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FARRUGIA, ERIC-JEAN. "Hypergammaglobulinemies polyclonales : etude de cent cas dans un service de medecine interne." Toulouse 3, 1991. http://www.theses.fr/1991TOU31072.
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Massinga, Loembé Marguerite. "Caractérisation phénotypique et fonctionnelle des lymphocytes B dans la lymphocytose polyclonale chronique B." Thesis, Université Laval, 2004. http://www.theses.ulaval.ca/2004/22193/22193.pdf.
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La lymphocytose polyclonale chronique B (LPCB) est un syndrome peu connu caractérisé par une élévation polyclonale du nombre de lymphocytes B et de l’IgM sérique. Elle se distingue des pathologies lymphoïdes classiques par son origine polyclonale, sa grande stabilité ainsi que sa symptomatologie discrète et affecte majoritairement des femmes fumeuses. La présence de caractéristiques morphologiques et cytogénétiques distinctives, notamment cellules binucléées et anomalies génétiques (réarrangements bcl2/Ig multiples, isochromosome +i(3q)), guide le diagnostic initial. Ces particularités associées à un processus de transformation maligne contrastent avec l’apparente bénignité de la LPCB. Néanmoins, elles n’ont pas permis la délimitation précise de la population B impliquée dans la lymphocytose. Nos travaux avaient pour but d’identifier la population et les mécanismes impliqués dans l’émergence du syndrome, et éventuellement d’estimer les risques de progression clinique. En premier lieu, l’évaluation détaillée du profil immunologique des lymphocytes sanguins chez plusieurs patientes nous a permis de circonscrire formellement la lymphocytose aux cellules B IgD+IgM+CD27+. Mettant à profit les récentes avancées techniques et théoriques concernant la biologie du développement chez le lymphocyte B mature, nous avons entrepris l’analyse moléculaire des régions variables des gènes des immunoglobulines. Ces investigations ont confirmé le statut mémoire des cellules B en expansion dans la LPCB. Elles n’ont toutefois pas révélé la signature moléculaire résultant de sélection antigénique, processus central de la réponse immunitaire T-dépendante. Parallèlement, nos études fonctionnelles ont attesté de l’intégrité des molécules CD40 et AID, deux régulateurs clés de la maturation chez le lymphocyte B. Il ressort de nos analyses qu’un défaut dans la régulation de la réponse immunitaire, permettant le contournement de la sélection antigénique dans les centres germinatifs, plutôt qu’un blocage de différenciation cellulaire, serait probablement à l’origine de la lymphocytose. Alternativement, ces cellules pourraient être dérivées d’une population nouvellement caractérisée, les lymphocytes B mémoires de la zone marginale splénique, aussi retrouvés dans le sang, provenant présumement d’une voie de diversification indépendante des centres germinatifs. En conclusion, nos résultats ont permis de préciser le portrait diagnostique de la LPCB et de délimiter de nouvelles pistes de recherche touchant tant les aspects cliniques que la biologie fondamentale du syndrome.Persistent polyclonal B cell lymphocytosis (PPBL) is an unusual haematological disorder, mainly detected in adult female smokers, that shares features of both benignity (polyclonal expansion, polyconal IgM secretion, lack of clinical symptoms, stable and mostly uneventful course); and features of malignancy (atypical binucleated cells, multiple bcl-2/Ig translocations, chromosome 3 anomalies, bone marrow involvement). Still, these morphological and clonal genetic anomalies have not been restricted to a distinctive B cell subset, and the apparent heterogeneity of the involved cellular population has long impeded further characterization of the syndrome. The aim of our research was to formally identify the population involved in the lymphocytosis, to gain some insight into the mechanisms at play in its development and to evaluate the risk for subsequent transformation in patients. Over the recent years, technical inputs from the molecular field have largely contributed to a better discrimination of the various B cells subsets and, by extension, of B cell lymphoid disorders. Thus, detailed immunophenotypic studies conducted in numerous PPBL patients allowed us to definitely circumscribe the disorder to IgD+IgM+CD27+ B lymphocytes, whereas exhaustive molecular analysis of immunoglobulin genes’ variable regions has corroborated the memory status of these cells. Yet, molecular signature of the antigenic selection process, the characteristic of a T-dependent immune response, was not detected. Sequencing of the CD40 and AID genes, key regulators in the diversification and affinity maturation of the immunoglobulin receptor, was additionally carried out and expression of both molecules was assessed. No anomaly was evidenced for either gene. In light of those observations, we conclude that a differentiation block in PPBL B lymphocytes is unlikely. Rather, we propose that defects in the affinity maturation process, namely impairment of the antigenic selection mechanism, allows the survival of low affinity IgD+IgM+CD27+ memory B lymphocytes in PPBL patients. Conversely, these cells could be related to the as yet scantily characterized IgD+IgM+CD27+ memory B cell subset from the splenic MZ, also found in the blood, and presumably derived from a germinal centre independent diversification pathway. Altogether, our results contributed to the elaboration of an accurate clinical definition for PPBL, and delineated avenues for future investigations regarding both the pathological aspects of the disorder and its purely fundamental biologic ramifications.
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Dueymes, Maryvonne. "Approche expérimentale du traitement des glomérulonéphrites lupiques par immunomodulation de la stimulation polyclonale." Lyon 1, 1989. http://www.theses.fr/1989LYO1H082.
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Delamotte, Pierre. "Deciphering the metabolic bases of Drosophila intestinal tumors." Electronic Thesis or Diss., université Paris-Saclay, 2024. http://www.theses.fr/2024UPASL064.
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Le métabolisme tumoral est étudié en raison de son importance dans la compréhension du fonctionnement des tumeurs et du développement de traitements anticancéreux. L’état des recherches actuelles prévoit, suivant le tissu et le stade de développement, le métabolisme des cellules tumorales, recensant ainsi les voies métaboliques empruntées par les tumeurs. Ces modèles restent néanmoins limités à des situations précises, et une compréhension globale des capacités métaboliques des cellules tumorales ainsi que leurs évolutions au cours du développement des tumeurs demeurent incompris. Mon projet de thèse consiste à caractériser les besoins métaboliques d’un modèle de tumeurs intestinales de drosophile. Ces tumeurs sont génétiquement induites par recombinaison somatique des cellules souches intestinales de façon contrôlée et reproductible. Premièrement, un criblage ARNi par cytométrie en flux a révélé, à différents niveaux, le besoin de plusieurs voies métaboliques pour soutenir la croissance tumorale. Une expérience de séquençage d’ARNm de cellules uniques de tumeurs isolées a confirmé ces résultats tout en révélant l’existence de groupes très conservés de cellules métaboliquement spécialisées au sein des tumeurs. L’utilisation de sondes métaboliques exprimées par les cellules tumorales nous a permis de confirmer l’hétérogénéité des tumeurs en imagerie d’une part, et de mettre en évidence une détermination métabolique des cellules avant la formation de tumeurs. Deuxièmement, la nature polyclonale des tumeurs a été démontrée par à l’aide d’outils de traçage du lignage cellulaire. Enfin, l’utilisation combinée d’un criblage ARNi par cytométrie en flux et d’outils de traçage du lignage cellulaire a mis en évidence l’importance du processus de migration cellulaire dans la croissance tumorale. Notre étude propose un modèle décrivant la formation des tumeurs, et leurs choix métaboliques – à l’échelle de la cellule – dans le cadre de tumeurs du tube digestif de la drosophile. Le concept de fusion de cellules tumorales issues d’évènements mutationnels différents, permettant l’obtention de tumeurs polyclonales pourrait constituer une nouvelle étape de progression tumorale dans certains types de cancers. Les résultats obtenus concernant les voies métaboliques préférentielles de ces cellules tumorales, leur besoin de migrer de de s’associer en tumeurs polyclonales constituent tout autant de pistes afin de développer ou de perfectionner des approches thérapeutiques contre le cancerTumor metabolism is extensively studied since its understanding is a milestone in developing efficient anti-cancer treatment. The current assumption in the field of tumor metabolism is a defined metabolism and tumor behavior for each type of tissue, providing a broad repertoire of cancer metabolic options. This vision is, however, limited to specific tumoral contexts so that the full metabolic capabilities of tumor cells or their metabolic evolutions throughout time remain unclear. My project aims to characterize metabolic requirements in a Drosophila midgut tumor model. These tumors are genetically induced by somatic recombination of intestinal stem cells in a controllable and reproducible manner. First, a cytometry-based RNAi screening has pointed, to various degrees, several metabolic pathways relevant to tumor growth. A single-cell RNA sequencing performed on isolated tumoral tissue not only confirmed this result but also showed metabolically specialized, highly conserved cell clusters. The use of genetically-encoded biosensors, allowed us to show metabolic heterogeneity within tumors and metabolic choices in cells before tumor formation. Second, the use of cell lineage tools on tumor cells reveals an obligatory polyclonality in this tumor model. Third, the combined use of cytometry-based RNAi screening and microscopy cell lineage tools demonstrate cell motility is a required process to form these tumors. Our study proposes a model for tumor formation and describes the metabolic pathways - at the cell resolution - performed in a Drosophila midgut tumor model. Importantly, the gathering of newly emerged cancer cells could constitute a new and critical step in tumor progression for polyclonal tumors. The experiments addressing preferential metabolic routes in tumors at early and late stages, cell motility, and cell gathering to tumors constitute as many potential targets to enrich current anti-cancer treatment and develop novel curative and preventing drugs
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Ryckman, Carle. "Analyse moléculaire de l'expression du virus Epstein-Barr dans la lymphocytose polyclonale chronique B." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp03/MQ31792.pdf.
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Massinga, Loembé Marguerite. "La lymphocytose polyclonale chronique B, étude in vitro des propriétés biologiques des lymphocytes T et B." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape7/PQDD_0012/MQ41959.pdf.
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Ouedraogo, David Eric. "Exploration du réservoir EBV chez les patients infectés par le VIH : implications pathologiques." Thesis, Montpellier 1, 2013. http://www.theses.fr/2013MON1T001.
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Les lymphocytes B mémoires circulants incluant ceux infectés par EBV de façon latente retournent périodiquement vers les territoires lymphoïdes secondaires où ils subissent une différenciation en cellules productrices d'immunoglobulines permettant au virus d'initier la réplication virale. Cependant, le suivi et la gestion de la réactivation de EBV et son association avec des néoplasies lymphoïdes chez les sujets infectés par le VIH restent un sujet de controverse et nécessite une meilleure compréhension des mécanismes impliqués. Dans cette étude, nous avons proposé de nouveaux outils biologiques pour la quantification de l'ADN EBV permettant la discrimination entre le réservoir latent et le cycle lytique du virus. Nous avons montré que la taille de ces réservoirs est étroitement associée à une activation polyclonale plus ou moins importante des cellules B. Nous avons également observé une association entre les marqueurs d'activation immunitaire et de réactivation de EBV avec la survenue de lymphome B. En outre, nous avons décrit l'évolution de gammapathies monoclonales chez des sujets infectés par le VIH sous traitement antirétroviral, et la persistante du pic monoclonal d'immunoglobulines était associée à des charges virales EBV plus élevés. Par conséquent, l'activation des lymphocytes B et subséquemment la réactivation EBV joueraient vraisemblablement les rôles principaux dans la survenue de tumeurs bénignes ou malignes des lymphocytes B au cours de l'infection VIHIt is assumed that circulating memory B cells including those latently infected by EBV return periodically to lymphoid nodes where they are stimulates and undergo differentiation into immunoglobulin-producing cells allowing the virus to initiate viral replication. However, the monitoring and the management of EBV reactivation and it association with lymphoid malignancies in HIV-infected patients are still being controversies and need a better understanding of the probable mechanisms involved. In this study, we proposed novel biological tools for EBV DNA quantification allowing discriminating latent and lytic reservoir. We showed that the EBV reservoir levels are closely associated with the polyclonal B-cell activation. We also observed an association between immune activation and EBV reactivation markers with the occurrence of B-cell lymphoma. Moreover, we described a long term evolution of monoclonal gammapathies in HIV-infected subjects and the persistence of the immunoglobulis monoclonal pike was found to be associated with higher EBV reservoir levels. Therefore, the B-cell activation and subsequently EBV reactivation likely play the main roles on the occurrence of B lymphocytes malignancies during HIV infection
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Chamond, Nathalie. "Quand un mitogène est une enzyme. ." Paris 6, 2003. http://www.theses.fr/2003PA066048.
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Leclercq, Lise. "Analyse du mode d'action du lymphocyte T "helper" : son rôle dans les phases précoces de l'activation de la cellule B et sa contribution à la régulation isotypique." Paris 7, 1985. http://www.theses.fr/1985PA077058.
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Lors d'une stimulation par un antigène protéique soluble, les lymphocytes B reconnaissent l'antigène grâce à leurs immunoglobulines (Ig) de surface et collaborent avec des lymphocytes T helper (Th) également spécifiques de cet antigène. Ces interactions cellulaires complexes conduisent la cellule B à produire des anticorps qui représentent la forme soluble de l'Ig de surface. Bien que tous dirigés contre l'antigène, ces anticorps peuvent néanmoins appartenir à différentes classes que l'on appelle isotypes et qui se distinguent par la nature de leur région constante. Notre travail réalisé dans un modèle murin utilisant des clones de lymphocytes Th spécifiques d'un antigène synthétique, le GAT (poly (G1u60 Ala30 Tyr ¹º)), a tenté d'analyser le mode d'action du lymphocyte Th, en particulier son rôle dans les phases précoces de l'activation de la cellule B et sa contribution à la régulation isotypique. Après avoir testé différents clones de lymphocytes Th pour leur capacité à induire une stimulation polyclonale de lymphocytes B vierges, nous avons concentré nos efforts sur le clone le plus actif, appelé 52. 3. Nous avons préparé du surnageant (SN) de culture du clone 52. 3 activé et montré que les lymphocytes B au repos, syngéniques ou allogéniques, prolifèrent en présence de ce SN et en l'absence de tout autre stimulus tel que celui délivré par des anticorps anti-Ig. Par cytométrie de flux, nous avons démontré qu'après stimulation avec du SN du clone 52. 3, la quasi-totalité des lymphocytes B hyperexpriment les antigènes Ia, augmentent de taille (30% d'entre eux devenant blastiques) et passent de G₀ en G₁, mais que seuls 20% d'entre eux atteignent les phases S et G₂/M du cycle cellulaire. Ces résultats nous ont conduits à postuler l'existence dans le SN d'une lymphokine agissant sur les lymphocytes B au repos en provoquant leur passage du stage G₀ au stade G₁ du cycle cellulaire et ceci d'une manière qui n'est pas restreinte par les gènes du complexe majeur d'histocompatibilité (CMH). Après stimulation avec le clone de lymphocytes Th 52. 3, des lymphocytes B spléniques IgM⁺ IgG⁻ (isolés par passage au trieur de cellules) sont capables de produire, en plus de l'isotype dominant IgM, des Ig appartenant aux différentes sous-classes d'IgG (avec une nette prédominance des IgG1). Des résultats analogues concernant la sécrétion des IgA ont été obtenus avec des lymphocytes IgM⁺ IgA⁻ isolés par adhérence sur une boîte recouverte d'anticorps anti-IgA. La stimulation de cellules B IgM⁺ IgG⁻ IgA⁻ par du SN du clone 52. 3 induit une réponse plus faible que celle induite par les cellules 52. 3 elles-mêmes, mais elle conduit néanmoins à une production des isotypes IgM, IgG et IgA. Parmi l'isotype IgG la sous-classe IgGi est dominante. Les expériences conduites sur des cellules B IgM⁺ IgG⁻ IgA⁻ semblent indiquer que des cellules Th spécifiques d'isotype ne sont pas indispensables pour qu'une cellule B primaire stimulée par une cellule Th spécifique d'un antigène produise des anticorps d'un isotype autre que IgM.
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Callet-Bauchu, Évelyne. "Apport des techniques de cytogénétique moléculaire dans le démembrement des lymphomes malins non hodgkiniens diffus à petites cellules B et des lymphocytoses B polyclonales à lymphocytes binuclées." Lyon 1, 1998. http://www.theses.fr/1998LYO1T073.
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Ozeren, Muserref. "Catalysis by polyclonal antibody." Thesis, University of Bath, 1997. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.246006.
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Lapage, John M. J. "Polyclonal architecture of the mammalian head." Thesis, University of Warwick, 2015. http://wrap.warwick.ac.uk/77488/.
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While much of modern developmental biology has focussed upon molecularly defined cell populations, relatively little is understood about how clonal groups within these broad cell populations organise complex tissues. In this thesis, I explore the clonal architecture of the jaws and teeth, and of the dermal bones of the calvaria, revealing cryptic modules as novel developmental features in both. I combine Confetti multicolour genetic lineage labelling with novel analytical techniques in order to map clonal populations in 3D and provide quantitative parameters for clonal expansions. Tooth identity within the mandible is thought to be encoded by the an initial proximodistal position within the branchial arch, which implies that cells do not undergo migration. I observe distal Hand2-Cre labelled cells in the proximal territory, which necessitates migration. These distal cells give rise to the mandible, alveolar bone and a small proportion of odontoblasts, while unlabelled proximal cells were found in the distal territory of the incisors in different proportions. The clonal composition of teeth and jaw bones is dissected by novel analysis of mixed cell populations. I find odontogenic and alveolar bone populations to share a common lineage, comprising a cryptic developmental unit distinct from the mandible, a feature that I can also verify in another transgenic for the upper jaw. I also find that the initial tooth composition radically changes in ontogenetic time. Starting from similar compositions of distal and proximal cells I find that in incisors the distal population expands while the proximal wanes, while in molars the opposite occurs. This is the first evidence for a temporally changing cell population structure underlying the well defined heterodonty between incisors and molars and allows a reinterpretation of early tooth specification events. The dermal bones of the calvaria are thought to grow in thickness by static osteoblasts depositing matrix appositionally and growth is supposed to occur exclusively at sutures. Whole calvaria single-cell clonal lineage analysis of cranial neural crest cells with Wnt1-Cre and Confetti labelling reveals an extensively dynamic program of invasive growth distributed throughout all parts of the dermal bone. Cryptic clonal modules grow laterally, with invasion through and into the bone primarily organised around the centres of these `patches'. The process of bone maturation is revealed to consist of a series of invasions between the three layers of the bone, with the innermost compacta layer driving initial thickness growth by invasion into the middle spongy layer, and the outermost (dermis-adjacent) compacta layer driving later growth. Conversely there is no evidence to suggest that the sutures are principal generative regions, as they do not share a common clonal lineage with adjacent bone. I also investigate muscle attachment regions and find that the same clones traverse tissue boundaries from bone into muscle connective tissues, thus a clonal model predicated on joint 'attachment point precursor cells' can now explain patterns of skeletomuscular connectivity previously found at the population level by my supervisor. A novel generative model of bone growth from cryptic clonal patch modules is proposed, allowing us for the first time to understand thickness growth and the evolutionary transition from a micromeric to a macromeric dermal bone condition, events first visible in crown gnathostomes (placoderms).
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Boushaba, Rihab. "Strategies for therapeutic polyclonal antibody fragments manufacture." Thesis, University of Cambridge, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.616154.
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Alder, Louise B. A. "Immunoregulatory properties of polyclonal immunoglobulin for therapeutic use." Thesis, University of Newcastle Upon Tyne, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.361937.
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Simms, Caroline Sarah. "Characterisation of anti-nitrophenylphosphate and anti-tropanyl phenylphosphonate catalytic antibodies." Thesis, University of Brighton, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.299216.
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Plante, Hendrick. "Production d'anticorps polyclonaux contre les récepteurs AT1 humain et ATp de poulet." Sherbrooke : Université de Sherbrooke, 1997.
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Lee, Yun-Kyung. "Production and characterization of polyclonal antibodies against chicken myostatins." Thesis, University of Hawaii at Manoa, 2003. http://hdl.handle.net/10125/7041.
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Myostatin is a new member of the transforming growth factor-beta superfamily of secreted growth and differentiation factors. Myostatin is almost exclusively expressed in skeletal muscle and act as a negative regulator of skeletal muscle growth. Because anti-myostatin antibodies will be useful in investigating the mechanism of action of myostatin, an experiment was designed to produce polyclonal anti-chicken-myostatin antibodies. A PCR amplified prepro-myostatin composed of the prodomain plus mature myostatin and a C- terminal fragment containing only the mature myostatin were cloned into an expression vector, and these proteins were expressed in E. coli. The recombinant proteins were fractionated by SDS-PAGE, then the myostatin bands were cut out and electro-eluted to obtain purified myostatins. Rabbits were immunized with the purified myostatins to produce polyclonal anti-myostatin antibodies. IgGs were separated from the rabbit sera using Protein A affinity chromatography. Antibody binding characteristics were then examined using Western blot analysis of various chicken tissues. Both polyclonal antibodies demonstrated a strong affinity to both form of recombinant myostatin in Western blot analysis. The anti-mature myostatin antibody showed binding affinity to proteins at 50, 30 and 20 kD in skeletal muscle and liver. No specific binding in skeletal muscle was demonstrated by the anti-mature myostatin antibody, suggesting that the above proteins are non-myostatin proteins that have affinity to anti-mature myostatin antibody. In contrast, the anti-prepro-myostatin antibody showed affinity to a 37 kD band only in skeletal muscle in addition to the 50, 30 and 20 kD bands. Since the molecular weight of the myostatin prodomain is known to be close to 37 kD, it is postulated that the 37 kD protein specifically recognized by the antiprepro- myostatin in skeletal muscles is likely to be a myostatin prodomain with further validation of its binding specificity, the anti-prepro-myostatin antibody generated in this study will potentially be useful in investigating the mechanism of action of myostatin.x, 101 leaves
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Coeur, Vera Mae Fran 1960. "Characterization of polyclonal antibodies raised against DNA attachment proteins." Thesis, The University of Arizona, 1991. http://hdl.handle.net/10150/277854.
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The eukaryotic nucleus is thought to contain an internal protein scaffolding structure which is present in interphase and metaphase cells. We were interested in identifying some of the protein components of the scaffolding structure by isolating proteins which have DNA attached to them (DNA attachment proteins). DNA attachment proteins have been previously described by our group, and are referred to as proteins 1, 2, 3, and 4, respectively. Their apparent molecular weights (Mr) and isoelectric values (pI) are 70,000, 4.3; 70,000, 5.3; 58,000, 5.3; and 57,000, 4.8, respectively. My project was to produce and characterize polyclonal antibodies made against these four proteins. The DNA attachment proteins were injected into rabbits. The serum was affinity purified using Protein-A sepharose chromatography. Antibody preparations 1, 2, and 4 recognized the specific DNA attachment proteins and did not cross react with the others suggesting that the polyclonal sera recognized distinct and separate epitopes.
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Macovei, Cristian-Paul. "Identification de catalyseurs à l'aide de criblages à haut débit basés sur des techniques immunoenzymatiques." Phd thesis, Université Paris Sud - Paris XI, 2008. http://tel.archives-ouvertes.fr/tel-00447202.
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Le travail décrit dans ce manuscrit présente l'utilisation de deux techniques immunoenzymatiques pour le criblage à haut débit de catalyseurs chimiques et biologiques. Ces méthodes, jusqu'à présent réservées au monde du diagnostic sont employées ici pour la découverte de nouveaux catalyseurs pour des réactions énantiosélectives ainsi que pour des réactions de couplage. Les dosages immunologiques « par compétition » faisant appel aux anticorps monoclonaux se sont avérés des excellents outils pour le criblage à haut débit de catalyseurs asymétriques pour les réactions d'insertion de carbénoïdes dans la liaison OH de l'eau tandis que celles utilisant des anticorps polyclonaux ont été utilisées avec succès pour le criblage de biocaytalyseurs pour la réaction d'ouverture énantiosélective des oxazolones par l'eau. Un autre type de méthode utilisant des anticorps monoclonaux, appelée « sandwich » nous a permis de réaliser le criblage à haut débit de catalyseurs pour la réaction de cycloaddition alcyne-azoture.
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Sonkaria, Sanjiv. "Kinetic characterisation of polyclonal hydrolytic catalytic antibodies and analogous enzymes." Thesis, Queen Mary, University of London, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.271023.
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Boucher, Guillaume. "Kinetic studies of alternative substrates for existing polyclonal catalytic antibodies." Thesis, University of Brighton, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.271976.
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Croll, A. D. "The regulation of polyclonal mitogen-stimulated human gamma-interferon production." Thesis, University of Warwick, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.377094.
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Dawkes, Adrian Charles. "Immunochemical studies on myelin basic protein using monoclonal polyclonal antibodies." Thesis, Open University, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.234745.
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Woolley, Stephen Terry. "Preparation and characterization of monoclonal and polyclonal antibodies to gamma interferon." Thesis, Oxford Brookes University, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.293559.
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Philp, Rebecca L. "The polyclonal antibody response to FMDV in cattle and African buffalo." Thesis, University of Glasgow, 2017. http://theses.gla.ac.uk/8660/.
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Protection against the highly contagious foot and mouth disease virus (FMDV) coincides with neutralising antibody titres. Infection in cattle is characterised by 100% morbidity of an acute vesicular disease whilst infection of their closest relative, the African buffalo, is sub-clinical despite having diverged only 5.7 – 9.3 million years ago (Glanzmann et al., 2016; 1). The germline and antibody repertoire in African buffalo has not previously been characterised and so the cause of their differential disease response may be the production of a more specific and / or avid antibody response to FMDV than cattle. The cattle and African buffalo antibody germline was sought to characterise the recombinatorial potential of the antibody loci and their subsequent primary antibody repertoire. Expression of the antibody heavy chain (IGH) and antibody lambda light chain (IGL) was investigated with qPCR and RNA-seq. The antibody repertoire in response to FMDV infection was interrogated in African buffalo infected with SAT1 FMDV and compared to the cattle IGH repertoire inoculated with highly purified SAT1 FMDV antigen. The recombinatorial potential of the cattle and African buffalo IGH and IGL is severely limited compared to other species such as mice and human. The characterisation of the cattle IGH and IGL is the most accurate to date and reveals internal duplications of the IGH, disrupting the expected IGHV-IGHD-IGHJ-IGHC ordering seen in mammalian immune loci and resulting in four IGHD regions, containing long and ultra-long IGHD. These IGHD provide a novel diversification mechanism that can compensate for limited germline diversity by forming long and ultra-long CDR3H loops that are highly diverse in their length and amino acid composition. The African buffalo antibody repertoire also forms highly diverse long and ultra-long CDR3H, despite lack of evidence for the existence of the duplications in their IGH. Limited variability is seen in the length and amino acid composition of the IGL in both species, suggesting they are playing a structural role to support these unusual long and ultra-long CDR3H. In response to FMDV infection in African buffalo, a dramatic increase in specific long and ultra-long CDR3H sequence abundance occurs but this change in frequency of specific transcripts is absent in cattle. The differential antibody response may account for the protection of African buffalo against FMD.
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Petch, Geoffrey Michael. "Aspects of the detection and discrimination of members of the fungal genus Pythium by serological and molecular methods." Thesis, University of Birmingham, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.368651.
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Semra, Yemane Kurban. "Endogenous ouabain-like immunoreactive substance (OLIS) : characterisation and physiological studies." Thesis, King's College London (University of London), 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.313282.
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Haidar, ahmad Hamad. "Pro-inflammatory activity and adjuvant effect of Neutrophil Extracellular Traps in physiological and pathological context." Electronic Thesis or Diss., Paris 13, 2024. http://www.theses.fr/2024PA131004.
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Les neutrophiles activés (PNNs) expulsent des "Neutrophil extracellular traps" (NETs). Les NETs constituent un mécanisme de défense contre les agents pathogènes. Cependant, le rôle des NETs va au-delà de leur fonction antimicrobienne, avec des implications dans divers processus physiologiques et pathologiques, y compris les troubles auto-immuns. Une augmentation de la formation des NETs a été signalée dans la polyarthrite rhumatoïde (PR); une maladie inflammatoire chronique touchant les articulations. Nous avons précédemment montré que les NETs sont pro-inflammatoires sur les macrophages au repos. En effet, les NETs contiennent plusieurs molécules ayant des propriétés immunostumulatrices. Nous suggérons que les NETs agissent comme des motifs moléculaires associés aux dommages (DAMPs) sur les lymphocytes B en induisant leur activation polyclonale, indépendamment de la spécificité de l'antigène ; un nouveau mécanisme par lequel les NETs pourraient contribuer à la dérégulation immunitaire, en particulier dans le contexte de la PR. Nous avons démontré par cytométrie de flux, ELISA et séquençage des ARN (RNAseq) que les NETs pouvaient directement activer les cellules B totaux et naïves vers un profil pro-inflammatoire robuste, caractérisé par une production élevée de cytokines pro-inflammatoires et une surexpression de HLA-DR et des molécules de co-stimulation CD40 et CD86 qui sont importantes pour la fonction de cellules présentatrices d'antigènes (CPA). Cette activation est amplifiée chez les patients atteints de PR. Nous avons également montré que ce mécanisme est modulé par la présence des protéines C1q et de LL-37 mais qu'il ne nécessite pas le "Toll-like receptor 9" (TLR9). Au-delà de l'activation des cellules B, nos résultats mettent en lumière l'effet domino initié par les NETs. Les cellules B activées par les NETs agissent ensuite comme CPA et déclenchent l'activation des cellules T, renforçant ainsi la réponse immunitaire adaptative. Les cellules B activées par les NETs induisant également le recrutement et l'activation des neutrophiles. Cette interaction potentielle met en évidence la nature polyvalente des NETs au-delà de leur rôle conventionnel dans la défense antimicrobienneActivated neutrophils (PMNs) expel neutrophil extracellular traps (NETs). NETs serve as adefense mechanism against pathogens. However, the role of NETs extends beyond their antimicrobial function, with implications in various physiological and pathological processes, including autoimmune disorders. Increased NET formation has been reported in rheumatoid arthritis (RA); a chronic inflammatory disease affecting joints. We have previously shown that NETs are pro-inflammatory on resting macrophages. Indeed, NETs contain several molecules with immunostumulatory properties. We suggest that NETs act as damage-associated molecular patterns (DAMPs) on B lymphocytes inducing their polyclonal activation, independently of antigen specificity; a new mechanism by which NETs might contribute to immune dysregulation, particularly in the context of RA. We demonstrate by flow cytometry, ELISA and RNA sequencing (RNAseq) that NETs could directlyactivate total and naïve B cells toward a robust pro-inflammatory profile, characterized by high productionof pro-inflammatory cytokines and the upregulation of HLA-DR and the co-stimulatory molecules CD40and CD86 which are important for antigen presenting cell (APC) function. This activation is amplified in RA patients. We show also that this mechanism is modulated by the presence of C1q and LL-37 proteins, and didn't required the Toll-like receptor 9 (TLR9). Beyond B cell activation, our findings shed light on the domino effect initiated by NETs. NET-activated B cells subsequently act as APCs and trigger T cell activation, bolstering the adaptive immune response. NET-activated B cells also induce the recruitment andactivation of neutrophils. This potential crosstalk highlights the versatile nature of NETs beyond their conventional role in antimicrobial defense
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Cleaton-Roberts, Melanie. "Development and characterisation of polyclonal and monoclonal antibodies raised against cathepsin K." Thesis, University of Liverpool, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.250227.
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Phillips, Christopher. "Production and assessment of ovine polyclonal antibodies to treat Clostridium difficile Infections." Thesis, Cardiff Metropolitan University, 2016. http://hdl.handle.net/10369/8323.
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The clinical manifestations of Clostridium difficile infections (CDI) are due to the release of two powerful exotoxins, TcdA and TcdB. The aim of this study was to raise polyclonal antibodies (PcAb) in sheep that bind to and neutralise these toxins with a view to developing potent therapeutic agents for use in humans. Dose response studies were performed with recombinant fragments of each of the toxins, TxA4 and TxB4, in separate flocks of sheep. The PcAb produced showed high binding titres in an immunoassay and protected Vero cells against the two natural toxins in-vitro. TcdB was considerably more cytotoxic than TcdA with LC50 of 16 pg/mL and 1000 pg/mL respectively. The TxA4 immunogen stimulated a greater toxin neutralising immune response than TxB4. Thus, 1 mL of anti-TxA4 sera neutralised 1,800 μg of natural TcdA, whereas 1 mL of anti-TxB4 neutralised only 15 μg of TcdB. There was no correlation between binding titre and neutralising potency, suggesting that toxin inactivation was dependant on PcAb binding to specific regions on the natural toxins. Affinity purified PcAb, directed against distinct regions of TcdB, were fractionated and those directed to the central region possessed neutralising potencies of up to 250% greater than those directed elsewhere. These investigations identified the quantity and potency of specific PcAb directed to each of three distinct TcdB regions. A novel cell based assay, simulating the colonic epithelial barrier, was developed to investigate the translocation of TcdA and TcdB from simulated colonic lumen to the systemic circulation and to determine the effects of various toxin neutralising strategies. TcdA was shown to cause colonic epithelial damage allowing TcdB to translocate to the systemic circulation. Addition of neutralising PcAb either directly to simulated colonic lumen or systemic circulation provided protection to the intestinal epithelial layer and reduced or prevented toxin translocation.
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Howlett, Jay Richmond Carleton University Dissertation Biology. "Enhancement of immunoaffinity chromatography processes using polyclonal antibodies and high pressure elution." Ottawa, 1993.
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Fujita, Kohei. "Association between polyclonal and mixed mycobacterial Mycobacterium avium complex infection and environmental exposure." Kyoto University, 2014. http://hdl.handle.net/2433/188673.
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Ozimek, Paulina. "Pooling of rabbit antisera to reduce lot to lot variability of polyclonal antibodies." Thesis, KTH, Skolan för bioteknologi (BIO), 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-215021.
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Al-Fadhli, Suad M. "Characterization of site-directed monoclonal and polyclonal antipeptide antibodies to human estrogen receptor." Thesis, Boston University, 1994. https://hdl.handle.net/2144/37996.
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Thesis (M.A.)--Boston UniversityPLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you.
Monoclonal and polyclonal antibodies were developed against a synthetic peptide derived from a sequence in the A/B transactivation domain (residues 140-154) of the human estrogen receptor (hER). These antibodies were characterized with respect to site specificity, receptor specificity and species specificity and ability to bind to the van ous receptor forms. The antibodies were site specific since binding to estrogen receptor (ER) was displaced with the free peptide. The antibodies did not recognize progestrone, and androgen receptors. The antibodies cross-reacted with the ER from calf, rat and mouse uteri and human breast tumors, suggesting that the epitope for these monoclonal antibodies (MAbs) is conserved among vanous spectes. The interaction of the antibody with the functional estrogen receptor has been characterized by sucrose density gradient analysis. The MAbs did not recognize the molybdate-stabilized, untransformed, oligomeric (8S), suggesting that this epitope may be masked by interaction with heat shock proteins or conformationaly inaccessible. The antibodies reacted with the activated (4S) and the transformed (5S) forms of the ER, suggesting that the epitope is accessible in the 4S and the 5S forms of the ER and that dimerization of the 4S ER into 5S ER does not interfere with the epitope accessibility. Immunoblot analysis using polyclonal and monoclonal antibodies demonstrated cross-reactivity with a 55 kDa nuclear protein (NP55). This protein was detected only in the nuclear-KCI extracts but not the cytosolic extracts . It lacked estradiol binding activity, and is expressed in steroid-hormone target tissues from different species. This protein was also detected in some ER positive human breast tumors but was not detected in ER negative tumors. This protein (NP55) was not detected with polyclonal or monoclonal anti bodies developed against different regions of the ER, indicating that it is not a proteolyzed form of the ER. The availability of site-directed antibodies to ER-functional domains represent a valuable tool, particularly, m studying ER in its different forms, and in the analyses of the structural integrity of human breast tumor estrogen receptors. The observation that one of these antibodies recognizes a umque nuclear protein may prove useful to identify this ER-related protein.
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Poirier, David. "Évaluation du pouvoir discriminatif d’anticorps créés à partir de peptides synthétiques issus de gluten de blé, d’orge, de seigle et d’avoine." Master's thesis, Université Laval, 2021. http://hdl.handle.net/20.500.11794/68974.
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Les symptômes de la maladie cœliaque sont déclenchés par l'ingestion de céréales contenant du gluten comme le blé, l'orge, le seigle et, dans de rares cas, l'avoine. Afin d’éviter ceux-ci, les personnes touchées doivent adopter un régime sans gluten. Pour être déclaré sans-gluten, un aliment ne doit pas contenir plus de 20 mg/kg de gluten. Ainsi, ces denrées doivent être analysées afin de garantir leur innocuité. Actuellement, des tests immunochimiques basés sur la reconnaissance de séquences protéiques de gluten sont utilisés. Cependant, des problèmes de sur- et de sous-estimation de la teneur exacte en gluten surviennent en fonction de la source de gluten analysée, c’est-à-dire selon la céréale. En cas de surestimation, cela réduit l’offre alimentaire des personnes cœliaques, mais en cas de sous-estimation, de graves conséquences affectent cette population. De plus, la réglementation canadienne exige une déclaration de la source du gluten sur l'étiquetage des produits préemballés, ce qui ne peut être réalisé adéquatement avec les moyens actuels. Ce travail porte sur le développement de nouveaux anticorps visant à discriminer les sources de gluten en utilisant des peptides synthétiques comme stratégie d'immunisation. Pour ce faire, 14 peptides synthétiques sélectionnés de séquences protéiques de gluten ont chacun été bioconjugués à une protéine porteuse et l'immunisation de lapins a été réalisée. Les anticorps polyclonaux (pAbs) résultants distinguent avec succès le gluten de blé, d'orge et d'avoine. Les pAbs dirigés contre le seigle réagissent également avec le gluten de blé et de seigle. Les résultats obtenus démontrent que la discrimination des sources de gluten peut répondre à la législation canadienne, mais aussi complémenter les tests immunochimiques actuels en nivelant les problèmes de sur- et sous-estimation de la teneur en gluten et améliorer davantage la sécurité des aliments destinés aux patients cœliaques.Symptoms of celiac disease are triggered by ingesting grains that contain gluten like wheat, barley, rye, and, in rare cases, oats. In order to avoid symptoms, those affected should adopt a gluten-free diet. To be declared gluten-free, a food must not contain more than 20 mg / kg of gluten. Thus, these foods must be analyzed in order to guarantee their safety. Currently, immunoassays based on the recognition of gluten protein sequences are used. However, problems of overestimating and underestimating of the exact gluten content arise depending on the type of gluten being analyzed (wheat, barley, rye or oat). If gluten is overestimated, this reduces the food supply of celiac patients, but if underestimated, serious consequences then affect this population. In addition, Canadian regulations require a declaration of the source of gluten on the labelling of prepackaged products, which cannot be done adequately with current means. This work focuses on the development of new antibodies aimed at discriminating between sources of gluten using synthetic peptides as immunization strategy. To proceed, 14 synthetic peptides selected from gluten protein sequences were each bioconjugated to a carrier protein and rabbit immunization was performed. The resulting polyclonal antibodies (pAbs) successfully distinguish gluten from wheat, barley and oats. Rye pAbs react evenly with wheat and rye gluten. The results obtained demonstrate that the discrimination of gluten sources can meet Canadian legislation, but also complement current immunoassays by levelling the problems of over and underestimation of gluten content and further improve the safety of food intended for celiac patients.
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Wong, Raymond, and 黃偉文. "Generation and characterization of polyclonal antibodies specific to the mouse homeodomain protein HOXB-3." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 1999. http://hub.hku.hk/bib/B31221968.
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O'Hehir, Robyn Elizabeth. "Polyclonal and monoclonal analysis of the human T lymphocyte immune response to Dermatophagoides spp." Thesis, Imperial College London, 1989. http://hdl.handle.net/10044/1/47596.
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Xia, Jing. "Immunocytochemical localisation of arabinoxylans in the wheat (Triticum aestivum) caryopsis using a polyclonal antibody." Thesis, University of Cambridge, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.396093.
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Canelle, Quentin. "Real Time Surface Plasmon Resonance Biosensors, a Powerful Technology to Assess Polyclonal Antibody Avidity." Doctoral thesis, Universite Libre de Bruxelles, 2015. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/216754.
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The present research focused on the development of a new methodology to assess the strength of the interaction between vaccine antigens and elicited polyclonal antibodies through SPR biosensors. Quantifying the binding strength of polyclonal antibodies is of first importance to evaluate the quality of the vaccine as well as to increase the scientific knowledge of immune protection mechanisms. To now the development of such tool has been complicated by the non-specific binding caused by high protein abundance in the blood and serum samples but also by the way of interpreting the data resulting from multi-interaction events measured at the same time. At first, we unsuccessfully tried to segregate the individual affinity contribution of each antibody population by measuring the signal as the sum of singular interactions. Differentiation of the singular contribution would have needed the fulfillment of the “additivity” hypothesis, meaning that each antibody bind identically alone or in mixture with other antibody. This hypothesis was not met and mathematical assessment by the sum of singular contribution led to fitting results that did not reflect the biological reality. It was therefore decided to switch the analysis method and to measure the end association binding level reached by the different samples injected at the same specific antibody content. The dissociation behavior was interpreted by the percentage of binding after long and fixed dissociation time. In a first application, we compared the antibodies elicited by two different commercially available vaccines and we showed that the binding interaction was not concentration dependent as, highly different levels were reached when injecting identical antibody concentration. No statistical significant difference was observed between both vaccines. Research firstly focused on the decrease of the non-specific binding and we found that ionic strength was a key parameter, increasing the buffer salt concentration reduced the non-specific binding without diminishing the binding strength. The sample composition was also a key parameter and purifying the IgG allowed to decrease dramatically the undesired binding events. A second application aimed at showing the equivalence between two different antigen constructions for two antibodies population. Even if identical antigen level immobilization is a challenge, the methodology is completely suitable to perform a 2-dimensional comparison (ligand and analyte). A last application was dedicated to the comparison between D and Q-pan Flu vaccines, and results showed that there was no statistical evidence of significant differences between both vaccines. End association level correlated well with haemagglutination inhibition assay at least when serum samples were not diluted at the same antibody content. This last application also showed that throughput may be extended to more than 50 samples per 80 hoursDoctorat en Sciences agronomiques et ingénierie biologique
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Lynch, Brittany. "Investigating the therapeutic potential of polyclonal antibodies in a murine model of allergic asthma." Thesis, Lynch, Brittany (2012) Investigating the therapeutic potential of polyclonal antibodies in a murine model of allergic asthma. Honours thesis, Murdoch University, 2012. https://researchrepository.murdoch.edu.au/id/eprint/41661/.
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Using the OVA mouse model of allergic airway disease, a histological grading system was established to evaluate four cardinal features of allergic asthma: Perivascular Inflammatory Cuffing, Peribronchial Inflammatory Cuffing, Goblet Cell Hyperplasia, and Tissue Eosinophilia. Using histochemical methods, this grading system was applied to identify a shallow dose response between allergen dose and resultant histopathology. This trend reached maximal effect with low doses of allergen and was observed in both perivascular and peribronchial cuffing thickness. A degree of goblet cell hyperplasia was observed in response to a low dose of allergen administration, but the dose response tended to plateau over the mid-range doses of allergen. Tissue eosinophilia was increased response to higher doses of allergen. In an investigation of the immune exclusionary capacity of Ruminant Polyclonal Antibodies (RPA), the histological grading system was applied to evaluate whether histopathological changes could be influenced or decreased by polyclonal antibodies. A non-specific action of allergen-specific Immunoglobulin G was identified. A pro-inflammatory tendency of an antibody:allergen complex was identified, and lung inflammation occurred as a result. These studies indicate the possibility that antibodies are able to act in an exclusionary manner at mucosal surfaces.
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Rai, Deepa Kumari. "Tracking total polyclonal CD8 T cell responses in inbred and outbred hosts after infection." Thesis, University of Iowa, 2010. https://ir.uiowa.edu/etd/877.
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Misaghi, Ali. "Development of monoclonal and polyclonal antibodies to the flagellar and somatic antigens of Bacillus cereus." Thesis, University of Strathclyde, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.248721.
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Figueroa, Z. E. F. "Specificity and protective effect of polyclonal antibodies to antigens of Plasmodium berghei and Plamodium chabaudi." Thesis, Open University, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.304217.
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Roustiau-Sévère, Sylvie. "Préparation et caractérisation d'un anticorps polyclonal antipeptide spécifique des jonctions "Gap" du myocarde du rat." Aix-Marseille 2, 1987. http://www.theses.fr/1987AIX22050.
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Un peptide synthetique correspondant a la region n terminale hydrophile du polypeptide jonctionnel myocardique (28kda) est couple a une proteine porteuse. Des lapins sont immunises, les igc antipeptide sont purifiees par chromatographie d'affinite. Ces anticorps reconnaissent specifiquement le polypeptide de 28 kda sous forme de jonctions gap. Les resultats obtenus en immunoreplique sur des jonctions gap purifiees indiquent que la proteine jonctionnelle cardiaque native a un pm de 43 kda et que la partie amino-terminale de cette proteine (28 kda) est cytoplasmique
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Petit, Robert G. "The development of polyclonal antisera which inhibits purified Epstein-Barr virus (B95-8) DNA polymerase /." The Ohio State University, 1987. http://rave.ohiolink.edu/etdc/view?acc_num=osu148732966214544.
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Roustiau-Sévère, Sylvie. "Préparation et caractérisation d'un anticorps polyclonal antipeptide spécifique des jonctions "Gap" du myocarde de rat." Grenoble 2 : ANRT, 1987. http://catalogue.bnf.fr/ark:/12148/cb376095645.
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Hedeen, Heather A. "A Kinetic Study of anti-VEGF-A Polyclonal Antibodies and anti-VEGF-A ssDNA Aptamers." DigitalCommons@CalPoly, 2012. https://digitalcommons.calpoly.edu/theses/754.
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A new detection reagent that could possibly augment or replace antibodies research and diagnosis methods are aptamers. Aptamers are ssDNA, RNA or polypeptide constructs that function like active antibodies. Antibodies and aptamers both specifically bind to selected target molecules, and as such they enable the detection or targeting of the presence or absence of a specific antigen.
In order to ensure that ssDNA aptamers perform similarly to antibodies, anti-VEGF-A polyclonal antibody and anti-VEGF-A ssDNA aptamer were evaluated against vascular endothelial growth factor A (VEGF-A) using Surface Plasmon Resonance (SPR). It was hypothesized that the anti-VEGF-A aptamer had the same, if not better, binding kinetics than the anti-VEGF-A polyclonal antibody, and as such offers an ideal replacement for use in in field, real-time testing assays.
SPR revealed that both the polyclonal antibody and ssDNA aptamer bound the target antigen, VEGF-A. Additionally, from the SPR kinetic analysis, the anti-VEGF-A aptamer had KD values of 20-28 nM and the anti-VEGF-A antibody had KD values of 16-127 uM. The binding efficacy of the aptamer was several orders of magnitude better than that of the antibody. The aptamer was also stable in solution for a longer amount of time than the antibody, which denatured in solution after two weeks.
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Carvalho, Carlos Eduardo Brantis de [UNESP]. "Caracterização funcional da proteína MxA: estudo do seu envolvimento com a mauinaria de SUMOilação." Universidade Estadual Paulista (UNESP), 2014. http://hdl.handle.net/11449/110838.
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000777406.pdf: 2095617 bytes, checksum: c1c25558e3e61efd44b8465985d10f95 (MD5)A proteína MxA humana é membro da superfamília de GTPases dinaminas. De maneira geral, as chamadas proteínas Mx estão presentes na grande maioria dos organismos vertebrados, estudados até o momento, e possuem dois domínios estruturais, chamados GTPase e CID-GED (stalk), além da capacidade de homooligomerização e associação com membranas intracelulares. Além disso, as proteínas Mx são produzidas unicamente após a sensibilização celular por interferons do tipo I e III. Entre as propriedades funcionais de MxA, destaca-se a sua vasta atividade contra diferentes vírus de RNA e DNA, incluindo o vírus influenza e membros da família dos buniavírus. Além disso, o silenciamento gênico de MxA está associado ao fenótipo de imortalização celular em uma série de neoplasias. Assim, MxA desperta o interesse por ser uma das proteínas chave nas respostas mediadas por interferons e por estar envolvida no controle da proliferação celular. Recentemente, por meio de rastreamento de duplo-híbrido, utilizando uma biblioteca de cDNA preparada a partir de cérebro fetal humano, foi possível mostrar que MxA interage com fatores envolvidos no processo de SUMOilação de proteínas e na formação de corpúsculos nucleares denominados PMLNB e com uma série de proteínas relacionadas com o controle da transcrição e apoptose. Neste estudo, foi investigada a interação entre MxA e as proteínas envolvidas nos processo de SUMOilação. Assim, foi possível confirmar a interação física entre a proteína MxA e os ligantes Ubc9 e SUMO1, por ensaios de coimunoprecipitação. A seguir, através de ensaios de duplo-híbrido, foi possível determinar que a região EIL (E67-interacting Loop) de SUMO1 e o domínio CID-GED de MxA estão envolvidos na interação entre essas proteínas e que esta interação independe de sequências SIM (SUMO-interacting motif) presentes em MxA. Ainda, foi possível determinar que Ubc9 interage diretamente com ...
The human MxA protein is a member of the superfamily of GTPases dynamins. The so called Mx proteins are present in the majority of the vertebrate organisms investigated so far and contain two structural domains, named GTPase and CID-GED (stalk), besides the capabilities of homo-oligomerization and association with intracellular membranes. Moreover, the Mx proteins are strictly produced upon cell sensibilization with type I and III interferons. The vast antiviral activity against RNA and DNA viruses, including the Influenza virus and members of the bunyaviridae family, is among the functional properties of MxA. Moreover, MX1 epigenetic silencing is associated with cellular immortalization in neoplasias. Therefore, the study of MxA is of great interest as it is a key component of the Interferon-mediated pathways and cell proliferation control. Recentely, in a two-hybrid screen using a fetal brain cDNA library, it was possible to reveal that MxA interacts with proteins related to the post-translational modification process named SUMOylation and to the assemble of the nuclear bodies named PML-NBs and with proteins implicated in the control of transcription and apoptose. In this study, it was investigated the interaction between MxA and the components of the protein SUMOylation pathway. It was possible to confirm the physical interaction between MxA and Ubc9 and SUMO1, using co-immunopreciptation assay. Then, using the yeast two-hybrid system, it was possible to determine that the EIL (E67-interacting loop) region on SUMO1 interacts with the CID-GED domain of MxA without the requirement of the SIM sequences (SUMO-interacting motif) present in MxA. Moreover, it was determined that Ubc9 interacts with the GTPase domain of MxA and that MxA homo-oligomerization is important for its interaction with SUMO1 and Ubc9. Also, we were able to demonstrate for the first time that the protein MxA undergoes SUMOylation by SUMO1, SUMO2 and SUMO3. Finally, it ...
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Carvalho, Carlos Eduardo Brantis de. "Caracterização funcional da proteína MxA : estudo do seu envolvimento com a mauinaria de SUMOilação /." Araraquara, 2014. http://hdl.handle.net/11449/110838.
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Orientador: Sandro Roberto ValentiniCo-orientador: Cleslei Fernando Zanelli
Banca: Alexandra Ivo de Medeiros
Banca: Andréia Machado Leopoldino
Banca: Mari Cleide Sogayar
Banca: Érico Tosoni Costa
Resumo: A proteína MxA humana é membro da superfamília de GTPases dinaminas. De maneira geral, as chamadas proteínas Mx estão presentes na grande maioria dos organismos vertebrados, estudados até o momento, e possuem dois domínios estruturais, chamados GTPase e CID-GED (stalk), além da capacidade de homooligomerização e associação com membranas intracelulares. Além disso, as proteínas Mx são produzidas unicamente após a sensibilização celular por interferons do tipo I e III. Entre as propriedades funcionais de MxA, destaca-se a sua vasta atividade contra diferentes vírus de RNA e DNA, incluindo o vírus influenza e membros da família dos buniavírus. Além disso, o silenciamento gênico de MxA está associado ao fenótipo de imortalização celular em uma série de neoplasias. Assim, MxA desperta o interesse por ser uma das proteínas chave nas respostas mediadas por interferons e por estar envolvida no controle da proliferação celular. Recentemente, por meio de rastreamento de duplo-híbrido, utilizando uma biblioteca de cDNA preparada a partir de cérebro fetal humano, foi possível mostrar que MxA interage com fatores envolvidos no processo de SUMOilação de proteínas e na formação de corpúsculos nucleares denominados PMLNB e com uma série de proteínas relacionadas com o controle da transcrição e apoptose. Neste estudo, foi investigada a interação entre MxA e as proteínas envolvidas nos processo de SUMOilação. Assim, foi possível confirmar a interação física entre a proteína MxA e os ligantes Ubc9 e SUMO1, por ensaios de coimunoprecipitação. A seguir, através de ensaios de duplo-híbrido, foi possível determinar que a região EIL (E67-interacting Loop) de SUMO1 e o domínio CID-GED de MxA estão envolvidos na interação entre essas proteínas e que esta interação independe de sequências SIM (SUMO-interacting motif) presentes em MxA. Ainda, foi possível determinar que Ubc9 interage diretamente com ...
Abstract: The human MxA protein is a member of the superfamily of GTPases dynamins. The so called Mx proteins are present in the majority of the vertebrate organisms investigated so far and contain two structural domains, named GTPase and CID-GED (stalk), besides the capabilities of homo-oligomerization and association with intracellular membranes. Moreover, the Mx proteins are strictly produced upon cell sensibilization with type I and III interferons. The vast antiviral activity against RNA and DNA viruses, including the Influenza virus and members of the bunyaviridae family, is among the functional properties of MxA. Moreover, MX1 epigenetic silencing is associated with cellular immortalization in neoplasias. Therefore, the study of MxA is of great interest as it is a key component of the Interferon-mediated pathways and cell proliferation control. Recentely, in a two-hybrid screen using a fetal brain cDNA library, it was possible to reveal that MxA interacts with proteins related to the post-translational modification process named SUMOylation and to the assemble of the nuclear bodies named PML-NBs and with proteins implicated in the control of transcription and apoptose. In this study, it was investigated the interaction between MxA and the components of the protein SUMOylation pathway. It was possible to confirm the physical interaction between MxA and Ubc9 and SUMO1, using co-immunopreciptation assay. Then, using the yeast two-hybrid system, it was possible to determine that the EIL (E67-interacting loop) region on SUMO1 interacts with the CID-GED domain of MxA without the requirement of the SIM sequences (SUMO-interacting motif) present in MxA. Moreover, it was determined that Ubc9 interacts with the GTPase domain of MxA and that MxA homo-oligomerization is important for its interaction with SUMO1 and Ubc9. Also, we were able to demonstrate for the first time that the protein MxA undergoes SUMOylation by SUMO1, SUMO2 and SUMO3. Finally, it ...
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Bhagwat, Bhagyashree Yogesh. "Design and synthesis of aryl hydrocarbon receptor fusion proteins for polyclonal antibodies production and cellular delivery." Scholarly Commons, 2001. https://scholarlycommons.pacific.edu/uop_etds/558.
Full textAbstract:
Polycyclic aromatic hydrocarbons are environmental chemicals that are produced during incomplete combustion of coal, oil, gas, and garbage. Toxic effects of these compounds are mediated via the ligand activated Aryl Hydrocarbon Receptor (AHR) signaling pathway. To enable the study of the AHR signaling mechanism, our lab has generated many human proteins using recombinant DNA technology. This thesis documents the design and synthesis of a number of proteins of the AHR deletion construct CΔ553. The bacterial expressed and purified fusion proteins could be utilized as antigen to generate antibodies and be used for cellular delivery. The purified protein was immunogenic in rabbits and produced significant amount of polyclonal antibodies. In western blot analysis, the antibodies were able to the detect baculovirus expressed AHR and different recombinant proteins of the AHR. The polyclonal antibodies were also used in the gel-shift assay to show the AHR dependent gel shift. Cellular delivery CΔ553 was achieved using the protein transduction domain from the HIV-1 virus transactivating protein (TAT). In order to deliver the CΔ553 into mammalian cells, an expression vector was constructed to generate the TAT-CΔ553 fusion protein. The TAT-CΔ553 fusion protein was successfully transduced into two mammalian cells-HeLa and HepG2. The in vivo function of TAT-CΔ553 was determined using the luciferase reporter plasmid assay. The transduced protein was functional; it competed with the AHR and heterodimerize with ARNT in both HeLa and HepG2 cells at a concentration of 3.8x103 nM and 18 nM respectively. Since there an apparent similarity between the basic region of TAT-PTD and CΔ553, we examined the transduction potential of CΔ553. Western blot analysis indicated that the extent of denatured protein transduction was comparable for CΔ553 and TAT-CΔ553 in HepG2 cells. Thus CΔ553 might have intrinsic transduction capability.