonal anti-digoxin Fab fragments are commercially available. Our goal was to obtain variants of monoclonal anti-digoxin Fab fragments by phage display technology, which allows the generation of high affinity and specificity antibody fragments. Anti-digoxin Fab fragments combinatorial library was constructed into pComb3X vector from total RNA of anti-digoxin hybridoma. Six clones were isolated and the heavy chain presented the same sequence. For the light chain, 2 clones were identical, one was a pseudogene and other one presented a distinct amino acid in the CDR2. Four clones presenting variations in the framework 1 were induced to express soluble Fab fragments, all positive for anti-digoxin binding in ELISA assays and Western blotting. The specific binding of the antibody was further confirmed by BIAcore, which allowed ranking of the clones.
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Utilização de anticorpos monoclonais como diferenciais de meningites bacterianas e virais pela técnica de Imuno-histoquímica. / Use of monoclonal antibodies such as bacterial and viral meningitis differentials by Immuno-histochemistry.Lataro, Thais Regina Brienza 01 November 2016 (has links)
Meningite é um grupo de doenças infecciosas que constituem um problema global sério à saúde, tendo uma grande relevância social pelas altas taxas de mortalidade que esta doença pode causar. A descrição da progressão da doença, do agente causador e dos sintomas está bem estabelecida, no entanto, as fases iniciais da doença e todo o processo de infecção ainda não é compreendido. O desenvolvimento de diagnósticos laboratoriais mais rápidos e efetivos têm sido promissores. Este trabalho aborda a utilização de anticorpos monoclonais para o diagnóstico por meio da técnica de imunohistoquímica. Os anticorpos monoclonais foram obtidos durante fusões utilizando-se células esplênicas, linfócitos B contra antígenos de N.meningitidis. Os anticorpos foram utilizados em estudo imuno-histoquímico (IHQ) utilizando amostras de tecidos fixados em formol de pacientes com suspeita de meningite ou meningococcemia, no período de 2009 a 2015. Nosso intuito com este projeto é incrementar o diagnóstico histopatológico da meningite meningocócica, sobretudo em situações em que não houve confirmação por técnicas biomoleculares, como o PCR, da presença do agente causador desta doença. Estabelecemos um protocolo para a pesquisa de antígenos de N.meningitidis, conforme padronizado para a reação de IHQ. Nosso trabalho obteve bons resultados, os dois anticorpos monoclonais quando aplicados na reação de IHQ, não apresentaram reatividade cruzada com meningite bacteriana e viral, causadas por outros agentes etiológicos quando utilizamos DAB ou Fast Red como cromógenos. Portanto o uso dos anticorpos em conjunto com a técnica de IHQ se mostrou uma ferramenta de saúde pública auxiliando a vigilância epidemiológica. / Meningitis is a group of infectious diseases that compose a serious global health problem, having a great social relevance by high mortality rates that this disease can cause. The description of the disease progression, the causative agent and the symptoms is well established, however, the early stages of the disease and the infection is not yet understood. The development of faster and effective laboratory diagnoses have been promising. This paper discusses the use of monoclonal antibodies for diagnosis by means of the technique of Immunohistochemistry. Monoclonal antibodies were obtained during mergers using splenic cells, B lymphocytes against antigens of N. meningitidis. The antibodies were used in studies immunohistochemical (IHC) using formalin-fixed tissue samples from patients with suspected meningitis or meningococcemia, during the period from 2009 to 2015. Our aim with this project was to increase the histopathological diagnosis of meningococcal meningitis, especially in situations where there has been no confirmation by biomolecular techniques, such as PCR, of the presence of the causative agent of this disease. We have established a protocol for detecting antigens of N. meningitidis, as standardized to the IHC. The result obtained was very promising, the two monoclonal antibodies obtained good results. There was no cross-reactivity with bacterial and viral meningitis, caused by other etiological agents when using DAB or Fast Red as chromogens. Therefore the use of antibodies in conjunction with IHQ technique proved to be a public health tool aiding the epidemiological surveillance.
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Evaluation de l’effet protecteur de protéines du système de sécrétion de type III de bactéries entéropathogènes pour la vaccination et l’immunothérapie. / Evaluation of the protective efficacy of Type III Secretion System proteins of enteropathogenic bacteria in vaccination and immunotherapy.Jneid, Bakhos 24 November 2016 (has links)
Les bactéries entéropathogènes du genre Salmonella et Shigella sont transmises par les aliments ou l’eau et sont responsables de nombreuses infections entériques chez les animaux et les humains. Ces maladies infectieuses restent une cause importante de morbidité et de mortalité dans les pays en voie de développement. L’existence de multiples sérotypes de Salmonella et de Shigella ainsi que l’émergence de souches résistantes aux antibiotiques, nécessite le développement de vaccins efficaces et large spectre. Ces bactéries utilisent un système d’injection de leurs protéines effectrices, appelé injectisome ou encore Système de Sécrétion de Type III (SST3), nécessaire à leur pathogénicité. Alors que les protéines effectrices injectées au moyen de cet injectisome sont variées et dépendent essentiellement du type cellulaire cible et donc de la spécificité du pathogène, certaines des protéines structurales composant l’injectisome sont relativement bien conservées parmi les différentes bactéries pathogènes, notamment les protéines de l’aiguille : PrgI et MxiH, et celles de la coiffe de l’aiguille : SipD et IpaD, respectivement de Salmonella et Shigella. Ces protéines, fortement impliquées dans la virulence des bactéries, semblent donc être des cibles de choix pour lutter contre des infections opportunistes impliquant ces bactéries pathogènes.Le premier objectif de cette thèse était d’évaluer l’immunogénicité et l’effet protecteur des protéines structurales de l’injectisome citées précédemment contre les infections à Salmonella et Shigella. Les protéines recombinantes préparées et produites au laboratoire ont été utilisées de façon séparée ou en combinaison pour immuniser des souris par différentes routes. Ensuite, les réponses immunitaires des souris ainsi immunisées ont été analysées par des tests immunométriques. Enfin, le potentiel immunogène et vaccinant de ces protéines structurales a été évalué en infectant les souris immunisées avec 100 DL50 de Salmonella par voie orale ou de Shigella par voie intranasale. Le meilleur résultat a été obtenu en utilisant la voie intra-gastrique pour les immunisations avec environ 70% de protection. Cette stratégie a permis également d’évaluer la pertinence de cette approche vaccinale dans un modèle murin de protection croisée (entre 25 et 60%). Le deuxième objectif de cette thèse était d’évaluer le pouvoir protecteur d’anticorps monoclonaux murins reconnaissant les régions conservées des protéines SipD et IpaD. Les anticorps obtenus ont été caractérisés et leur pouvoir neutralisant a été évalué in vivo dans un modèle murin d’infection avec Salmonella ou Shigella (jusqu’à 60% de protection).L’ensemble de ces travaux montre que l’utilisation de certaines protéines structurales conservées de l’injectisome de bactéries entéropathogènes présente un intérêt vaccinal et immunothérapeutique pour aider au traitement de certaines salmonelloses et shigelloses. / Salmonella and Shigella species are food and water borne pathogens that are responsible for enteric infections in both humans and animals. These infectious diseases are still the major cause of morbidity and mortality in the emerging countries. The existence of multiple Salmonella and Shigella serotypes as well as the emergence of antibio- resistant strains, require the development of protective and broad-spectrum vaccines. All these bacteria utilize a system for injection of their effectors, called injectisome or Type III Secretion System (T3SS), necessary for their pathogenicity. While effector proteins are varied and depend essentially on the cellular target and thus on the specificity of the pathogen, the structural proteins that form the injectisome are common to all virulent Salmonella and Shigella spp., particularly the needle proteins PrgI and MxiH and the needle-tip proteins SipD and IpaD of Salmonella and Shigella respectively. These proteins, strongly involved in the virulence of the bacteria, appear to be ideal candidate antigens for a subunit-based, broad spectrum vaccine.The first aim of my PhD was to evaluate the immunogenicity and protective efficacy of structural proteins of the above-mentioned injectisome against Salmonella and Shigella infections. The recombinant proteins were prepared and produced in the laboratory and were used alone or in combination to immunize mice using different routes. The immune responses of immunized mice were then analyzed by immunometric assays. Finally, the protective efficacy was evaluated in a mouse model of intestinal (Salmonella) or pulmonary (Shigella) challenge. The best result was obtained by orogastric immunization with 70% of protection. This strategy also allowed to estimate the relevance of this approach in a mouse model of crossed protection (from 25 to 60%). The second objective of my PhD was to evaluate the protective efficacy of murine monoclonal antibodies recognizing conserved regions of SipD and IpaD proteins. The obtained antibodies were characterized and their therapeutic effect was evaluated in vivo with a Salmonella and Shigella infection murine model (up to 60% of protection).To conclude, this work showed that some conserved structural proteins composing the injectisome of enteropathogenic bacteria is of interest for treatment of enteric diseases caused by Salmonella and Shigella.
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Nanostructured Membranes Functionalized with Gold Nanoparticles for Separation and Recovery of Monoclonal AntibodiesSoldan, Giada 11 1900 (has links)
The need of purified biomolecules, such as proteins or antibodies, has required the biopharmaceutical industries to look for new recovering solutions to reduce time and costs of bioseparations. In the last decade, the emergent field of membrane chromatography has gained attention as possible substituent of the common used protein A affinity chromatography for bioseparations. In this scenario, gold nanoparticles can be used as means for offering affinity, mainly because of their biocompatible and reversible binding behavior, together with their high surface area-to-volume ratio, which offers a large number of binding sites.
This work introduces a new procedure for purification of monoclonal antibodies based on polymeric membranes functionalized with gold nanoparticles. This novel approach shortens the process of purification by promoting selective binding of antibodies, while separating a mixture of biomolecules during a filtration process. The effects of gold nanoparticles and the surrounding ligand on the proteins adsorption and filtration are investigated.
The results confirm that the functionalization helps in inducing a selective binding, preventing the non-selective one, and it also improves the selectivity of the separation process.
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Impact de la galectine-9 exogène sur les lymphocytes T humains et caractérisation de nouveaux anticorps monoclonaux à visée thérapeutique / Impact of exogenous galectin-9 on human T cells and characterization of new monoclonal antibodies for therapeutic useLhuillier, Claire 12 February 2016 (has links)
La galectine-9 (gal-9) est une lectine multifonctionnelle se liant à des glycoprotéines ou des glycolipides possédant des liaisons β-galactosides. Elle est impliquée dans plusieurs pathologies telles que le carcinome nasopharyngé associé au virus d’Epstein-Barr ou les infections chroniques par les virus des hépatites B et C.La gal-9 possède une activité immunosuppressive prédominante liée notamment à une stimulation de l’expansion et de l’activation des lymphocytes T (LTs) régulateurs. Ses effets sur les LTs conventionnels sont plus complexes : elle induit un phénomène d’apoptose précoce dans une fraction des LTs conventionnels mais aussi une activation et une prolifération dans une autre fraction incluant des LTs CD4+ Th1. D’où la nécessité d’explorer les événements de signalisation déclenchés par la gal-9 exogène dans les LTs humains.Notre recherche a été centrée sur des LTs CD4+ humains provenant de lignées leucémiques ou de PBMCs. Nos résultats montrent que le complexe TCR-CD3 et la kinase Lck sont requis pour la mobilisation de Ca2+ et la production de cytokines dont l’expression est induite en aval par la gal-9 (IL-2 et IFN-γ). A l’inverse, l’apoptose déclenchée par la gal-9 n’est pas réduite lorsque les cellules sont invalidées pour l’un de ces composants. Ces données indiquent que la gal-9 agit sur les LTs par deux voies de signalisation distinctes: l’une mimant l’activation classique du TCR avec une contribution majeure des éléments proximaux du complexe TCR, notamment Lck, et l’autre qui aboutit à l’apoptose des cellules et qui est indépendante de ce complexe.Parallèlement à ce volet fondamental de notre recherche, nous avons caractérisé de nouveaux anticorps monoclonaux (AcM) dirigés contre la gal-9. Ceux-ci neutralisent certains effets de la gal-9 sur les LTs humains tels que l’apoptose. Nous avons défini certaines propriétés immunochimiques de nos AcM en vue de leur utilisation potentielle en thérapeutique. Nous pensons que ces travaux pourraient ouvrir la voie à de nouvelles approches dans le champ actuellement en pleine expansion des thérapeutiques qui visent à une restauration de la réponse immunitaire anti-tumorale. / Galectin-9 (gal-9) is a multifunctional lectin binding some glycoproteins and glycolipids containing β-galactoside-bounds. It is involved in several pathologies such as Epstein-Barr virus-associated nasopharyngeal carcinoma or chronic infections by the hepatitis B and C viruses.Gal-9 has predominant immunosuppressive functions notably supported by its capacity to stimulate the expansion and activation of regulatory T cells. Its effects on conventional T cells are more complex: it induces apoptosis in a fraction of them, but it also activates and stimulates proliferation of another fraction including CD4+ Th1 cells. Hence the need to explore the signaling events triggered by exogenous gal-9 in human T cells.Our study was mainly focused on human CD4+ T cells from leukemic cell lines and PBMCs. We found that the TCR-CD3 complex and the Lck kinase were required for Ca2+ mobilization and subsequent cytokines production (IL-2 and IFN-γ) induced by gal-9 in T cells. By contrast, gal-9-triggered apoptosis was not reduced by the knocking-out of these TCR components. These data demonstrate that gal-9 acts on T cells by at least two distinct pathways: one mimicking the classical activation of the TCR with a mandatory contribution of proximal elements of the TCR complex, especially Lck, and another resulting in apoptosis which is independent of this complex.In parallel to this basic research, we characterized new monoclonal antibodies (mAb) targeting gal-9. We demonstrated that our mAb can neutralize the apoptosis and other gal-9 effects in human T cells and we defined some of their immunochemical properties in view of their potential therapeutic use. We believe that this work could pave the way for new approaches in the rapidly expanding field of therapeutics aiming at a restoration of the anti-tumor immune response.
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Étude d’agrégats d’anticorps monoclonaux sous écoulement microfluidique / Study of monoclonal antibodies aggregates under microfluidics flowDuchêne, Charles 13 November 2018 (has links)
La formation d'agrégats d'anticorps monoclonaux en solution est difficile à empêcher. Même si la présence de gros agrégats est assez rare, leur existence peut avoir des effets dramatiques dans les systèmes d'injection, en menant à des situations de colmatage partiel ou total de la restriction dans ce dernier. Cela entraîne une injection mal contrôlée ou même une obstruction totale du système d'injection. Très peu est connu sur le rôle de la taille des agrégats et de la pression appliquée sur de tels évènements de colmatage. Dans cette thèse, nous présentons un système microfluidique modèle, imitant les systèmes médicaux d'injection afin de comprendre fondamentalement le colmatage de restrictions d'une taille donnée. Des solutions très concentrées en anticorps monoclonaux nous permettent de créer des agrégats de protéines (plus grands que 50 micromètres) en utilisant un stress mécanique ou thermique. Nous montrerons que le colmatage a lieu quand les agrégats atteignent la taille de la restriction et peut dans certains cas être défait en augmentant la pression appliquée. La possibilité observée d'éjecter des agrégats de la restriction via une augmentation en pression indique le rôle important de la déformabilité des agrégats de protéines, à ce jour complètement inexplorée. Nous réalisons des expériences systématiques pour différentes tailles relatives d'agrégats et de pressions appliquées, et nous mesurons le débit en sortie. Malgré leurs formes et densités différentes, nous pouvons prédire le nombre d'évènements de colmatage pour une taille donnée de restriction par des mesures utilisant le Flow Imaging Microscopy (MFI). De plus, notre système peut détecter l'occurrence de très gros agrégats (très rares) souvent non détectés par d'autres techniques. Avec un modèle mécanique simple, nous pouvons estimer pour la première fois un ordre de grandeur du module d'Young et un diamètre effectif de pores pour des agrégats d'anticorps monoclonaux. Nous avons également développé une autre expérience modèle dans un canal hyperbolique couplé avec un flow focusing afin d'observer la déformation d'agrégats sous écoulement élongationnel. Nous décrirons leur comportement en analysant leurs trajectoires qui sont pour la plupart d'entre eux du tumble et de l'alignement avec l'écoulement. De plus, nous développerons un modèle mécanique qui tient compte de la force de friction dans une expérience modèle contrôlée avec une solution polymérique de PEGDA. Nous étudierons ainsi le rôle d'une différence de pression minimale à appliquer pour remettre la particule en mouvement dans la restriction, et ainsi relier cela aux agrégats de protéines. / The formation of aggregates in solutions of monoclonal antibodies is difficult to prevent. Even if the occurrence of large aggregates is rather rare, their existence can have dramatic effects in injection devices, as they can lead to partial or total clogging of constrictions in the latter. This leads to badly controlled injection or even total obstruction of the device. Little is know on the role of aggregate size and applied pressure on such clogging events. In this thesis, we present a microfluidic model system, mimicking medical injection devices to gain fundamental understanding of the clogging of constrictions of given size. Highly concentrated solutions of monoclonal antibodies allow us to create protein aggregates (bigger than 50 micrometers) using mechanical or heat stress. We show that clogging occurs when aggregates reach the size of the constriction and can in some cases be undone by increasing the applied pressure. The observed possibility to eject aggregates from constrictions via an increase in pressure indicates the important role of protein aggregate deformability, so far completely unexplored. We perform systematic experiments for different relative aggregate size and the applied pressure, and measure the flow-rate. Despite their different shapes and density, we can predict the number of clogging events for a given constriction size by Flow Imaging Microscopy (MFI) measurements. In addition our device can detect the occurrence of very rare big aggregates often overlooked by other detection techniques. With a simple mechanical model where we neglected the friction, we could estimate for the first time an order of magnitude for the Young modulus and a porous diameter for monoclonal antibodies aggregates. We also develop another model experiment with an hyperbolic channel coupled with a flow focusing to observe deformation of the aggregates under extensional flow. We describe their behavior by analyzing their trajectories which are for most of them tumbling and alignment with the flow. Moreover, we develop a mechanical model which took into account the friction force in a controlled model experiment with polymeric solution. We thus investigate the role of a minimal applied pressure to generate the particle movement into the constriction, and then link it with protein aggregates.
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Intracellular delivery of rabbit monoclonal antibodyQian, Qi 01 January 2007 (has links)
In the past decades, a series of small peptides, Protein Transduction Domain (PTD), were discovered to be able to facilitate the delivery of small proteins into living cells. With the specific feature, researchers have successfully delivered some functional proteins into living cells. To fully explore and understand the functions and structures of intracellular proteins, more powerful tools are under demand. Recently, an increasing number of rabbit monoclonal antibodies (RabMAbs) have been approved to able to recognize subtle distinctions between the changes of intracellular proteins status. They could be good tools for researchers with the ability to traverse through cell membrane into living cells.
In this dissertation, a novel delivery technology for RabMAbs was established. Transcriptional activator of transcription (TAT) peptide was utilized as a delivery carrier for RabMAbs. It was demonstrated that RabMAbs could be delivered into living cells by conjugating with TAT peptide. Different cell lines, including adherent and suspension cells, were tested for the delivery of RabMAbs. The delivery process was studied in terms of incubation concentration and time, and an optimal delivery condition was established.
To investigate the biological function of delivered RabMAbs inside cytoplasm, three RabMAbs against actin, procaspase-3 and NF-κB respectively were studied. Their binding activities after delivery were verified via sandwich-ELISA data. The immunofluorescent staining of the delivered RabMAb against actin showed it specifically bound to the actin filament in its native morphology. The quantitative analysis of the delivered RabMAb against procaspase-3 showed that approximately 60% of delivered antibody bound to the antigen proteins. The delivered RabMAb against NF-KB apparently blocked the nuclear translocation of NF-KB introduced by TNF-a. The success of delivering the three rabbit monoclonal antibodies with binding or inhibiting functions demonstrated the feasibility of delivering various RabMAbs into living cells by TAT peptide for studying the biological functions of intracellular proteins.
Furthermore, to overcome the efficiency and cost issues of the RabMAb delivery system, a universal delivery platform for RabMAbs was developed. This platform uses goat-anti-rabbit polyclonal antibody conjugated with TAT peptide as delivery vehicle. It was confirmed that the goat-anti-rabbit polyclonal antibody modified with TAT peptide was able to capture RabMAbs and deliver RabMAbs into living cells by the conjugated TAT peptide. The results provide a promising delivery platform for all RabMAbs.
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Use of a monoclonal antibody to detect gray mold (Botrytis cinerea) in strawberryMohr, Alexandra. January 2001 (has links)
No description available.
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Modulation of cell-cycle associated antigen expression by the B16 melanoma : multiparameter analysis using monoclonal antibodies and flow cytometry /Trimpe, Kevin L. January 1985 (has links)
No description available.
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Monoclonal Antibody Expression and Novel Purification in Nicotiana benthamianaFulton, Andrew Dale 28 June 2011 (has links)
Over the past few decades researchers and industrial professionals alike have realized the vast potential of monoclonal antibodies to treat diseases ranging from arthritis, immune and infectious diseases to cancer. There are a number of antibodies on the market that constitute a large portion of the biopharmaceutical niche in the drug industry. Blockbuster drugs (selling greater than $1 billion/year), include antibodies such as Avastin (bevacizumab), Herceptin (trastuzumab), Rituxan (rituximab), Humira (adalimumab) and Remicade (infliximab), which are cornerstones in this type of sector. With the cost of development to market approval rising astronomically for a new drug, new ways to produce and process these molecules becomes a paramount objective to ultimately help both patients and drug developers.
Plants, such as Nicotiana benthamiana, offer a unique production platform due to their recently found ability to produce large amounts of therapeutic proteins in a quick manner. While production would be simple and cheap, purification would not be due to the presence of toxic compounds in ground plant tissue. The current methods to purify these molecules from plant extract include expensive affinity column steps (Protein A/G) that are difficult to scale-up to bed volumes that would be necessary for this technology.
In the following paper, a method to purify a monoclonal antibody by non-Protein A/G resins is accomplished and compared to purification by Protein A. The modified process involved an UF/DF step, a precipitation of native impurities step using a charged polymer, hydrophobic interaction chromatography and hydrophobic charge induction chromatography. The yield of this modified process was 19.0%. This process compared favorably with Protein A due to the fact that even with washing steps including NaCl and Tween-20, the Protein A elution fraction still contained a large portion of host cell impurities. A chromatography step would need to be included before Protein A to both protect the column resin and provide a more purified immunoglobulin. / Master of Science
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