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Yang, Jie. "Characterization of bovine granzymes and studies of the role of granzyme B in killing of Theileria-infected cells by CD8+ T cells". Thesis, University of Edinburgh, 2012. http://hdl.handle.net/1842/6487.
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Previous studies have shown that cytotoxic CD8+ T cells are important mediators of immunity against the bovine intracellular protozoan parasite T. parva. The present study set out to determine the role of granule enzymes in mediating killing of parasitized cells, first by characterising the granzymes expressed by bovine lymphocytes and, second, by investigating their involvement in killing of target cells. Experiments using the perforin inhibitor concanamycin A confirmed that CD8+ T cell killing of T. parva-infected cells is dependent on granule exocytosis, a process that involves release of granzymes into the target cell, resulting in activation of apoptotic pathways. Analysis of the bovine genome sequence identified orthologues of granzymes A, B, H, K and M, as well as another gene O, most closely related to granzyme A. The genes were found within 3 loci in the genome. Using specific PCR assays, all of these granzymes were shown to be expressed in Theileria-specific CD8+ T cells. Further studies were undertaken to study the role of granzyme B in killing. DNA constructs encoding functional and non-functional forms of bovine granzyme B were produced and the proteins expressed in COS cells were used to establish an enzymatic assay to detect and quantify expression of functional granzyme B protein. Using this assay, the levels of killing of different T. parvaspecific CD8+ T cell clones were found to be significantly correlating with levels of granzyme B protein expression. Moreover, the granzyme B inhibitor III, Z-IETDFMK was shown to inhibit killing by CD8+ T cell clones.
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Tinangon, Maria M. "Strategies to identify granzyme J /". abstract and full text PDF (UNR users only), 2001. http://0-gateway.proquest.com.innopac.library.unr.edu/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:1404986.
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Koot, Gretchen E. "Serine and cysteine protease inhibitors for blockade of cell mediated cytotoxicity /". abstract and full text PDF (UNR users only), 2002. http://0-gateway.proquest.com.innopac.library.unr.edu/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3121138.
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Ben, Safta Thouraya. "Implication de la protéine suppresseur de tumeurs p53 dans la mort cellulaire induite par les lymphocytes T cytotoxiques et les cellules NK : rôle dans la régulation de l’apoptose dépendante du granzyme B". Thesis, Université Paris-Saclay (ComUE), 2017. http://www.theses.fr/2017SACLS162/document.
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Les lymphocytes T cytotoxiques (CTL) et les cellules tueuses naturelles (NK) éliminent leurs cellules cibles tumorales grâce à l’exocytose et à la libération du contenu des granules cytotoxiques contenant une protéine formant des pores, appelée perforine (PFN), et une famille de serine-protéases induisant la mort cellulaire, appelés granzymes (Gzms). Ces Gzms, pénètrent dans les cellules cibles de manière dépendante de la PFN et activent diverses voies de signalisation apoptotiques aboutissant à la mort de la cellule cible. Au cours de ce travail, nous avons étudié le rôle de la protéine suppresseur de tumeurs p53 dans la cascade moléculaire conduisant à l’apoptose induite par les effecteurs cytotoxiques via la voie PFN/Granzyme B (GzmB). Nous avons ainsi pu montrer qu’en réponse au GzmB ou à des effecteurs cytotoxiques, la forme sauvage de p53 s’accumule dans les cellules cibles au niveau des mitochondries afin d’interagir avec la protéine anti-apoptotique Bcl-2 et de réguler positivement la perméabilisation de la membrane mitochondriale externe induite par le GzmB. L’activité non transcriptionelle de p53 au niveau des mitochondries joue donc un rôle clé dans le contrôle de l’apoptose induite par les CTL et les NK (Ben Safta et al. J Immunol 2015). Etant donné que le gène TP53 est muté dans plus de 50% des tumeurs humaines, nous avons également cherché à déterminer si la restauration d’une p53 sauvage dans des cellules tumorales portant une p53 non fonctionnelle pourrait potentialiser la réponse cytotoxique antitumorale. Nos résultats montrent qu'effectivement, la réactivation pharmacologique de l’activité sauvage de p53 dans une lignée d'adénocarcinome mammaire possédant une p53 mutée sensibilise ces cellules tumorales à la lyse induite par les cellules NK via l’activation d’un processus d’autophagie et d’une cascade d’événements moléculaires qui sont en cours d’identificationCytotoxic T lymphocytes (CTL) and natural killer (NK) cells eliminate their tumor target cells through exocytosis and release of the cytotoxic granules (CG) content. These CG contain a pore-forming protein called perforin (PFN), and a family of cell death inducing serine-proteases, called granzymes (Gzms). Gzms enter the target cells in a PFN-dependent manner and activate various apoptotic signaling pathways leading to the death of the target cell. In this work, we studied the role of the tumor suppressor protein p53 in the molecular cascade leading to apoptosis induced by cytotoxic effectors via the PFN/Granzyme B (GzmB) pathway. We have shown that in response to GzmB or to cytotoxic effectors, wild-type p53 accumulates on target mitochondria in order to interact with the anti-apoptotic protein Bcl-2 and to positively regulate the GzmB-induced mitochondrial outer membrane permeabilization. Thus, the non-transcriptional activity of p53 in the mitochondria plays a key role in the control of apoptosis induced by CTL and NK (Ben Safta et al J Immunol 2015). Because the TP53 gene is mutated in more than 50% of human tumors, we also aimed to determine whether the restoration of a wild-type p53 fonction in tumor cells carrying a non-functional p53 could potentiate the cytotoxic antitumor response. Our results show that the pharmacological reactivation of a wild-type p53 activity in a mammary adenocarcinoma cell line harboring a mutated p53 sensitize these tumor cells to the NK cell lysis via the activation of autophagy and a cascade of molecular events that are being identified
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LAFAURIE, CLOTILDE. "Etude de la regulation de la transcription et de l'expression des genes des granzymes b et h humains". Paris 11, 1997. http://www.theses.fr/1997PA112327.
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Les granzymes sont une famille de serine esterases exprimee de facon specifique dans les lymphocytes t actives. Nous avons etudie la regulation de l'expression des granzymes b et h humains. Deux axes ont ete choisis : la caracterisation des evenements genetiques necessaires a la regulation de l'expression de ces deux genes dans les lymphocytes t actives et l'etude de l'expression des proteines codees par ces deux genes dans differentes populations cellulaires. Nous avons caracterise un promoteur minimal qui controlait specifiquement la transcription du gene du granzyme b dans les lymphocytes t cytotoxiques. Ce promoteur contient des sites specifiques des proteines ap-1, cbf et ikaros qui sont necessaires a l'activation de la transcription du gene du granzyme b. Nous avons utilise ce modele pour etudier le mecanisme utilise par la dexamethasone pour inhiber l'expression du gene du granzyme b. Nous avons mis en evidence que cette inhibition etait transcriptionnelle et impliquait l'inhibition de la liaison des facteurs de transactivation sur les sites ap-1 et ikaros. Nous avons montre que la dexamethasone etait capable d'inhiber l'expression nucleaire des isoformes actives d'ikaros. Nous avons caracterise une region d'adn en amont du site de l'initiation de la transcription du gene du granzyme h responsable de l'expression specifique de ce gene dans les cellules yt-2c2. Cette region est capable de lier une proteine nucleaire specifiquement exprimee dans les noyaux des cellules yt-2c2. Nous avons montre que le granzyme b etait exprime dans des cellules non t. Nous avons produit un anticorps monoclonal dirige contre le granzyme h afin d'etudier son expression.
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Mouchacca, Pierre. "Granzyme B-td TOMATO, un nouvel outil fluorescent pour le suivi de la cytolyse chez la souris". Thesis, Aix-Marseille, 2012. http://www.theses.fr/2012AIXM4008/document.
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La fonction de cytolyse est un mécanisme majeur des effecteurs du système immunitaire pour éliminer les cellules infectées ou tumorales. Cette fonction associe l'activité de la perforine, qui forme des pores dans la membrane d'une cellule cible, à la sécrétion de protéases: les granzymes. Ces dernières sont des molécules pro-apoptotiques qui induisent la mort de la cellule cible. Les granzymes et en particulier granzyme B ciblent plusieurs voies intracellulaires complémentaires pour assurer l'efficacité de la cytolyse. Or il est difficile d'observer directement la fonction de cytolyse au cours de réponse immunitaire in vivo dans des conditions physiologiques. Dans les travaux présentés dans cette thèse, nous avons développé un nouveau modèle qui permet de suivre la fonction de cytolyse en temps réel par l'expression d'une protéine de fusion fluorescente GZMB-tdTomato. Les résultats obtenus par expression rétrovirale ont montré que la protéine de fusion est correctement exprimée dans les vésicules cytolytiques qui deviennent fluorescentes. Dans un second temps, nous avons réalisé un nouveau modèle murin qui exprime GZMB-tdTomato de manière substituée au GZMB natif par recombinaison homologue (Knock In). Nous avons mis en évidence que la protéine de fusion conserve l'activité catalytique de la protéine native et ses caractéristiques (conditions d'expression, de maturation, de sécrétion et demeure active après le passage dans la cellule cible lors de la cytolyse). En utilisant un modèle murin exprimant un TCR transgénique nous avons pu suivre le déroulement de la fonction de cytolyse de lymphocytes cytotoxiques en temps réel par video microscopiesCytolysis is a major function used by the immune system's effectors to kill infected or tumor cells. Cytolysis depends on the pore forming protein perforin and the secretion of proteases of the granzyme family. Granzymes, including granzyme B (GZMB) have pro-apoptotic features and induce target cell death. Several complementary pathways are triggered by granzymes to ensure efficient cytolysis. It remains difficult to directly observe cytolysis during in vivo immune responses under physiological conditions. In this PhD we developed a new model to visualize cytolytic function in real time by expression of a fusion protein: GZMB-tdTomato. Results obtained from retroviral transduction showed that the fusion protein is correctly expressed in cytolytic vesicles, which became fluorescent. We then constructed a new mouse model by homologous recombination (Knock In) that express GZMB-tdTomato substituted for the native GZMB. The fusion protein conserves the catalytic activity of GZMB and its features (expression, maturation, secretion conditions) and remains active after its passage into target cells. Using TCR transgenic OTI cells, we followed the sequence of events of cytolysis from lymphocytes in real time by videomicroscopy. We also observed the cytolytic vesicles relocalization towards the cell contact zone and the death of target cell by cytolysis. Finally, we studied in vivo differentiation of naïve lymphocyte to cytolytic effector cells (the acquisition of cytolysis) and target cell death after bacterial infection
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Sumaria, Nital. "The relevance of specific molecular and cellular effectors during murine cytomegalovirus infection". University of Western Australia. School of Biomedical, Biomolecular and Chemical Sciences, 2008. http://theses.library.uwa.edu.au/adt-WU2008.0116.
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[Truncated abstract] The design and development of effective anti-viral immunotherapies requires a comprehensive understanding of the cellular and molecular processes that are involved in the generation and regulation of immune responses. The fundamental objective of the immune system is to successfully complete the task of eliminating/controlling the invading pathogen without causing overt pathology. Cytomegaloviruses (CMVs) are large DNA viruses that are able to evade immune attack and persist lifelong within the host. In a healthy host, CMV causes an asymptomatic infection, but in instances of decreased immune functions, such as in newborns, acquired immunodeficiency syndrome (AIDS) patients and transplant recipients, the infection can result in serious morbidity and mortality. Thus, human CMV (HCMV) is a clinically important pathogen and an understanding of the pathogenesis, mechanisms of immune subversion and, importantly the cascade of immune events that ensue following infection is highly relevant. The studies presented in this thesis have provided useful insight into various aspects of viral immunity and it is hoped that they will assist in the design of more effective therapies against viruses of clinical importance. Genetic variability in humans can greatly influence anti-viral immune responses and the outcome of viral infection. ... Furthermore, these studies provide novel evidence that NK cells are also crucial for the control of virus in some organs of susceptible mice during early acute infection. The data reveals that both NK cells and CD8+ T cells utilise perforin- and IFN-? dependent control of MCMV. Furthermore, these studies provide novel evidence that protection mediated by Ly49H+ NK cells in resistant mice is dependent on perforin. Chapter 3 focuses on the biological relevance of Grz during MCMV infection. These studies found that GrzA and GrzB are essential components of the machinery involved in limiting MCMV during acute infection. These analyses also provide the first evidence suggesting that GrzM plays a role, albeit minor, in controlling MCMV replication. Furthermore, the current studies suggest that Grz can mediate direct antiviral activities independent of the induction of cell death in conjunction with perforin. Interestingly, in the absence of both GrzA and GrzB (GrzAB), mice were as susceptible to MCMV infection as perforin-deficient mice. However, unlike perforin-deficient mice, GrzAB-deficient mice controlled and survived the infection. In Chapter 4 the roles of perforin, GrzA and GrzB in anti-viral immunity and immunopathology during MCMV infection were examined. These studies show that NK cell-derived perforin is required to eliminate infected targets as well as activated effector cells, suggesting that NK cells are crucial not only in defensive immunity but also in limiting the immune activation that follows MCMV infection. In summary, the studies presented in this thesis define the significant role played by specific effector molecules in limiting MCMV replication during different stages of this viral infection. Furthermore, these studies provide novel evidence that perforin, GrzA and GrzB play distinct roles in defensive immunity and limiting immunopathology during MCMV infection.
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Benechet, Alexandre. "Dynamic of the effector T cells egress from secondary lymphoid organs after infection". Paris 7, 2014. http://www.theses.fr/2014PA077126.
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Une réponse immunitaire efficace va dépendre de la migration des cellules immunitaires à l'intérieur et entre les tissus lymphoïdes. Notre compréhension des facteurs impliqués dans la migration des lymphocytes T effecteurs antigen-spécifiques après une infection reste incomplète. La sortie des T effecteurs du ganglion drainant est une des étapes essentielles dans l'éradication potentielle du virus au site de l'infection. Bien qu'il soit connu que le récepteur au shingosine-1- phosphate (S1PR1) contrôle la sortie des T naïfs, son influence sur l'émigration des T effecteurs après infection reste obscure. Dans cette étude, nous avons dans un premier temps utilisé la technique de marquage au complexe d'histocompatibilité majeur (CHM) Tétramérique in-situ, ainsi que l'imagerie intra vitale d'un modèle de souris rapporteur granzyme B (GzmB) YFP afin de décrire le panel migratoire des T antigène-spécifiques dans le ganglion drainant après une infection virale localisée. Les T effecteurs localisent premièrement au paracortex tôt après l'infection puis enfin migrent à la périphérie du ganglion. Une fois en périphérie, les effecteurs vont emprunter les sinus lymphatiques corticaux ainsi que medullaires pour émigrer du ganglion. De plus, afin d'investiguer le role de S1PR1 dans la dynamique des T effecteurs nous avons développé une souris déficiente spécifiquement dans les T effecteurs dont la délétion est contrôlée dans le temps après infection. En utilisant ce modèle unique nous avons clairement démontré que même, en l'absence de signaux de rétention comme CCR7 et CD62L, S1PR1 est le signal essentiel à la sortie des T effecteurs du ganglion drainantAn effective immune response depends on the large-scale, but carefully regulated migration of cells within and between lymphoid tissues. Our understanding of the factors that regulate the anatomical program followed by antigen-specific T cells during an infection remains incomplete. Egress of effector T cell from the draining lymph node (dLN) is one of the essential steps for the eventual eradication of the pathogen at the infection site. Although it is known that sphingosine-1-phosphate receptor 1 (S1PR1) controls naive T cell exit, how S1PR1 influences the emigration of effector T cells after infection is not well understood. Herein, by using both in-situ major histocompatibility complex (MHC)-tetramer staining and intravital imaging of a granzyme B (GzmB) YFP reporter mouse, we mapped the endogenous antigen-specific CD8 T cell response after localized viral infection in the dLN. In fact, we observed the localization of effector T cells in the paracortex early after infection, followed by the migration to the periphery. Notably, they exit the dLN via the medullary and cortical lymphatic sinuses. Furthermore, to assess the role of S1PR1 in their dynamic behavior, we generated a conditional GzmB YFP deficient mice to disrupt S1PR1 signals specifically and temporally in effector CD8 T cells after infection. Using this unique model we clearly demonstrate that after infection, even in the absence of retention signals such as CCR7 and CD62L S1PR1 signaling is the overriding factor that regulates effector T cell emigration from the dLN
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Chollat-Namy, Marie. "Effet de l’inactivation du gène suppresseur de tumeur p53 et de sa réactivation pharmacologique sur la réponse cytotoxique anti-tumorale The Pharmalogical Reactivation of p53 Function Improves Breast Tumor Cell Lysis by Granzyme B and NK Cells Through Induction of Autophagy Mutant P53 Gain of Function Stimulates PD-L1 Expression". Thesis, université Paris-Saclay, 2020. http://www.theses.fr/2020UPASL032.
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Le système immunitaire joue un rôle important dans le contrôle et l'éradication du cancer. Des acteurs majeurs de la réponse immune antitumorale sont les cellules tueuses naturelles (ou cellules NK) et les lymphocytes T cytotoxiques (ou CTL), capable de reconnaitre et détruire des cellules tumorales par l’exocytose de perforine et de granzymes contenus dans leur granule cytotoxique. Il a été montré au sein du laboratoire l’implication de la protéine suppresseur de tumeur p53 dans cette voie apoptotique. Or, plus de 50% des tumeurs humaines présentent des mutations inactivatrices de p53 ce qui favorise le développement tumoral. De ce fait, l’inactivation fréquente de p53 dans les tumeurs humaines pourrait leur permettre d’échapper à la destruction par les CTL et les cellules NK.Dans ce contexte, mes travaux de thèse ont montré que la réactivation pharmacologique de la fonction de p53 sauvage dans des cellules tumorales exprimant une p53 mutée augmente leur susceptibilité à la lyse induite par les cellules NK grâce à l’induction d’un processus d’autophagie. De plus, j’ai cherché à déterminer le lien entre les mutations de p53 et l’expression à la surface des cellules tumorales de PD-L1 qui empêche l’activation optimale des cellules cytotoxiques et conduit à leur épuisement. Mes travaux actuels suggèrent que l’expression de p53 mutantes induits une surexpression de PD-L1 à la surface des cellules cancéreuses. Les mécanismes expliquant ce phénomène sont en cours d’étudesImmune system plays an important role in the control and destruction of cancer cells. The major effectors of antitumor immune response are Natural Killer (NK) cells and the cytotoxic T lymphocytes, which recognize et destroy tumor cells by exocytosis of perforin and granzymes contained in cytotoxic granules. It has been previously shown in the laboratory that the tumor suppressor p53 plays an important role in this apoptotic pathway. However more than 50% of human tumors have p53 inactivating mutations which favor tumor development. Consequently, frequent p53 inactivation in human tumor could enable them to escape from destruction by cytotoxic immune cells. In this context, my thesis work has shown that the pharmacological reactivation of wild type p53 function in cancer cells expressing a mutated p53 increased their susceptibility to NK cell-mediated apoptosis cells through the induction of an autophagic process. Moreover, I tried to determine the link between p53 mutations and the expression of the immune checkpoint ligand PD-L1 which prevent efficient activation of cytotoxic cells and promote immune cells exhaustion. My work suggests that the expression of p53 mutants promotes an the expression of PD-L1 at the cancer cell surface. The study of the underlying mechanisms is still in progress
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Musembi, Susan Mbithe. "Immunological assays relevant to definition of bovine theileria parva-specific cytotoxic CD8+ T cell responses". Thesis, Brunel University, 2012. http://bura.brunel.ac.uk/handle/2438/7171.
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A major objective in Theileria parva subunit vaccine development is to induce a vaccine antigen specific response mediated by cytotoxic CD8+ T cells (CTL). Therefore it is essential to be able to measure the frequency of the responding CD8+ T cells after vaccination and correlate it with a clinical outcome on challenge. Recently concluded immunogenicity and efficacy studies of T. parva specific CTL antigens showed successful induction of CTL responses in some animals, which correlated with reduced disease severity after challenge. To provide correlates of immunity antigen-specific CD8+ T cell mediated IFN-γ responses and CTL lytic responses were measured over the course of the experiments. Several challenges presented in these trials aimed at optimising vaccine efficacy. While the IFN-γ ELISPOT is a sensitive and reliable assay widely used in vaccine research, the use of chromium/indium release assay remains to be the only assay in use that measures T. parva-specific CTL activity. Hence the overall goal of the study was to develop novel reagents and novel assays to identify parasite-specific CD8+ T lymphocytes with lytic potential. To address this objective, bovine perforin, granzymes A and B, as specific effector proteins expressed in activated CTL were cloned and expressed using a baculovirus expression system. Sequence analysis of the cloned cDNAs showed the isolated cDNA belonged to the perforin and granzyme sub-families respectively. Perforin cDNA demonstrated 85% homology to human perforin with presence of conserved regions resembling calcium binding motif, membrane attack complex component as well complement protein. The sequences encoded by the cloned granzyme A and B cDNAs have the features of a trypsin like serine protease and demonstrates over 70% homology to the human cDNA over the active enzyme region as well catalytic residues characteristic of serine proteases. The expressed polypeptides of all three proteins were used to produce specific antibodies for use as reagents in immunoassays including ELISpot and intracellular staining for flow cytometric analysis. While the antibodies showed reactivity to the recombinant proteins, these reagents displayed different functionality in the recognition of the native protein. Peptide-major histocompatibility complexes (MHC) class I tetrameric complexes (tetramers) are proving invaluable as fluorescent reagents for enumeration, characterisation and isolation of peptide-specific CD8+ T cells and have afforded advantages to phenotype antigen-specific T cells with minimal in vitro manipulation. Fluorescent bovine tetramers were shown to specifically stain antigen-specific CTL by directly binding the T cell receptor (TCR). Analyses of CD8 T-cell responses in live-vaccine immunised cattle also showed that this method is robust and demonstrates changes in the kinetics and specificity of the CD8+ T cell response in primary and secondary infections with T. parva. On average, results of functional assays and tetramer staining followed parallel trends, measured roughly the same populations and allowed for surface and intracellular staining for CD8 T cell marker and perforin, respectively, demonstrating a method that reliably quantifies the frequency, phenotype and function of specific CD8+ T cells. The technical simplicity, rapidity and ability of the flow cytometric technique described in this thesis to measure low frequency antigen-specific responses suggests that tetramer staining, combined with functional assays could be broadly applicable to the valuation of vaccination efficacy to determine which protocols are most successful in inducing CTL responses.
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Bruno, Alain. "Effets cellulaires des rayonnements ionisants sur les cellules hématopoïétiques immatures". Paris 11, 2000. http://www.theses.fr/2000PA11T021.
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A partir de lignées représentatives de la différenciation hématopoïétique, nous avons montré que les cellules hématopoïétiques immatures CD34+ sont moins sensibles que les cellules hématopoïétiques matures CD34- aux radiations. Dans les cellules CD34-, les radiations activent une mort par apoptose, en rapport avec la stimulation d'une sphingomyélinase neutre responsable de l'hydrolyse de la sphingomyéline nucléaire, et génération intranucléaire de céramide. Dans les cellules CD34+, les radiations n'entraînent aucune production de céramide et pas d'apoptose, et activent un processus de mort reproductive différée. Ces résultats suggèrent que la sphingomyélinase neutre nucléaire joue un rôle critique dans l'orientation de la réponse cellulaire aux rayonnements. Nous avons ensuite tenter de déterminer la nature des évènements régulant négativement la sphingomyélinase nucléaire dans les cellules CD34+, en envisageant l'implication d'évènements protéolytiques intranucléaires. Nous avons ainsi montré : la présence de granzyme B, une sérine protéase, dans le noyau des cellules CD34+; que les radiations augmentent l'expression de granzyme B dans le noyau et dans le cytoplasme des cellules CD34+; que la surexpression de granzyme 8 n'est pas responsable de l'absence de réponse apoptotique des cellules CD34+ aux radiations ; que la surexpression de granzyme 8 confère à ces cellules un fort potentiel cytotoxique vis-à vis de diverses cibles cellulaires d'origine myéloïde ou lymphoïde. Nos résultats suggèrent que les cellules CD34+ irradiées présentent un avantage de survie, et acquièrent une cytototoxicité vis-à-vis des cellules de l'environnement médullaire. Par contre, sur les lymphocytes T cytotoxiques, les radiations utilisées à des doses infratoxiques inhibent l'expression de granzyme 8, phénomène corrélé à l'effondrement de la fonction cytotoxique. Ces résultats suggèrent un effet immunosuppresseur des radiations lié à leur capacité de moduler l'expression de granzyme BUsing cell lines representative of hemopoietic differentiation, we showed that immature hemopoietic CD34+ cells are less sensitive to ionizing radiation than mature CD34- cells. Ln immature cells, ionizing radiation activate apoptotic cell death related to neutra! sphingomyelinase stimulation responsible for nuclear sphingomyelin hydrolysis and nuclear ceramide generation. Ln CD34+ cells, ionizing radiation do not produce neither ceramide nor apoptosis, and activate delayed reproductive cell death (mitotic cell death). These results suggest that nuclear neutra! sphingomyelinase plays a pivotai role in the cellular response of ionizing radiation. Ln a second part, we tried to determine the mecanisms responsible for negative regulation of nuclear sphingomyelinase in CD34+ cells. We first considered nuclear proteolytic events. Thus, we provided evidences for the presence of the serine-protease, Granzyme B, in the nucleus of CD34+ cells. We showed that ionizing radiation up-regulates Granzyme B expression both in the nucleus and the cytoplasm of these cells. This overexpression is not responsible for the lack of apoptotic response of CD34+ cells to ionizing radiation. Lnterestingly, we observed that Granzyme B overexpression confers a potent cytotoxic ability to these cells towards target cell lines of myeloid and lymphoid origin. Our results suggest that when irradiated, CD34+ cells acquire cytotoxic potential toward cells of the medullar environment. Conversely, in cytotoxic T lymphocytes, ionizing radiation triggers down-regulation of Granzyme B expression correlated with the loss of cytotoxic function. These results suggest immunosuppressor effects of ionizing radiation, related to their capacity to modulate Granzyme B expression
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HADDAD, PATRICK. "Etude de la structure et de la regulation de genes codant pour des serine esterases (granzymes a et h) chez l'homme : relations a la cytotoxicite a mediation cellulaire et applications a la clinique". Paris 7, 1990. http://www.theses.fr/1990PA077202.
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Au cours du mecanisme de cytotoxicite mediee par les lymphocytes t ou les cellules natural killer, la delivrance du coup letal resulterait de l'exocytose des granules cytoplasmiques des cellules cytotoxiques. Dans ce modele, la perforine, molecule formant des pores, et une famille de serine esterases ou granzymes, sont relarguees lors de l'interaction de la cellule effectrice avec sa cellule cible et joueraient un role important dans la lyse de la cellule cible. Nous avons isole le gene codant pour le granzyme b humain ainsi qu'un autre gene tres proche, designe granzyme h. Ces deux genes forment avec la cathepsine g, un complexe de genes de serine esterases a proximite du locus des chaines alpha et delta du recepteur t a l'antigene sur la bande 14q11-q12 du chromosome 14 de l'homme. La comparaison des organisations genomiques et des sequences en acides amines montre que les granzymes constituent une famille multigenique chez l'homme et la souris. Nous avons montre que les genes des granzymes b et h sont preferentiellement exprimes dans les cellules cytotoxiques, in vitro. Une etude d'hybridation in situ, a l'aide de sondes arn, sur des biopsies ou des ponctions de cellules de patients ayant recu une allogreffe de rein, de cur ou de moelle osseuse a permis de detecter la presence d'arnm de la perforine et du granzyme b in vivo, dans les cellules infiltrant la greffe. Ainsi l'expression des genes de la perforine et du granzyme b pourrait servir de marqueur dans les phenomenes d'activation des cellules infiltrant le greffon pour suivre la prise (ou le rejet) d'une allogreffe chez l'homme
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Hariss, Fatima. "Etude du rôle des Lymphocytes Intraépithéliaux innés dans la réponse immune protectrice contre Cryptosporidium parvum". Thesis, Université de Lille (2018-2021), 2021. http://www.theses.fr/2021LILUS047.
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Les lymphocytes intraépithéliaux (LIE) résident entre les cellules épithéliales (CE) intestinales et sont donc les premières cellules immunitaires à entrer en contact avec les agents pathogènes. En plus de répondre rapidement à l'infection, ils régulent l'homéostasie intestinale et maintiennent la barrière épithéliale. Ces fonctions sont assurées par différentes sous-populations de lymphocytes T et innés. Les LIE innés ont été identifiés récemment. Ils sont majoritaires dans l'épithélium intestinal à la naissance et lorsque l'immunité adaptative est compromise. Leur rôle dans la réponse immunitaire contre les pathogènes intestinaux reste cependant peu étudié.Au cours de ma thèse, j'ai étudié le rôle des LIE innés dans l'infection à Cryptosporidium, un parasite opportuniste qui infecte l'épithélium intestinal. L'infection est bénigne chez les individus immunocompétents, mais peut être sévère chez les individus immunodéprimés et les enfants.Pour étudier le rôle spécifique des LIE innés, nous avons développé un modèle in vitro qui consiste à co-cultiver des organoïdes intestinaux murins infectés par C parvum avec des LIE innés de souris RAG2-/-. Grâce à ce modèle original, nous avons démontré que les LIE innés contrôlent la prolifération du parasite et bien qu’ils sécrètent l'IFN-ƴ en réponse à C parvum, cette sécrétion n’est pas suffisante pour inhiber la prolifération du parasite. L'effet protecteur des LIE innés est en fait médié par un mécanisme cytotoxique dépendant des granzymes. L’analyse du transcriptome a révélé que les CE infectées régulent négativement la serpinb9b, un inhibiteur de granzyme, et pourraient ainsi être plus sensibles aux attaques cytotoxiquesIntraepithelial lymphocytes (IEL) reside between intestinal epithelial cells and thus are the first immune cells to contact intestinal pathogens. In addition to respond rapidly to infection, they regulate intestinal homeostasis and maintain the epithelial barrier. This wide range of functions is achieved by distinct subsets of T and innate lymphocytes. Innate IEL which share many features with NK/ILC1 cells have been identified recently. These cells dominate the gut epithelium at birth and when the adaptive immunity is compromised. Their role in the immune response against intestinal pathogens remains however poorly studied.During my PhD thesis, I have investigated the role of innate IEL subsets in Crysptosporidium infection. Crysptosporidium is a common parasite that infects the gut epithelium. The infection is self-limiting in immunocompetent individuals, but it can be severe in immunocompromised individuals and children in whom innate IEL dominate.To study the specific role of innate IEL, we have developed an in vitro model that consist to co-culture mice 3D intestinal organoids infected with C parvum with innate IELs from RAG2-/- mice. Thanks to this original model, we demonstrated that innate IELs control parasite proliferation. We further showed that although innate IEL secrete IFN-ƴ in response to C parvum infection, the IFN-ƴ secretion was not sufficient to inhibit parasite proliferation. The protective effect of innate IELs was in fact mediated by a cytotoxic, granzyme-dependent mechanism. Moreover, transcriptomic analysis revealed that infected epithelial cells down regulated serpinb9b, a granzyme inhibitor, and thus may be more sensitive to cytotoxic attack
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Boivin, Wendy Anne. "Extracellular granzyme B and pathophysiological implications". Thesis, University of British Columbia, 2012. http://hdl.handle.net/2429/42827.
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Granzyme B (GzmB) is a serine protease that contributes to immune-mediated elimination of
cells by initiating a tightly-regulated form of death known as apoptosis. However, during
inflammation, GzmB leaks out and accumulates in the extracellular space, retains its activity,
and proficiently cleaves extracellular matrix (ECM) proteins. I therefore hypothesized that
extracellular GzmB is capable of cleaving novel ECM substrates, contributing to dysregulated
ECM integrity and function in disease. In the present dissertation I identified eleven novel
extracellular GzmB substrates. Further investigations revealed that GzmB-mediated
proteoglycan cleavage was implicated in the dysregulation of active transforming growth factorbeta
(TGF-β) sequestration and bioavailability. GzmB cleavage sites were identified in biglycan
and betaglycan and active TGF-β was shown to be released from decorin, biglycan and
betaglycan. The pathophysiological role of my findings were further investigated and validated
using animal models of disesase in which inflammation and elevated GzmB are observed.
Evidence of fibrillin-1 and decorin cleavage were observed in atherosclerosis, abdominal aortic
aneurysm and in skin aging pathogenesis. I also assessed the activity of GzmB in advanced
atherosclerosis using perforin/apolipoprotein E- double knockout (Perf/apoE-DKO) and
granzyme B/apolipoprotein E-double knockout (GzmB/apoE-DKO) mice. Interestingly, unlike
our aneurysm findings whereby only GzmB/apoE-DKO mice were protected, both Perf/apoEDKO
and GzmB/apoE-DKO mice were protected from atherosclerosis compared to apoE-KO
controls, suggesting the intracellular Perf-dependent activities of granzymes are also important
in the pathogenesis of atherosclerosis. In summary, GzmB is a protease that functions both
intracellularly and extracellularly in disease. My findings suggest that the use of Perf knockout
mice alone to study the role of GzmB in disease should be re-evaluated given the increasing evidence in both animal models and in human disease showing elevated GzmB in bodily fluids is associated with inflammation and age.
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Hiebert, Paul Ryan. "Granzyme B in skin aging, injury and repair". Thesis, University of British Columbia, 2013. http://hdl.handle.net/2429/44226.
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Hendel, Alon. "Granzyme B in vascular remodeling and pathological angiogenesis". Thesis, University of British Columbia, 2013. http://hdl.handle.net/2429/44782.
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Granzyme B (GZMB) is a serine protease that is expressed by a variety of immune cells and is abundant in a large number of chronic inflammatory disorders. GZMB is highly expressed in cytotoxic lymphocytes where it serves as the main effector molecule of the granule exocytosis pathway by which cytotoxic immune cells mediate target cell death through intracellular delivery of GZMB, leading to activation of apoptotic signaling cascades. GZMB can also accumulate extracellularly during inflammation, where it can cleave a range of extracellular matrix (ECM) proteins that may disrupt cell-matrix interactions and modulate the bioavailability of matrix-bound growth factors. In this dissertation I have explored the intracellular and extracellular roles of GZMB in vascular remodeling in disease. By examining human atherosclerotic plaques, I discovered an imbalance between GZMB and its endogenous inhibitor, proteinase inhibitor 9 (PI-9). PI-9 expression by vascular smooth muscle cells (VSMC) in plaques was reduced with increased disease severity. Elevated levels of GZMB in advanced lesions were correlated with reduced PI-9 expression and increased VSMC apoptosis. These findings suggest that VSMC are more susceptible to GZMB-induced apoptosis in advanced lesions due to reduced PI-9 expression. While examining the extracellular activities of GZMB on vascular remodeling, I focused on the role of GZMB-mediated cleavage of fibronectin (FN), a known GZMB substrate. FN has a major role in regulating angiogenesis as it facilitates endothelial cell (EC) migration and capillary formation, as well as binding to angiogenic growth factors in the ECM including vascular endothelial growth factor (VEGF). VEGF is a potent vascular permeabilizing agent that is sequestered in the ECM by binding FN. GZMB-mediated FN cleavage resulted in reduced EC adhesion, migration and capillary tube formation. In addition, GZMB-mediated FN cleavage induced the release of VEGF from the ECM and promoted VEGF-dependent vascular leakage in vivo. Thus, GZMB may contribute to the progression and/or persistence of chronic inflammation by dysregulating angiogenesis and promoting vascular permeability. Collectively, the results of this work suggest that both intracellular and extracellular GZMB activities contribute to vascular remodeling and pathological angiogenesis.
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Estébanez, Perpiñá Eva. "Crystal Structure of Human Granzyme B. Modelling of the Granzyme B-Cation-Independent Mannose-6-Phosphate Receptor Complex. Crystal Structure of Human Pro-Granzyme K. Crystal Structure of the Procarboxypeptidase from Helicoverpa armigera". Doctoral thesis, Universitat Autònoma de Barcelona, 2002. http://hdl.handle.net/10803/3477.
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Estructura Tridimensional de Granzima B humanaGranzima B es la proteina prototípica de la familia de serina proteasas que se encuentran en células NK y CTLs. GzmB induce apoptosis mediante activación de las caspasas, y está implicada en la etiología de la artritis reumatoidea. Hemos cristalizado y resuelto la estructura 3D de la GzmB humana a una resolución de 3.1Å. GzmB muestra un plegamiento similar al de catepsina G y quimasa humanas. GzmB exhibe una especificidad de substrato muy inusual ya que corta tras Asp, debido a la presencia de Arg226 en bolsillo de especificidad S1. El sitio de unión del substrato está diseñado para acomodar y cortar hexapéptidos, i.e. IETD_SG, secuencia presente en el lugar de activación de la caspasa-3 y de Bid, sus substratos fisiológicos. Esta estructura ayudará en el diseño de inhibidores que podrian ser usados en la cura de enfermedades inflamatorias crónicas.
Granzyme B y el Receptor Manosa-6-Fosfato Independiente de Cationes
GzmB cristalizó como dímero, mediante la interdigitación de las cadenas de azúcares unidas a Asn65 en los dos monómeros. GzmB es captada por las células mediante el Receptor Manosa-6-Fosfato Independiente de Cationes. Sugerimos que la GzmB dimérica sea la forma internalizada por las células diana. Hemos modelado cómo posiblemente GzmB se une a su receptor celular.
Estructura Tridimensional de pro-Granzyme K humana
Hemos determinado la estructura 3D de la pGzmK a una resolución de 2.2Å. pGzmK se parece más a una serina proteasa activa, a pesar de ser un zimógeno. Esta proteina carece de triada zimogénica y utiliza un nuevo mecanismo de estabilización de la forma inactiva.
Estructura Tridimensional de una Procarboxipeptidasa de Helicoverpa armigera. H. armigera es un insecto cuya plaga afecta a un gran número de países. Hemos determinado la estructura 3D de una nueva carboxipeptidasa (PCPAHa) de larvas de H. armigera, la primera resuelta de un insecto hasta la fecha. El zimógeno de PCPAHa muestra una estructura similar a las ya resueltas de mamífero. Su sitio de activación presenta el motivo (Ala)5Lys. Es curioso apreciar similar motivo ((Ala)6Lys) cerca del extremo N-terminal del enzima activo. Ser255 agranda el bolsillo S1´de especificidad y influencia las preferencias de substrato de ésta enzima. Hemos modelado el extremo C-terminal del LCI dentro del sitio activo de PCPAHa.
Crystal Structure of Human Granzyme B
Granzyme B is the prototypic member of the granzymes, trypsin-like serine proteinases localized in activated NK cells and CTLs. GzmB triggers apoptosis by activating the caspases, and is implicated in the etiology of rheumatoid arthritis. Human GzmB has been crystallized and its structure has been determined to 3.1 Å resolution. GzmB overall fold is similar to that found in cathepsin G and human chymase. GzmB exhibits an unusual substrate specificity as it cleaves after Asp residues due to the presence of Arg226 at the back of the S1-specificity pocket. GzmB substrate binding site is designed to fit and cleave hexapeptides, i.e. IETD_SG, sequence present in the activation site of caspase-3 and Bid, physiological substrates of GzmB. This structure would help in the design of inhibitors for a treatment of chronic inflammatory disorders.
Granzyme B and the Cation-Independent Mannose-6-Phosphate Receptor
Our crystal structure of GzmB unexpectedly revealed a dimer, mediated by the interdigitation of the sugar chains attached to Asn65 in the two monomers. The uptake of GzmB is effected by the cation-independent mannose-6-phosphate (M6P) receptor. We suggest that the GzmB dimer would be the form preferentially recognized by its receptor. To investigate the probable binding mode of GzmB to its cell receptor we have modeled the binding of the GzmB dimer to the M6P-receptor.
Crystal Structure of Human Pro-Granzyme K
We have determined the crystal structure of human pGzmK at 2.2 resolution. The overall fold of pGzmK is most similar to that found in active serine proteinases rather than in zymogens. An unusual feature of pGzmK is that the residues Ser32, His40 and Asp194 do not form a zymogen triad, while pGzmK uses a novel mechanism for zymogen stabilization.
Crystal Structure of a Procarboxypeptidase from Helicoverpa armigera
H. armigera is one of the most serious insect pests worldwide. We present the 2.5 Å crystal structure from this novel procarboxypeptidase (PCPAHa) from H. armigera larvae, the first one reported for an insect. PCPAHa zymogen has a 3D structure similar to the corresponding mammalian digestive carboxypeptidases. The activation site contains the motif (Ala)5Lys. It is noteworthy the occurrence of the same (Ala)6Lys near the C-terminus of the active enzyme. Ser255 enlarges the S1' specificity pocket and influences the substrate preferences of the enzyme. The C-terminal tail of LCI was modeled into PCPAHa active site.
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Fellows, Edward. "Granzyme H: A novel cell-death-inducing serine protease". Diss., lmu, 2007. http://nbn-resolving.de/urn:nbn:de:bvb:19-78190.
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Chamberlain, Ciara M. "Granzyme B in abdominal aortic aneurysm and aortic dissection". Thesis, University of British Columbia, 2008. http://hdl.handle.net/2429/5584.
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An aneurysm is a permanent focal dilatation of an artery, often associated with
atherosclerosis and with a weakening of the vessel wall. An arterial dissection is when a tear
in the inner layer of the blood vessel causes blood to flow between the layers and force the
blood vessel apart. Aneurysms or dissections can result in rupturing of the vessel, leading to
excessive hemorrhaging and death if not immediately repaired. Granzyme B (GrB) is a
protein that is expressed and secreted by cytotoxic immune cells. GrB has been identified as
a key player in atherosclerosis both in the induction of apoptosis in target cells and the
degradation of extracellular matrix proteins. We generated GrB/apolipoprotein E (apoE)
double knockout (GrB/apoE-DKO) mice, and have shown that advanced atherosclerosis is
significantly reduced in GrB/apoE-DKO mice as compared to apoE-KO mice. Evaluation of
elastin staining indicated a loss of elastic tissue and medial thinning in the aortas of apoE-KO
mice that was restored when GrB was absent in the GrB/apoE-DKO mice.
Since medial thinning renders the aorta wall more susceptible to aneurysms or dissections,
we hypothesize that the absence of GrB will reduce the incidence of aneurysm formation and
dissection in GrB/apoE-DKO mice as compared to apoE-KO mice during angiotensin II
(angli) infusion.
To induce aortic dissections, 3-month-old C57 (wildtype), apoE-KO, and GrB/apoE-DKO
mice were implanted with an osmotic mini pump which released a dose of 1000ng/kg/min of
angli (or saline as a control) for 28 days. The mice were euthanized at 28 days, and the
aortas were removed and evaluated for gross morphology and, after cross sectional staining,
for histopathological lesions.
A significant reduction in aortic dissections and aneurysms was observed in GrB/apoE-DKO
mice as compared to apoE-KO mice treated with angII. Further, GrB deficiency
corresponds to a significant increase in survival at the 28 day time-point. Aneurysms and
dissections were not observed in the saline-treated groups or the angli-treated wild-type
controls. In conclusion, our studies suggest that GrB contributes to the loss of vessel wall
integrity and to the occurrence of aortic aneurysm and dissection.
20
Pinkoski, Michael J. "The role of granzyme B in target cell apoptosis". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/nq23057.pdf.
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Uhl, Dieter. "Einfluss körperlicher Ausdauerbelastung auf Perforin und Granzyme-B-exprimierende Lymphozytenpopulationen". [S.l.] : [s.n.], 2002. http://www.freidok.uni-freiburg.de/volltexte/440.
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Ang, Lisa Shouning. "The extracellular role of granzyme B in abdominal aortic aneurysm". Thesis, University of British Columbia, 2013. http://hdl.handle.net/2429/45649.
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Trzaska, Timo [Verfasser]. "The role of granzyme B in antigen-presenting cells / Timo Trzaska". Ulm : Universität Ulm, 2021. http://d-nb.info/1234554968/34.
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Hirst, Claire Elizabeth 1971. "Tissue distribution and regulation of the granzyme B inhibitor, proteinase inhibitor 9". Monash University, Dept. of Biochemistry and Molecular Biology, 2002. http://arrow.monash.edu.au/hdl/1959.1/8488.
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Babichuk, Charolyn Kim. "Transcriptional regulation of the murine granzyme B gene in cytotoxic T cells". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/nq22945.pdf.
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Haeryfar, Seyed Mohammad Mansour. "Antiestrogens modulate the perforin/granzyme pathway of natural killer cell-mediated cytolysis". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape8/PQDD_0001/MQ45052.pdf.
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Bannard, Oliver Michael. "The memory functions of CD8⁺ T cells that have expressed granzyme B". Thesis, University of Cambridge, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.611596.
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Yokoi, Akiko. "Selective Expression and Function of Granzyme D in the Lymphohematopoietic Stromal Cells". Kyoto University, 2000. http://hdl.handle.net/2433/181270.
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Schiffer, Sonja [Verfasser]. "Improving the therapeutic potential of human granzyme B and evaluation of granzyme M as novel effector molecules in cytolytic fusion proteins for the treatment of Serpin B9-positive cancer / Sonja Schiffer". Aachen : Hochschulbibliothek der Rheinisch-Westfälischen Technischen Hochschule Aachen, 2014. http://d-nb.info/1048671534/34.
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Dhenni, Rama B. S. "Role of Granzyme B in the Susceptibility to Secondary Bacterial Infection after Viral Infection". University of Cincinnati / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1460446984.
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Merkulova, Yulia. "Granzyme B inhibits keratinocyte migration by disrupting epidermal growth factor receptor (EGFR)-mediated signaling". Thesis, University of British Columbia, 2016. http://hdl.handle.net/2429/58050.
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Chronic skin ulceration is a common complication and cause of morbidity in the elderly, diabetic, obese and/or immobile populations. Effective therapies that adequately promote efficient closure and remodelling of chronic wounds are lacking. Inflammation and excessive protease accumulation and activity are thought to play key roles in the impairment of normal wound healing. Granzyme B (GzmB) is a serine protease that was, until recently, believed to function exclusively in cytotoxic lymphocyte-mediated apoptosis. However, during dysregulated and/or chronic inflammation, GzmB can accumulate in the extracellular milieu, retain its activity, and cleave a number of important extracellular proteins. Epidermal growth factor receptor (EGFR) is a transmembrane receptor involved in cellular processes such as proliferation and migration. EGFR signaling is integral to the wound healing process. I hypothesized that GzmB impairs EGF-induced keratinocyte migration by impairing EGFR signaling. The present study investigated the effects of GzmB on keratinocyte cell migration. Using Electric Cell Substrate Impedance Sensing (ECIS) and other in vitro wound healing assays, the present study demonstrates that GzmB inhibits keratinocyte migration by interfering with the EGFR pathway. GzmB limited cell transition into a migratory morphology and was found to reduce ligand-induced EGFR phosphorylation. Inhibition of GzmB reversed the aforementioned effects in HaCaT cells. In summary, data from the present study suggests a key role for GzmB in the pathogenesis of impaired wound healing through the reduction of EGFR signaling and cell migration.Medicine, Faculty of
Pathology and Laboratory Medicine, Department of
Graduate
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Sharma, Mehul. "Extracellular Granzyme K mediates endothelial inflammation through the cleavage of Protease Activated Receptor-1". Thesis, University of British Columbia, 2015. http://hdl.handle.net/2429/55402.
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Granzymes are a family of serine proteases that were once thought to function exclusively as mediators of cytotoxic lymphocyte-induced target cell death. However, non-lethal roles for granzymes, including Granzyme K (GzK), have been recently proposed. As recent studies have observed elevated levels of GzK in plasma of patients diagnosed with sepsis, we hypothesized that extracellular GzK induces a pro-inflammatory response in endothelial cells. In the present study, extracellular GzK proteolytically activated Protease Activated Receptor-1 (PAR-1) leading to increased IL-6 and MCP-1 production in Human Umbilical Venous Endothelial Cells (HUVEC). Enhanced expression of ICAM-1 along with an increased capacity for adherence of THP-1 cells was also observed. Characterization of downstream pathways implicated the MAPK p38 pathway for ICAM-1 expression, and both the p38 and the ERK1/2 pathways in cytokine production. GzK also increased TNFα–induced inflammatory adhesion molecule expression. Furthermore, the physiological inhibitor of GzK, IαIp, significantly inhibited GzK activity in vitro. In summary, extracellular GzK is not cytotoxic but promotes a pro-inflammatory response in endothelial cells.Medicine, Faculty of
Pathology and Laboratory Medicine, Department of
Graduate
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Schwesinger, Elisabeth [Verfasser]. "Charakterisierung Granzym-B-sezernierender B-Zellen / Elisabeth Schwesinger". Ulm : Universität Ulm. Medizinische Fakultät, 2012. http://d-nb.info/1020022434/34.
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Panitz, Verena Michaela [Verfasser]. "Regulation of human granzyme B-producing plasmacytoid dendritic cells by viral stimuli / Verena Michaela Panitz". Ulm : Universität Ulm, 2017. http://d-nb.info/1122644876/34.
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Seignez, Cédric. "Etude des mécanismes d'action d'une immunothérapie par un lipide A, seul ou associé à l'oxaliplatine, dans des modèles de cancers coliques". Thesis, Dijon, 2013. http://www.theses.fr/2013DIJOMU01/document.
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Le cancer colorectal est un problème de santé publique majeur pour lequel la recherche de nouveaux traitements est indispensable. Notre équipe a démontré l’efficacité d’une immunothérapie par un lipide A dans un modèle de cancer du colon chez le rat. Lorsque les rats sont porteurs de petites carcinomatoses, le lipide A induit la guérison de 95% des rats. L’étude des mécanismes d’action de cette immunothérapie nous a permis de montrer que l’effet antitumoral du lipide A est dépendante de la cytotoxicité induite par le granzyme B produit par les neutrophiles intratumoraux. En effet, nous avons montré que, dans le microenvironnement tumoral, les neutrophiles produisent du granzyme B et présentent un phénotype de type N2 protumorigène. Lorsque les rats sont traités par lipide A, il y a modification du phénotype des neutrophiles en type N1 antitumoral et libération du granzyme B qui induit l’apoptose des cellules tumorales. Lorsque les rats développent des tumeurs beaucoup plus volumineuses, l’efficacité du lipide A est diminuée et seul 40% des animaux sont guéris. L’injection préalable d’oxaliplatine permet alors de maintenir l’efficacité de l’immunothérapie par le lipide A. Nous avons montré que l’oxaliplatine induit la sénescence des cellules tumorales, générant ainsi un microenvironnement propice au recrutement au sein des tumeurs des neutrophiles, lesquels sont activables par l’immunothérapie subséquente. L’association de l’induction de la sénescence et de l’activation des cellules immunitaires par immunothérapie est une approche efficace et originale sur laquelle les recherches doivent se poursuivreColorectal cancer is a major public health concern in France. Resistance to standard chemotherapy requires development of novel therapeutic approaches. In the past decades, our team showed the immunotherapeutic properties of lipid A in a model of colon cancer in rats. 95% of rats bearing small carcinomas were cured following treatment by lipid A. The study of mechanisms underlying this immunotherapy allowed us to show that the antitumor effect of lipid A was dependent on cytotoxicity induced by granzyme B produced by intratumoral neutrophils. Indeed, we have shown that, in the tumor microenvironment, neutrophils produced granzyme B and had a pro-tumorigenic N2 phenotype. When rats were treated with lipid A, neutrophils shifted to an antitumor N1 phenotype and released granzyme B, thus inducing apoptosis of tumor cells. In rats bearing advanced carcinoma, the effectiveness of lipid A was reduced and only 40% of animals were cured. An injection of oxaliplatin prior to lipid A treatment allowed sustaining the effectiveness of lipid A immunotherapy. In the present study, we showed that oxaliplatin injection induced tumor cell senescence. The microenvironment produced by senescent cells enabled then the recruitment of neutrophils within tumors, subsequently activated by lipid immunotherapy.Combining the induction of tumor cells senescence and activation of immune cells by an immunotherapeutic agent constitute an original and interesting therapeutic approach, but still studies must be carrying out to better understand underlying mechanisms
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Hirayasu, Hirofumi. "Studies on the role of a lymphocyte protease granzyme A in the intestinal epithelial-cell turnover". Kyoto University, 2010. http://hdl.handle.net/2433/131909.
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Kyoto University (京都大学)0048
新制・論文博士
博士(農学)
乙第12509号
論農博第2743号
新制||農||986(附属図書館)
学位論文||H22||N4574(農学部図書室)
28300
京都大学大学院農学研究科食品生物科学専攻
(主査)教授 伏木 亨, 教授 井上 國世, 教授 安達 修二
学位規則第4条第2項該当
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Pashkovska, N. V. "The role of perforin/granzyme-induced apoptosis in the development of cognitive impairment in diabetes mellitus". Thesis, БДМУ, 2021. http://dspace.bsmu.edu.ua:8080/xmlui/handle/123456789/18836.
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Dahlke, Karen [Verfasser]. "Die regulatorische Rolle Granzym B-sezernierender B-Lymphozyten / Karen Dahlke". Ulm : Universität Ulm. Medizinische Fakultät, 2013. http://d-nb.info/1036454460/34.
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Nishie, Mariko. "Downregulated ATP6V1B1 expression acidifies the intracellular environment of cancer cells leading to resistance to antibody-dependent cellular cytotoxicity". Kyoto University, 2021. http://hdl.handle.net/2433/261614.
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Ngan, David Allen. "Discovering inflammatory biomarkers in chronic obstructive pulmonary disease and cystic fibrosis : a case study of granzyme B". Thesis, University of British Columbia, 2011. http://hdl.handle.net/2429/36292.
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Granzymes, and particularly granzyme B (GzmB), are classically known to be involved in cell-mediated immunity and the induction of apoptosis through cell-specific targeting activity of cytotoxic T-lymphocytes in conjunction with perforin. However, recent literature has emerged that describes a largely overlooked role for GzmB in potentially mediating disease progression. This pathogenic role has been based on findings that GzmB can cleave extracellular matrix proteins while functioning independently of perforin. In chronic inflammatory lung states such as chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF), there are important implications including the degradation of extracellular matrix leading to airway remodelling, reduced lung integrity, and emphysema. The generation of autoantigens may also result from cleavage of extracellular matrix proteins, contributing to inflammation.
We measured GzmB levels in plasma and lung tissue homogenates of COPD subjects by enzyme-linked immunosorbent assays (ELISAs) to determine the relationship with lung function, measured by FEV₁% predicted and FEV₁/FVC ratio, and clinical COPD severity. We found that GzmB levels in the lung were positively associated with lung function. The data raise the possibility that the GzmB we measured may be part of a protective inflammatory response in the microenvironment, or it may be pathogenic in the early but not the later phases of COPD.
In CF subjects, we measured levels of GzmB and other inflammatory biomarkers in plasma samples to determine their relationship with lung function parameters and hospitalization status. While plasma levels of GzmB were not related to lung function or hospitalization status, we found that IL-6, IL-1β, and LPS levels were significantly higher in hospitalized patients, and CRP, IL-6, IL-1β, and LBP were significantly correlated with lung function impairment. The results provide evidence that systemic inflammation is an independent factor associated with disease progression in CF and suggests an important role for chronic bacterial colonization in the lungs.
Further research is needed to validate the pathogenic contributions of GzmB in diseases with chronic inflammatory lung states and to delineate the mechanisms of such potential contributions.
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Al, Absi Antoun. "Role of the actin cytoskeleton in breast cancer cell resistance to natural killer cells". Thesis, Strasbourg, 2018. http://www.theses.fr/2018STRAJ038/document.
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L'évasion immunitaire tumorale joue un rôle central dans la progression tumorale et représente un obstacle majeur au succès des immunothérapies. Dans cette Thèse nous avons étudié le rôle du cytosquelette d’actine dans la résistance des cellules de cancer du sein à la lyse induite par les cellules "natural killers" (NKs). Nous avons trouvé que les cellules de cancer du sein résistantes échappent à l’attaques des cellules NKs par une accumulation importante et rapide d’actine près de la synapse immunologique, un processus que nous avons nommé "réponse actine". Nos analyses mécanistiques suggèrent que la réponse actine induit la polarisation d’autophagosomes vers la synapse immunologique et facilite ainsi la dégradation des molécules cytotoxiques sécrétées par les cellules NKs, tel que le ganzyme B, par autophagie. De plus, la réponse actine est associée au regroupement de ligands inhibiteurs à la synapse, suggérant qu’elle est au centre de plusieurs mécanismes de résistance. Dans leur ensemble, nos résultats constituent une base pour le développement d’approches thérapeutiques visant à interférer avec la réponse actine et à restaurer une réponse immunitaire anti tumorale efficaceTumor immune evasion plays a central role in cancer progression and is a major hurdle to effective immunotherapy. In this Thesis, we examine the role of the actin cytoskeleton in breast cancer cell resistance to natural killer (NK) cell-mediated cell lysis. We found that resistant breast cancer cells escape from NK-cell attack through a rapid and prominent accumulation of actin near the immunological synapse, a process we termed the “actin response”. Our mechanistic investigations suggest that the actin response drives autophagosome polarization toward the immunological synapse and thereby facilitates the autophagy-mediated degradation of NK cell-derived cytotoxic molecules such as granzyme B. In addition, the actin response was associated with inhibitory ligand clustering at the immunological synapse, suggesting that it is a common driver of different immune evasion mechanisms. Taken together, our data lays the groundwork for therapeutic approaches aimed at interfering with the actin response and restoring an effective anti-tumor immune response
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Vargas, Hernández Giovanni [UNESP]. "Linfomas cutâneos em cães: estudo epidemiológico, morfológico, imunofenotípico e seroproteico". Universidade Estadual Paulista (UNESP), 2017. http://hdl.handle.net/11449/151209.
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O linfoma cutâneo (LC) em cães é uma doença que se caracteriza pela proliferação clonal de linfócitos atípicos na pele (MILLER et al., 2013). É um linfoma não-Hodgkin, formado por um grupo de doenças neoplásicas malignas de linfócitos T e B e células Natural Killer (NK), cuja primeira manifestação clínica é a presença de lesões cutâneas, sem existir lesão extra cutâneas no momento do diagnóstico (RUEDA; CORTES, 2008). A apresentação clínica é inespecífica e pode mimetizar muitas dermatites (FONTAINE et al., 2010). Nas fases inicias da doença torna-se difícil diferenciar a condição neoplásica de quadro inflamatório linfocítico cutâneo (MURPHY; OLIVRY 2000). Descrita pela primeira vez em 1972 (KELLY et al., 1972), e para muitos autores a doença rara e de etiologia desconhecida (FONTAINE et al., 2009; WITHROW et al., 2013). Diversos estudos mencionaram novas variedades e formas de apresentação do LC na espécie canina baseadas principalmente nos resultados de reações imuno-histoquímicas, comportamento clínico da neoplasia e nas frequentes mudanças da classificação desta doença na espécie humana, que tem levado na classificação em novas variantes de LC (WILLEMZE et al., 2005; DE BOSSCHERE; DECLERCQ, 2008; AFFOLTER et al., 2009; FONTAINE et al., 2009; MILLER et al., 2013; MOORE et al., 2012). Nas últimas três décadas, a classificação morfológica e imunofenotípica do LC em medicina veterinária baseou-se nos critérios de classificação utilizados em humanos (VALLI et al., 2011). O objetivo desta revisão é definir quais são as atuais variantes, tipos e subtipos do LC em cães e estabelecer as principais semelhanças e diferenças com a classificação existente na espécie humana.
Cutaneous lymphoma (LC) in dogs is a disease characterized by the clonal proliferation of atypical lymphocytes in the skin (Miller et al., 2013). It is a non-Hodgkin's lymphoma, consisting of a group of malignant neoplastic diseases of T and B lymphocytes and Natural Killer (NK) cells, whose first clinical manifestation is the presence of cutaneous lesions, with no extra-cutaneous lesion at the time of diagnosis (WHEEL . The clinical presentation is non-specific and may mimic many dermatitis (FONTAINE et al., 2010). In the early stages of the disease, it is difficult to differentiate the neoplastic condition of cutaneous lymphocytic inflammatory disease (MURPHY; OLIVRY 2000). It was first described in 1972 (KELLY et al., 1972), and for many authors, the rare disease of unknown etiology (FONTAINE et al., 2009, WITHROW et al., 2013). Several studies have mentioned new varieties and forms of LC presentation in the canine species based mainly on the results of immunohistochemical reactions, clinical behavior of the neoplasia and the frequent changes in the classification of this disease in the human species, which has led to classification in new variants of LC (Muller et al., 2009), and in the literature on the use of this method (Macker et al., 2009). In the last three decades, the morphological and immunophenotypic classification of CL in veterinary medicine was based on the classification criteria used in humans (VALLI et al., 2011). The objective of this review is to define the current variants, types and subtypes of LC in dogs and establish the main similarities and differences with the existing classification in the human species.
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Woodsworth, Daniel. "Characterizing the granzyme-perforin pathway and its utility as a cell-to-cell delivery system for cellular therapeutics". Thesis, University of British Columbia, 2017. http://hdl.handle.net/2429/62073.
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Alongside small molecules and biologics, cell-based therapies are emerging as a third class of medical therapy. Additional sensors, actuators and control circuits would greatly expand the range of function and application of cellular therapeutics. To this end, a cell-to-cell delivery module has been developed by investigating and re-engineering the granzyme-perforin pathway of cytotoxic lymphocytes. A computational biophysical model of this process was developed and implemented using a spatial stochastic simulation algorithm, which indicated that hindered diffusion in the immune synapse is critical to ensure reliable granzyme internalization and that large amounts of granzyme escape the synapse, but should not have toxic effects due to rapid spatiotemporal dilution. Additionally, these results indicated that passive diffusion is sufficient for granzyme entry into the target cell, which motivated efforts to use granzyme as a molecular chaperone to transfer exogenous payloads from effector to target cells. Using a fluorescent protein payload, the subcellular localization of several granzyme B derived chaperones was characterized using fluorescence microscopy, and then their capacity to transfer the payload to target cells was evaluated in co-culture experiments. The results indicated that the motifs in granzyme B that are required for lytic granule loading are only functional and contiguous in the folded protein. Additionally, these experiments demonstrated that full length granzyme B is a suitable chaperone for delivering protein payloads to target cells via the granzyme-perforin pathway. Attempts were then made to use this system to deliver potent orthogonal toxins to apoptosis and lymphocyte resistant tumor cells. A range of granzyme B toxin fusion proteins were constructed, all of which retained toxic activity to varying degrees. To render target cells resistant to lymphocyte attack both small molecule and protein based inhibitors of apoptosis were tested in several cell lines, which delayed cell death, but did not stop it. Using effector target dose response curves, a moderate increase in target cell death was observed in cells targeted by lymphocytes expressing granzyme toxin fusion proteins, as compared to wild type lymphocytes, but the biological significance of this effect is uncertain. Approaches to improve this granzyme-perforin mediated delivery system and its therapeutic utility are discussed and explored.Science, Faculty of
Graduate
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Doss, Brigitte. "Generation of an expression system for human granzyme B and analysis of the in vitro and in vivo efficiency". Diss., lmu, 2010. http://nbn-resolving.de/urn:nbn:de:bvb:19-124132.
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Stahnke, Bettina [Verfasser]. "Expression and in vitro characterisation of human Granzyme B-based immunotherapeutics for specific targeting of CD64+ malignancies / Bettina Stahnke". Aachen : Hochschulbibliothek der Rheinisch-Westfälischen Technischen Hochschule Aachen, 2011. http://d-nb.info/1018204113/34.
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Cheng, Wei. "Development of carbamate modified PEI for delivery of a granzyme B inhibitor gene to protect against cytotoxic lymphocyte killing". Thesis, Imperial College London, 2013. http://hdl.handle.net/10044/1/23983.
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Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells protect vertebrates by killing infected or transformed cells using granzyme B (GrB) to induce apoptosis. However, GrB-induced apoptosis causes inflammatory disease and allograft rejection and is an important disease target. The aim of this project is to prevent apoptosis of the target cells by delivering a plasmid encoding GrB inhibitor proteinase inhibitor-9 (PI-9) using cationic polymers. Polyethylenimine (PEI, branched, Mw 25 kDa) gives high gene transfection efficiency in many cell lines, but it is highly cytotoxic. To reduce its cytotoxicity, we modified PEI by blocking primary amines through nucleophilic addition between primary amine and a protected functionalized cyclic carbonate, generating a carbamate linkage through the ring-opening of the cyclic carbonate. Different hydrophobic-groups or sugar functionalized carbonates were substituted onto PEI and the optimum substitution ratios to achieve high gene transfection efficiency and minimal cytotoxicity in various cell lines were determined. Among these polymers, PEI with 7 or 20 of 56 primary amine groups substituted by mannose had similar gene binding ability as unmodified PEI, leading to almost 100% transfection efficiency of a GFP plasmid in HEK293T cells as well as a large decrease in cytotoxicity. However, PEI with all primary amine groups blocked was unable to form complexes with DNA, and gene transfection was negligible. The PI-9 encoding plasmid was transfected into HEK293T cells effectively using the optimally modified PEIs, protecting up to 80% HEK293T cells from killing by human natural killer-like YT cells. Furthermore, PEI with 25 primary amine groups substituted by mannose successfully delivered PI-9 plasmid into Balb/3T3 fibroblasts and protected them from killing by GrB. Therefore, the carbamate-mannose modified PEI/PI-9 encoding plasmid complexes have potential clinical utility in the prevention of allograft rejection and inflammatory disease caused by GrB.
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Lindner, Stefanie [Verfasser]. "Funktion und pathophysiologische Bedeutung Granzym B-exprimierender regulatorischer B-Zellen / Stefanie Lindner". Ulm : Universität Ulm. Medizinische Fakultät, 2015. http://d-nb.info/1074196171/34.
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Thorpe, Michael. "Haematopoietic Serine Proteases : A Cleavage Specificity Analysis". Doctoral thesis, Uppsala universitet, Institutionen för cell- och molekylärbiologi, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-221891.
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Mast cells are innate immune cells, historically involved in allergy responses involving IgE. Through this, they have earned a reputation as a fairly detrimental cell type. Their beneficial roles remain somewhat enigmatic although they clearly have the ability to modulate the immune system. This is due to their ability to synthesise many cytokines and chemokines as well as immediately release potent granule-stored mediators. One such mediator is a serine protease, chymase, which has been targeted by pharmaceutical companies developing inhibitors for use in inflammatory conditions. In order to address roles of the proteases, information regarding their cleavage specificity using substrate phage display can help find potential in vivo substrates. The human chymase cleaves substrates with aromatic amino acids in the P1 position and has a preference for negatively charged amino acids in the P2’ position. The molecular interactions mediating this P2’ preference was investigated by site-directed mutagenesis, where Arg143 and Lys192 had a clear effect in this selectivity. As humans express one chymase and rodents express multiple chymases, extrapolating data between species is difficult. Here, the crab-eating macaque was characterised, which showed many similarities to the human chymase including a near identical extended cleavage specificity and effects of human chymase inhibitors. Appropriate models are needed when developing human inhibitors for therapeutic use in inflammatory conditions. The effects of five specific chymase inhibitors in development were also tested. The selectivity of inhibitors was dependent on both Arg143 and Lys192, with a greater effect of Lys192. Identification of residues involved in specific inhibitor interactions is important for selective inhibitor development. Another innate cell type, the NK cell, is important in virus and tumour defence. In the channel catfish, a serine protease from an NK-like cell, granzyme-like I, was characterised. A strict preference for Met in the P1 position was seen, and caspase 6 was identified as a potential in vivo target. This may highlight a novel apoptosis-inducing mechanism from a similar cell type has been conserved for approximately 400 myr. Here, important residues mediating chymases’ specificity and interactions with inhibitors has been addressed, as well as finding a new animal model for providing ways to combat their roles in pathological settings.
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Sontheimer, Kai [Verfasser]. "Induktion von Granzym B in humanen B-Zellen durch virale Antigene / Kai Sontheimer". Ulm : Universität Ulm. Medizinische Fakultät, 2015. http://d-nb.info/1073211983/34.
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Thonel, d'Orgeix Aurélie de. "Réponse des cellules leucémiques immatures au ligand de Fas". Toulouse 3, 2003. http://www.theses.fr/2003TOU30003.
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